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the gluteus maximus (gm) plays a major role in functional activity as a trunk and hip extensor. weakness of the gm could cause dysfunction in the pelvic and hip joints and back pain1. prone hip extension (phe) is an exercise that can activate the gm effectively and is popularly used in clinics2. however, unwanted substitution motion, such as pelvic anterior tilt or excessive lumbar extension, could happen during phe due to stiffness of the hip flexor, dominance of the erector spinae (es), weakness of the gm, and so forth3. therefore, recent studies have focused on controlling unwanted substitution motion of the pelvis and lower back during phe4,5,6. many previous studies about phe have been performed at the 0 hip flexed position8,9,10. some other studies have used starting positions varying from 20 to 90 hip flexion for people with hip flexor contracture6, 7, 11.. however, little information exists concerning whether hip joint angles during phe selectively change the emg activity of the gm. therefore, the purpose of this study was to examine the effects of initial hip joint position on the muscle activity of the es, gm, and biceps femoris (bf) as measured by electromyography (emg) of the hip extensor as well as the effect of the angle of pelvic anterior tilt. through this process, we sought the most effective position for selective gm muscle activation during phe. fifteen healthy adults were enrolled in this study (age, 26.73.7 years [meansd ] ; height, 167.19.2 cm ; mass, 58.111.7 kg). exclusion criteria were neuromuscular or musculoskeletal dysfunction of the lower back and both legs, lower back or hip pain, contracture of the hip flexor, or a hip extensor strength below fair. all subjects agreed to participate and signed an informed consent form approved by the inje university ethics committee for human investigations. prior to testing, kinematic data of the pelvis were recorded at 100 hz using an eight - camera vicon mx - t20 motion capture system (vicon motion systems ltd., the respective muscle activities of the bilateral es, unilateral gm, and biceps femoris bf were collected using a delsys trigno wireless emg system (delsys inc., natick, ma, usa), which was synchronized with a vicon motion system. the emg signals were sampled at 2,000 hz and filtered at 20 to 450 hz, and the root mean square values of the signals were calculated. the emg data were expressed as a percentage relative to the maximal voluntary contraction for normalization. a newly developed two - segment model consisting of the lumbar spine and pelvis was used to measure the angle of pelvic anterior tilt. the lumbar segment was defined with four markers located on the subject s l1 and l2 spinous processes, and 1 cm lateral from the l2 spinous process on both sides. the pelvic segment was defined with three markers located on the mid - point of the posterior superior iliac spine and the bilateral anterior and superior iliac spines. the subjects lifted their right legs during phe at three starting positions arranged in random order (0, 20, and 45 hip flexed position). with phe at 0 hip flexion, each subject was positioned prone on a table consisting of 2 segments that were jointed in a way that it was possible to adjust the table s position so the subject s hip could be positioned at an initial hip flexion of 20 or 45. a bar placed over the table would come in contact with the achilles tendon when the subject s hip was extended to 30 from the initial position. emg activities and pelvic kinematics were measured when subjects extended from their initial position. all subjects performed 3 repetitions with a 10-second rest between trials and rested for 2 minutes between each testing position. pasw statistics for window (ver. 18.0 ; spss inc., chicago, il, usa) was used to analyze differences in muscle activities and pelvic kinematics. repeated measures anova and bonferroni - adjusted post hoc comparisons were used to compare the respective muscle activities of the es, gm, and bf and the amount of pelvic anterior tilt among the three initial positions. there were significant differences in es, gm and bf muscle activity on the left side among the three positions during phe. muscle activity for the es on the right side did not show any difference in emg among the three positions. the amount of pelvic anterior tilt showed a significant decrease at the 20 hip flexion position compared with that at the 0 angle (table 1table 1.normalized emg data and amount of pelvic anterior tilt (n=15)variableship joint position (meansd)02045lt. es (% mvic)49.515.344.112.431.713.2rt. bf (% mvic)36.59.433.39.638.711.1pelvic anterior tilt ()7.42.05.92.47.32.8bf : biceps femoris ; es : erector spinae ; gm : gluteus maximus ; lt. bf : biceps femoris ; es : erector spinae ; gm : gluteus maximus ; lt. : left ; mvic : muscle voluntary isometric contraction ; rt. : of the three positions, active phe at 45 resulted in a significant decrease in muscle activity of the contralateral es compared with the other positions. the role of the es as a global stabilizer was reduced at 45 because alteration of this position from the initial position to 30 hip extension induced pelvic anterior tilt. therefore, we consider that the 45 flexed position would be able to effectively prevent excessive lumbar motion and maintain an upright pelvis alignment we think that the 45 hip flexed position decreased internal momentum due to the short lever arm in comparison with other positions12. although the muscle activity of the contralateral es tended to decrease at 20, this decrease was not significant. this investigation revealed that the 20 hip flexed position needs an additional 10 hip extension for a total of 30 extension during phe, which may result in contraction of the es. however, this position increased gm activity compared with the other positions and decreased bf activity compared with that occurring with the 45 hip flexion angle. additionally, the amount of pelvic anterior tilt at 20 was lower than those at 0 and 45. we consider that the pelvic anterior tilt provided by 20 hip flexion led to the optimal length by elongation of the gm, which may contribute to increases in gm activity12. worrell.7 demonstrated that gm activity was greatest at the 0 angle and that hs muscle activity did not differ among all the positions they studied, which was not in line with the results of our study. they examined gm and bf activity with changes in hip and knee joint angles during maximal voluntary isometric contraction, whereas we investigated muscle activities with initial positions to 30 extensions of the hip joint during active phe with knee extension. chance - larsen.9 reported that restraint of pelvic anterior tilting during phe decreased the length of the bf and influenced the synergistic relationship between the gm and bf. of three positions, 0 could negatively impact the lumbar segment by increasing the es activity and 45 did not activate the gm in spite of the reduction of es activity. hence, the 45 position could be recommended for persons who need to minimize the weight - bearing load on the lumbar spine. in conclusion, we suggest that 20 is the optimal position to activate the gm selectively by minimizing additional pelvic anterior tilt. first, our sample size was small, so it is difficult to generalize the findings to all subjects. second, we examined the changes in emg - measured muscle activity at only three joint angles ; it is possible that the results do not indicate the optimal position for phe. therefore, further studies are needed to investigate the changes at the various phe joint angles. | [purpose ] the aim of this study was to identify the effects of initial position of the hip joint with changes in the hip joint angle on the respective muscle activities of the bilateral erector spinae (es), unilateral gluteus maximus (gm), and biceps femoris (bf) and the amount of pelvic anterior tilt during prone hip extension (phe). [subjects ] fifteen healthy volunteers were enrolled in this study. [methods ] the subjects performed phe in three positions : neutral, 20, and 45 flexed hip joint. the activities of the es, gm, and bf were measured using surface electromyography, and kinematic values for pelvic anterior tilt were calculated using a motion capture system. [results ] there was a significant decrease in muscle activity of the contralateral es at 45, and an increase in the gm muscle activity and decrease in the bf muscle activity at 20. the amount of pelvic anterior tilt was lower at 20. [conclusion ] these results suggest that a hip flexion position of 20 would have an advantage over the other measured positions. |
vitreoretinal pathology has rarely been observed after laser - assisted in situ keratomileusis (lasik) or photorefractive keratectomy (prk). posterior segment complications are more common in myopic eyes and no direct causal relationship with keratorefractive surgery has been established. retinal breaks, rhegmatogenous detachments, posterior vitreous detachments, choroidal neovascularization (cnv), and vitreous hemorrhage have all been reported [13 ]. central serous chorioretinopathy (cscr) is generally a self - limited disease with the majority of patients recovering visual acuity to 20/30 or better within three months. approximately 4% of patients will present with binocular involvement, while up to 40% may eventually have involvement of the fellow eye. increased permeability of the choroidal vessels and dysfunction of the retinal pigment epithelium (rpe) have been observed with neurosensory retinal detachment and pigment epithelial detachment. patients present with central visual aberrations including scotoma, metamorphopsia, dyschromatopsia, and micropsia. known risk factors for cscr include type a personality, pregnancy, and corticosteroid elevation either endogenously or through exogenous corticosteroid use. cscr has been reported in 4 separate case reports after lasik [912 ]. to date, there have not been any reports of cscr after prk. the goal of this study was to report the incidence of cscr after both prk and lasik through a retrospective chart review. from january 2005 to december 2010, an icd-9 code search for cscr (362.41) identified 250 cscr patients seen at the john a. moran eye center. patients ' age, sex, medication history, pre- and postrefractive surgery exam findings, onset of cscr symptoms, clinical course, treatment, and visual outcomes were analyzed. the university of utah hospital institutional review board approved the research protocol in accordance with the tenets of the declaration of helsinki. database billing records of the moran eye center were reviewed to determine the total number of lasik and prk procedures performed during the 6-year study period. prior to 2008, a hansatome microkeratome (bausch & lomb, inc, rochester, ny) was used for flap creation. thereafter, the intralase fs60 femtosecond laser (abbott medical optics (amo), santa ana, ca) was utilized. both prk and lasik were performed with either the visx star s4 customvue (amo), ladarvision customcornea (alcon inc., fort worth, tx), or the wavelight allegretto (alcon, inc.) excimer laser platforms. all moran eye center refractive surgery patients are routinely followed at a minimum of 6 visits at 1 day, 1 week, 1, 3, 6, and 12 months after surgery. any retinal complications that are discovered during postoperative exams are routinely referred to our institution 's retina service. because of our internal referral system, any cscr complication after lasik or prk would be identified in the review of retina record visits. a medline keyword search for cscr and prk or lasik identified all previously reported cases of cscr after lasik or prk in the literature. of the 250 cscr patients seen by the retina service, 7 patients were identified as having a previous history of lasik or prk at either the moran eye center or an outside facility (table 1). all 7 patients were male with an average age of 37 years, and all presented with unilateral cscr. all had myopic keratorefractive treatment, and none had a previous history of cscr or documented oral, topical skin, or inhaled corticosteroid use or other significant medical history. only 2 patients (patients 1 and 2) had onset of disease within the immediate postoperative period of 1 month. the remaining patients presented more than 4 years after refractive surgery and were not considered to have a potential causal relationship. all 7 patients were followed at the moran eye center from the time of initial cscr symptoms to resolution or stabilization of disease. two of the patients received treatment with focal laser photocoagulation, while 5 resolved spontaneously without treatment. all 7 patients improved to an uncorrected distance visual acuity (udva) of 20/30 or better. best corrected visual acuity (bcva) was not recorded in all patients. from january 2005 to december 2010, there were 2,728 eyes which had lasik, and 2,148 eyes that received prk at the moran eye center. only one of the combined 4,876 total lasik and prk cases at our institution developed cscr within one month. the annual incidence of cscr over the 6-year study period was calculated to be 3.4 per 100,000 eyes for both prk and lasik cases, and 7.8 per 100,000 eyes of only prk cases. a comprehensive literature search revealed 4 other reports of cscr after lasik ; however, long - term recovery was not available in all cases (table 1). two patients presented with bilateral cscr [10, 11 ] and 1 patient developed a rapid onset of cscr with cnv one week following lasik. this is a 37-year - old male with an unknown employment who underwent prk in september 2010 with a preoperative manifest refraction of 4.50 + 0.25 134 and 4.50 sphere, right and left eyes, respectively. spectral domain optical coherence tomography (sd - oct, heidelberg engineering, heidelberg, germany) demonstrated subretinal fluid accumulation consistent with cscr (figure 1) in the affected eye. consultation with a retina specialist led to the decision to keep the patient on standard corticosteroids drops with close observation. after one month, the patient was switched to fluorometholone 0.1% (allergan inc.) udva improved to 20/20 bilaterally 1 month after initial symptoms with complete resolution of metamorphopsia by 5 months after prk. anterior and posterior segment exam and sd - oct findings were normal and the patient was advised to monitor his symptoms without further intervention. this is a 28-year - old male lab technician who underwent prk at an outside facility in august 2009. his preoperative manifest refraction was 4.50 + 1.00 90 and 4.75 + 1.75 90, right and left eyes, respectively. the diagnosis of cscr was confirmed by fundus exam and oct subretinal fluid in the left eye, with a normal exam in the right eye. ten months after prk his udva was 20/15 in the left eye with no visual complaints. cscr as a complication of refractive surgery is rare with only 4 previously reported cases [912 ]. these reports identified 2 hyperopic [9, 10 ] and 1 myopic patient who developed cscr shortly after lasik and 1 patient who developed cscr with cnv. the annual age - adjusted incidence of cscr has been reported to be 5.8 per 100,000, with a male incidence of 9.9 and a female incidence of 1.7 per 100,000. although not directly comparable with population studies, our findings support a large male predominance with a low annual incidence of 7.8 per 100,000 eyes after prk and 3.4 per 100,000 eyes after both prk and lasik, based on analysis of the cases performed at our institution over 6 years. there were 5 patients who developed cscr 4 years or more after lasik or prk. these patients were excluded from our analysis as having cscr potentially caused by lasik or prk. limitations in our study include the retrospective nature, possible loss of followup, and development of cscr after the time period of medical chart review. chronic cscr often is bilateral, with symptoms lasting longer than 6 months and can occur with recurrent disease. the high percentage of spontaneously resolving cases is the reason conservative management is initially advised. corticosteroids should be discontinued and if resolution is not observed within 3 months more aggressive management can be considered. half - fluence photodynamic therapy (pdt) and photocoagulation result in more rapid resolution of subretinal fluid, and may also limit recurrences. previous studies that explored processes leading to vitreoretinal pathology following refractive surgery have identified two possible exposures. the first is potential shock wave damage in the posterior segment during excimer laser treatment. however, krueger. reported insignificant retinal stress wave amplitudes in experimental models after excimer photoablation. the second potential risk during lasik is the rapid change in intraocular pressure from the suction ring used to stabilize the microtome. although less vacuum is required with femtosecond - laser - assisted lasik, the longer flap creation time may still predispose patients to retinal pathology. both mechanisms can theoretically destabilize a structurally altered retina such as in the case of cscr. corticosteroids are often used after prk for their anti - inflammatory properties as an attempt to prevent long - term sequelae of haze and unpredictable refractive outcomes. although, some reports have shown no difference in outcomes following prk treated with or without topical corticosteroids [15, 16 ], most prk surgeons reported using some form of topical corticosteroids postoperatively. early corticosteroid treatment has also been reported more effective than nonsteroidal anti - inflammatory drugs. multiple routes of corticosteroid administration have been associated with cscr, including oral, intravenous, epidural, intra - articular, inhaled, topical skin application, and also following intravitreal triamcinolone injections. however, to our knowledge there have been no reported cases of cscr following topical ocular corticosteroid use. the lack of such cases may reflect low penetration of topical drugs in reaching the posterior segment, with even lower systemic absorption due to further dilution in the tear film. in rabbit eyes, difluprednate showed dramatically decreased concentrations in the posterior retina and barely detectable blood concentrations, suggesting that even a highly lipophilic topical corticosteroid with excellent corneal penetration may not significantly alter retinal pathology. alternatively, the stress and anxiety of refractive surgery inducing elevated levels of systemic cortisol may contribute to exacerbations in cscr patients. the dilemma for the surgeon is how to treat such a patient who develops cscr after prk. continuing topical corticosteroids may prolong or exacerbate cscr symptoms, whereas early removal may decrease control over inflammation potentially leading to long - term haze. whether topical corticosteroids are discontinued or changed to less potent formulations, worsening cscr may still require consideration of pdt or laser photocoagulation. the decision was made to continue our patient on topical corticosteroids with close clinical observation. the patient was noted to have mild rpe changes preoperatively which may have indicated prior cscr disease activity. a dilated fundoscopic exam is an important part of both the refractive screening process and postoperative evaluation when outcomes are not as expected. in summary, from review of the literature and analysis of over 4,800 consecutive lasik and prk cases at our institution, we report a low incidence of cscr after lasik or prk without a direct causal association. cscr after keratorefractive surgery may rarely be induced by extensive topical corticosteroids, elevated cortisol levels from stress of the surgery, or mechanical disturbances after excimer laser or lasik flap creation in susceptible patients. | purpose. to assess the incidence of central serous chorioretinopathy (cscr) following laser in situ keratomileusis (lasik) and photorefractive keratectomy (prk). methods. a chart review was performed to identify all patients with cscr and a previous history of lasik or prk. results. over the 6-year study period, 1 of 4,876 eyes which had lasik or prk at the moran eye center was diagnosed with cscr. one other patient was referred from an outside center, developed cscr symptoms one month after prk. both patients were managed conservatively with a final visual acuity of 20/20 or better. all other patients presented 4 or more years after refractive surgery. conclusions. we report the first 2 cscr cases developing within one month after prk. the low incidence argues against a causal association. topical corticosteroids or anxiety may elevate cortisol levels presenting therapeutic challenges for the management of cscr after prk or lasik. |
the global prevalence of overweight and obesity in children and adolescents has increased substantially over the past several decades. many studies done previously, suggest that children and adolescents with risk factors such as obesity, dyslipidemia, elevated blood pressure and impaired glucose metabolism are at increased risk of developing atherosclerosis in adulthood. high - sensitivity c - reactive protein (hscrp) has been emerged as a novel biomarker for vascular inflammation associated with atherosclerosis. accumulating evidence suggests that hscrp, which is also found within macrophages of atheromatous plaques. however, there is no much data available that guarantee its utility as a marker of cardiovascular risk in obese children and adolescents. hence, the present study was taken up to assess the metabolic abnormalities and its association with hscrp in obese children and adolescents. a total of 62 obese children and adolescents were consecutively recruited from our department of endocrinology and metabolism, nellore, andhra pradesh, india over a period of 14 months (october 2011 to december 2012). obesity was defined based on reference values stated in the centers for disease control and prevention growth chart (cdc charts). children with body mass index (bmi) greater than the 95 percentile for age and gender were classified as obese. those with bmi equal to or exceeding 85 percentile but, below the 95 percentile are defined as overweight. secondary causes of obesity such as hypothyroidism, cushing 's disease and other causes were excluded. none of the patients was taking any medication at the time of evaluation for the presence of insulin resistance (ir) and impaired glucose tolerance / diabetes mellitus. the criteria for healthy control group were no major medical illness and normal glucose tolerance test according to the american diabetes association criteria. the study was approved by the institutional ethical committee and informed consent was taken from parents. physical examination included measurements of height (by a stadiometer measuring with accuracy of 0.1 cm), weight (by electronic weighing machine) and blood pressure were obtained. bmi was calculated as the weight in kilograms divided by the height in meters squared. systolic blood pressure (sbp) and diastolic blood pressure (dbp) were measured at the right arm after 15-min rest in sitting position using manual sphygmomanometer. after a 10-h overnight fast, venous blood samples were collected for laboratory evaluation of fasting glucose and insulin, triglycerides, total cholesterol and high - density lipoprotein (hdl) cholesterol and hscrp. glucose levels were measured by glucose oxidase and peroxidase method ; triglycerides, total cholesterol were measured by the enzymatic and cholesterol oxidase methods respectively, hdl - cholesterol (hdl - c) were measured by direct immunoturbidimetry method. low - density lipoprotein (ldl) were calculated by friedewald 's formula (ldl = tc [tgl/5 + hdl ]). glucose and lipid levels were measured by commercially available in vitro assay kits from human gmbh on humastar 600 (fully automated chemistry analyzer), max - planck - ring 21, wiesbaden, germany. hscrp was measured by immunoturbidimetric method (aptec technologies) on humastar 600 (fully automated chemistry analyzer). insulin was measured by chemilumniscence using beckman immunoassays on beckman coulter access 2, ca, usa. both intra - assay and inter - assay coefficient of variations were lesser than 3.6% and 4.8%, respectively. ir was calculated using homeostatic model assessment - ir (homa - ir) according to the formula (homa - ir = plasma glucose [mg / dl ] insulin [miu / l]/405). pearson 's correlation analysis was performed to assess the relationship between hscrp and other metabolic variables. after a 10-h overnight fast, venous blood samples were collected for laboratory evaluation of fasting glucose and insulin, triglycerides, total cholesterol and high - density lipoprotein (hdl) cholesterol and hscrp. glucose levels were measured by glucose oxidase and peroxidase method ; triglycerides, total cholesterol were measured by the enzymatic and cholesterol oxidase methods respectively, hdl - cholesterol (hdl - c) were measured by direct immunoturbidimetry method. low - density lipoprotein (ldl) were calculated by friedewald 's formula (ldl = tc [tgl/5 + hdl ]). glucose and lipid levels were measured by commercially available in vitro assay kits from human gmbh on humastar 600 (fully automated chemistry analyzer), max - planck - ring 21, wiesbaden, germany. hscrp was measured by immunoturbidimetric method (aptec technologies) on humastar 600 (fully automated chemistry analyzer). insulin was measured by chemilumniscence using beckman immunoassays on beckman coulter access 2, ca, usa. both intra - assay and inter - assay coefficient of variations were lesser than 3.6% and 4.8%, respectively. ir was calculated using homeostatic model assessment - ir (homa - ir) according to the formula (homa - ir = plasma glucose [mg / dl ] insulin [miu / l]/405). pearson 's correlation analysis was performed to assess the relationship between hscrp and other metabolic variables. a p < 0.05 was considered to be statistically significant. table 1 shows anthropometrical and biochemical characteristics of the study subjects. there is no significant difference between the study groups in terms of age and fasting plasma glucose. bmi, sbp and dbp were significantly elevated in obese children and adolescents compared to healthy controls. fasting insulin levels (p < 0.001) and ir calculated by homa - ir (p < 0.001) and hscrp (p < 0.001) were significantly higher in obese children and adolescents when compared with the healthy control group. obese children and adolescents had significantly higher serum total cholesterol (p = 0.005), triglycerides (p < 0.001), ldl - cholesterol (ldl - c) (p = 0.006) and decreased hdl - c (p < 0.001) compared to control group. anthropometric and biochemical variables of the study subjects table 2 shows pearson 's correlation analysis between hscrp and metabolic variables. hscrp showed a positive correlation with bmi (r = 0.357 ; p = 0.028), total cholesterol (r = 0.367 ; p = 0.008), ldl - c (r = 0.356 ; p = 0.01), insulin (r = 0.311 ; p = 0.026) and not with homa - ir (r = 0.244 ; p = 0.084). no significant association was found with bmi, triglycerides, hdl - c and homa - ir. this study is first of its kind from the indian subcontinent, which shows the evidence for the presence of early cardiovascular risk factors in obese children and adolescents. many studies, have demonstrated a high cardiovascular risk profile in overweight and obese children and adolescents compared with a normal weight children. in this study, compared with age matched and normal weight subjects, obese children and adolescents had higher sbp and dbp, total cholesterol, triglycerides, ldl - c, insulin, ir and hscrp. in agreement with our study, meyer., showed higher sbp and dbp, higher concentrations of fasting triglycerides, insulin and hscrp in obese children compared with the normal weight controls., demonstrated that obese children had higher levels of blood pressure, triglycerides, glucose, insulin, ir and crp compared to the control group (100 obese children with 50 non - obese children). the probable mechanisms underlying this association with obesity is as follows ; the cytokines such as tumor necrosis factor- and interleukin-6 are released from adipose tissue and these cytokines stimulate the production of acute - phase proteins such as crp in the liver. elevated hscrp levels may be associated with the development of cardiovascular diseases and diabetes by means of a variety of mechanisms : altered sensitivity to insulin, increased liberation of adhesion molecules by endothelium, increase in hepatic production of fibrinogen and platelet coagulation factor. in our study, hscrp showed a positive correlation with bmi (r = 0.357 ; p = 0.028), total cholesterol (r = 0.367 ; p = 0.008) and ldl - c (r = 0.356 ; p = 0.01), insulin (r = 0.311 ; p = 0.026) and not with homa - ir (r = 0.244 ; p = 0.084)., showed a positive correlation between hscrp with bmi (r = 0.35 ; p 0.01) and insulin (r = 0.30 ; p = 0.01). however, in our study, there is no significant association between hscrp and blood pressure, triglycerides and hdl - c. in another study done by weiss., showed that there exists no statistical relationship between hscrp levels and metabolic risk factors. the limitations of this study are : (1) small sample size, (2) measurement of ir by homa - ir, not by the gold standard euglycemic glucose clamp technique. therefore, hscrp seems to be an excellent cardiovascular marker in obese children and adolescents and could be a useful tool for the early diagnosis of cardiovascular risk factors among this population. early intervention in these obese individuals can reduce the incidence of comorbidities in adulthood. | obesity in children and adolescents predispose to the development of obesity in adulthood and subsequent cardiovascular disease. high - sensitivity c - reactive protein (hscrp) is a marker of low grade inflammatory state, which characterizes an atherosclerotic process. the aim of this study was to assess the metabolic abnormalities and its association with hscrp in obese children and adolescents. a total of 62 obese children and adolescents and 24 healthy children and adolescents with a normal weight were recruited. in all subjects, anthropometric and biochemical parameters were measured. body mass index (bmi) and blood pressure were significantly higher in the obese children and adolescents than the control. obese children had significantly higher hscrp levels (p < 0.001), total cholesterol, triglyceride, low - density lipoprotein - cholesterol (ldl - c) and lower high - density lipoprotein - cholesterol than the control group. furthermore, homeostatic model assessment - insulin resistance (homa - ir) was significantly higher in obese children compared with the normal weight children. furthermore, hscrp showed a positive correlation with bmi (r = 0.357 ; p = 0.028), total cholesterol (r = 0.367 ; p = 0.008) and ldl - c (r = 0.356 ; p = 0.01), insulin (r = 0.311 ; p = 0.026) and not with homa - ir (r = 0.244 ; p = 0.084)). in conclusion, obese children and adolescents have significantly increased hscrp compared with a normal weight group. early intervention and prevention of obesity in children and adolescents decreases cardiovascular disease in later life. |
our device incorporates three key elements : patterned polymer brushes to present selected haptenic groups ; cofactors required for acwop ; and components for electrochemical detection and quantification of h2o2 (figure 1). details about the materials and methods used and additional control experiments are given in the supporting information. a fundamental feature of our device is the use of poly(oligoethylene glycol methacrylate) (poegma) polymer brushes (figure 2a) for anchoring a variety of hapten groups and for preventing nonspecific adsorption of other biomolecules that may be present in the test sample. oeg moieties are known to be resistant to protein adsorption and have long - term stability. this is due to the dense packing of neighboring chains which results in an increased entropic force and drives the brushes into a stretched state at high grafting densities to yield effective resistance to nonspecific binding. to produce the necessary high grafting density, we employed atom transfer radical polymerization (atrp) methods to grow brushes on either a silicon wafer chip or a gold - plated quartz crystal microbalance (qcm) crystal. by functionalizing the initiator end with either a silane or thiol group, polymer brushes have the capacity to be modified with a broad range of haptens for corresponding detection of antibodies with a broad range of specificities. for initial development and optimization of this platform, we used 2,4-dinitrophenyl (dnp) groups as a model hapten. this well - characterized hapten binds specific anti - dnp antibodies of several classes and species and can be conjugated to the ends of the brushes through a one - step process (figure 2a). the functionalized polymer brushes ranged in thickness from 13 to 35 nm, measured via ellipsometry, and two methods were utilized to confirm the presence of dnp groups. first, fluorescence imaging with fluorescently labeled anti - dnp igg antibodies verified the presence of the dnp groups when compared to a sample of unfunctionalized brushes (supporting information figure s1). second, dnp is electroactive, allowing measurement of its surface coverage through cyclic voltammetry by means of the reduction of nitro groups to the corresponding hydroxylamine (figure 2b). the surface coverage of dnp was typically on the order of 10 mol / cm, and as high as 1.5 10 mol / cm. inset : cyclic voltammogram of hydroxylamine formed upon reducing dnp. supporting electrolyte, 0.1 m h2so4 ; sweep rate, 100 mv / s. (c) frequency response of qcm crystal platform in pbs, ph 7.2, containing 1 mg / ml bsa solution at 25 c. rat anti - dnp igg antibody solution (resultant concentration 11 nm) was added at 9000 s. (a, b) grid patterns of 150 m wide lines surrounding 300 300 m square areas on silicon (a) and gold electrode of a qcm crystal (b). solid lines are for qcm crystal platform immersed in 0.5 mm [ru(v - bpy)3](pf6)2, showing electropolymerization of photosensitizer upon reduction. dashed lines are for an electropolymerized layer of [ru(v - bpy)3 ] in fresh solution. supporting electrolyte, 0.1 m tbapf6 in mecn ; sweep rate, 100 mv / s. quartz crystal microbalance (qcm) measurements were used to determine the surface coverage of the antibodies (figure 2c). the functionalized qcm crystal was placed in phosphate - buffered saline solution (pbs, ph 7.2), and the frequency was allowed to stabilize before the addition of a solution containing anti - dnp igg antibodies (figure 2c). acoustic impedance methods (supporting information figure s2) confirmed that the films were rigid, and the sauerbrey equation was used to relate the change in frequency to the mass of immobilized antibodies. the poegma brushes were tested for nonspecific adsorption in control experiments by incubating the qcm crystals modified with poegma - dnp in a solution of nonspecific antibodies. no significant change in the frequency was observed (supporting information figure s2), confirming that nonspecific antibodies do not bind to the brushes. furthermore, no significant increase in the amount of h2o2 was observed under these control conditions (vide infra) (supporting information figure s3). two types of platforms were used, one on silicon and another on a qcm crystal, in both cases presenting the polymer brushes adjacent to the photosensitizer on the same surface (figure 3a and b). our ultimate device is designed to employ silicon chips. however, for calibration purposes, qcm crystals were used to quantify the mass of the bound antibodies. for the silicon chips, gold was evaporated onto 1 2 cm silicon wafer pieces in a grid pattern. the photosensitizer was electropolymerized on the gold (vide infra), whereas the polymer brushes, grown as depicted in figure 2a, were confined to the silicon oxide squares (figure 3a). the same grid pattern was used for a qcm crystal (figure 3b). however, because both the polymer brushes and photosensitizer were polymerized from a gold surface on the qcm crystal, a series of steps was employed to prevent the two films from overlapping. first, a photoresist was spin coated onto the qcm crystals, exposed and developed to pattern the grid. next, the thiol functionalized atrp initiator was immobilized on the exposed gold surface, and the photoresist was subsequently removed. finally, the photosensitizer was electropolymerized on the sections where the photoresist was removed, and polymer brushes were then grown from the regions containing the immobilized atrp initiator. this specific order of patterning and immobilization of the different components allowed for the maximum yield of both photosensitizer and polymer brushes on the same surface. as described above, the acwop process requires singlet oxygen, o2, which can be generated from ambient oxygen o2 through the use of a photosensitizer, such as [ru(4-vinyl-4-methyl-2,2-bipyridine)3 ] ([ru(v - bpy)3 ]). we found that electropolymerized films of [ru(v - bpy)3 ] (figure 3c) on the gold electrode immediately adjacent to the brushes maximized production of h2o2. typical coverage of photosensitizer was 1.5(0.5) 10 mol / cm, corresponding to a thickness of ca. in addition to enhancing the signal - to - noise ratio, our results showed a significant increase in the acwop h2o2 signal for the adjacent, immobilized photosensitizer compared to photosensitizer in solution. this increase is likely the result of having the singlet oxygen generated in close proximity to the antibody. corresponding to a mean square distance (msd) diffusion of less than 0.5 m, assuming a diffusion coefficient of 2 10 cm / s. generating the singlet oxygen in close proximity to the antibody clearly enhances the sensitivity of the biosensor. during the course of developing this biosensor we tested a number of other photosensitizers, including those used previously in acwop experiments, and we found these to be suboptimal. while there may be more efficient singlet oxygen sensitizers in solution, in our view [ru(4-vinyl-4-methyl-2,2-bipyridine)3 ] represents an almost ideal sensitizer in that it can be readily polymerized, giving rise to the above - mentioned proximity advantage, and the resulting films are conformal and quite robust. moreover, the complex is easily synthesized and purified, and solutions can be used multiple times. to generate o2, and consequently h2o2, the qcm crystal containing the immobilized antibodies and electropolymerized sensitizer was immersed in 3 ml of pbs (ph 7.2) and exposed to uv irradiation for 60 min. we found that 60 min with a broad wavelength source was optimal, as longer exposure times resulted in a decrease in signal and increase in background, possibly due to uv damage of the antibody (supporting information figure s4). the h2o2 generated was quantified using square - wave voltammetry (swv) (figure 4), which provides high sensitivity at low analyte concentrations. amplex red (n - acetyl-3,7-dihydrophenoxazine) reacts with h2o2 in a 1:1 ratio in the presence of horseradish peroxidase (hrp) to produce resorufin (7-hydroxy-3h - phenoxazin-3-one), which is fluorescent and also exhibits a reversible redox response. the lowest concentration of h2o2 detected with swv was 0.33 nm (figure 4a, b). (a) square wave voltammetry of 10 m amplex red and 0.2 u / ml horseradish peroxidase (hrp) with varying concentrations of h2o2 to construct a (b) calibration curve. supporting electrolyte was pbs, ph 6.0. step size, 5 mv ; amplitude, 25 mv ; frequency, 25 hz. (c) square wave voltammograms showing detection of h2o2 via reduction of resorufin at a 3 mm glassy carbon working electrode after irradiation of the immunosensors surface with uv light. black short dashed line is 10 m amplex red ; red long dashed line is 10 m amplex red with 0.2 u / ml hrp in the absence of antibody ; blue solid line is 10 m amplex red with 0.2 u / ml hrp in the presence of adsorbed antibody. step size, 5 mv ; amplitude, 25 mv ; frequency, 25 hz. a 1.5 ml aliquot of each irradiated solution in pbs, ph 7.2, was diluted to 5.0 ml using pbs, ph 6.0, and then amplex red and hrp solutions were added after deaerating the pbs. square wave voltammetry was subsequently carried out. assuming a surface coverage of antibodies of 5 10 mol / cm, and a liquid thickness of 100 m (a value readily achievable in a microfluidic platform), a single turnover per immobilized antibody would give rise to a peroxide concentration of 500 nm, clearly well above our detection limit (less than 0.5 nm). in comparative measurements we confirmed that swv detection is much more sensitive than the fluorescence assay (supporting information figure s5). to quantify the number of mole equivalents of h2o2 generated per antibody, we evaluated h2o2 produced in the presence and absence of the immobilized antibody (figure 4c). an aliquot of the irradiated solution was diluted with ph 6.0 pbs, followed by addition of amplex red and hrp. swv was used to measure the amount of resorufin, which reduces near 0.2 v versus ag / agcl, at a glassy carbon electrode. a readily quantifiable increase in reduction current (i.e., the amount of h2o2 produced) resulted from the presence of adsorbed antibody. using a calibration curve, (figure 4a, b). the ratio of h2o2 per antibody was 6401200, which substantially exceeds the previously reported values. although this difference may be due, in part, to an increase in temperature, the biggest influence may be the generation of o2 in close proximity to the antibody (vide supra), such that this highly reactive species is more readily intercepted by the antibody to generate h2o2. for any particular application, further calibration with reference antibody stocks will allow the concentration of antibodies in the test sample to be determined. in summary, we have developed a general immunobiosensor platform, employing patterned polymer brushes with photosensitizer films, based on the electrochemical detection of h2o2 at clinically relevant concentrations generated through the acwop. we have shown that h2o2 at concentrations as low as 0.33 nm can be measured in a biosensor device. antibodies at a surface coverage of 5 10 mol / cm generate more than 25 10 mol h2o2/cm (or > 250 m h2o2, assuming a volume of 1 cm 1 cm 100 m) in 60 min. however, h2o2 can be readily quantified well below this concentration (see figure 4a), and our tests indicate that this device can detect less than 3 pg antibodies in a 10 l sample (2 pm). this compares quite favorably to the most sensitive elisas, which require secondary agents and additional procedural steps. as for the elisa, a challenge is to ensure sufficiently high signal - to - noise, which can be limited by nonspecific antibody binding. the poegma brushes in our platform appear to excel in this regard, and we will continue to develop these as we test samples including high concentrations of nonspecific antibodies, resembling typical blood serums. eliminating the use of secondary antibodies, as in the case of elisa, reduces the chances for nonspecific false positives and furthermore makes our platform appealing for low cost applications. moreover, since the acwop is a general characteristic of antibodies, our approach can, in principle, be applied to antibodies of virtually any specificity, class, or species. we found results obtained from antibodies adsorbed on the platform on a silicon chip to be similar to those obtained using the qcm crystal (supporting information figure s6). we are currently exploring the incorporation of this approach into a microfluidic platform, which would expedite wash steps to minimize nonspecific binding, as well as delivery of the final test solution for swv measurement of h2o2 concentration in a separate part of the device. in the long run this device may also be useful for quantitatively assessing affinities of specific antibodies in a way not possible with elisas because of their need for secondary antibodies that complicate the binding analysis. such a microfluidic platform, in combination with flexible electronics (again recall that [ru(4-vinyl-4-methyl-2,2-bipyridine)3 ] can be electropolymerized as conformal films) should eventually facilitate widespread use and field deployment. | infectious diseases, such as influenza, present a prominent global problem including the constant threat of pandemics that initiate in avian or other species and then pass to humans. we report a new sensor that can be specifically functionalized to detect antibodies associated with a wide range of infectious diseases in multiple species. this biosensor is based on electrochemical detection of hydrogen peroxide generated through the intrinsic catalytic activity of all antibodies : the antibody catalyzed water oxidation pathway (acwop). our platform includes a polymer brush - modified surface where specific antibodies bind to conjugated haptens with high affinity and specificity. hydrogen peroxide provides an electrochemical signal that is mediated by resorufin / amplex red. we characterize the biosensor platform, using model anti - dnp antibodies, with the ultimate goal of designing a versatile device that is inexpensive, portable, reliable, and fast. we demonstrate detection of antibodies at concentrations that fall well within clinically relevant levels. |
lendoprothse endobiliaire peut tre utilise substitut la localisation pancratique dans lutilisation de la tomographie par ordinateur faisceau conique (tofc) en radiothrapie externe. la prsente tude fait tat du dplacement interfraction de lendoprothse durant la radiothrapie externe pour le cancer du pancras localement avanc (cpla). six patients porteurs dune endoprothse endobiliaire ayant reu des traitements de radiothrapie externe pour un cpla ont t valus. les mesures pour laspect le plus suprieur de lendoprothse (sup stent) et son aspect le plus infrieur (inf stent) par rapport laspect le plus suprieur et infrieur de lapophyse pineuse centrale de la vertbre l1 ont t dtermins partir des traitements quotidiens de tofc et compars celles tablies lors de la tomographie de planification. les changements dans la mesure endoprothse - l1 ont t interprts comme des changements dans la position relative de lendoprothse. trois patients ont prsent un dplacement interfraction de lendoprothse de 1 cm lorsque les mesures de traitement ont t compares aux mesures de planification. pour le patient a, la mesure sup stent montre un dplacement vers la droite (2,662,77 cm) et le bas (3,03,12 cm), tandis que la mesure inf stent se dplaait vers la droite (1,922,02 cm), le bas (3,233,34 cm) et larrire (1,411,43 cm). la mesure inf stent pour le patient b montre un dplacement vers le haut (2,230,49 cm) et larrire (1,720,59 cm). les mesures sup et inf stent du patient f montrent un dplacement vers le bas (0,980,35 cm et 1,210,38 cm, respectivement). chez les trois autres patients (c, d et e), le dplacement interfraction est infrieur 1 cm. la migration et la dformation de lendoprothse endobiliaire ont t observes chez un petit sous - ensemble de patients. dautres tudes seront ncessaires avant de pouvoir confirmer leur utilisation comme substitut la localisation des cibles cpla durant la radiothrapie externe guide par limage. the 5-year survival rate in england for men and women diagnosed with pancreatic cancer is 3.9% and 4.4%, respectively. for those with pancreatic tumours deemed surgically unresectable with no distant metastases, their disease is classified as locally advanced pancreatic cancer (lapc). it is possible to treat lapc with either conformal radiotherapy or intensity - modulated radiotherapy though the presence of several critical organs near the pancreas (eg, spinal cord, kidneys, liver, and bowel) makes the planning and delivery of radiotherapy complex, especially if nodal volume inclusion creates a larger planning target volume (ptv). image - guided radiotherapy (igrt) can be used to ensure treatment accuracy during radiotherapy treatments and limit toxicity to organs at risk. while igrt using megavolt (mv) or kilovolt (kv) planar images can enhance accuracy, these images provide mostly bony anatomy information with minimal soft tissue definition. using bony anatomy to align organs such as the pancreas has proven to be a poor predictor of pancreatic location. cone - beam computed tomography (cbct) can provide additional soft tissue definition, but delineating the pancreas and pancreatic tumours on cbct remains difficult. although feng found that the abdominal wall and diaphragm were unsuitable surrogates for pancreatic tumour position, this has not excluded the use of other internal structures as surrogates. studies have sought to use fiducial markers, surgical clips, electromagnetic transponders, and endobiliary stents as pancreatic tumour surrogates to assist with image matching. endobiliary stents are commonly implanted in pancreatic cancer patients as a palliative measure to relieve symptoms associated with biliary strictures such as jaundice, thereby aiding chemoradiotherapy tolerability. as the common biliary duct runs through the pancreatic head, using an endobiliary stent as a surrogate for pancreatic localization can serve as a potential alternative to the implantation of fiducial markers, which are placed percutaneously under radiographic guidance, intraoperatively, or using endoscopic ultrasound. fiducial marker insertion for igrt use would be an additional procedure for patients with endobiliary stents already inserted for palliative purposes. it is also unknown how fiducial marker visibility may be affected if a stent is in place as well. endobiliary stents can be visualized on cbct, and it has been suggested that they can be used as a surrogate when determining pancreatic tumour positions. two studies have reported on cbct - determined interfraction motion of endobiliary stents during external - beam radiotherapy (ebrt) and found minimal stent position changes (sup stent. for the z component of the stent - l1 distance, five patients (a, b, c, e.1, and f) had inf stent > sup stent. of the six patients assessed, three patients (a, b, and f) exhibited pronounced and sustained stent position changes as determined by examining the individual mean position change for each patient in each direction (table 2) and by visualizing position change graphically over time (figure 2). patient a showed sustained stent position changes with the sup stent moving to the right (2.66 2.77 cm) and inferiorly (3.0 3.12 cm) and the inf stent moving to the right (1.92 2.02 cm) inferiorly (3.233.34 cm) and posteriorly (1.41 1.43 cm). patient b showed sustained stent position changes with the inf stent moving superiorly (2.23 0.49 cm) and posteriorly (1.72 0.59 cm). patient f showed sustained stent position changes with both the sup and inf stent moving inferiorly (0.98 0.35 cm and 1.21 0.38 cm, respectively). the sustained position change was apparent from the start of treatment for patients b and f but did not become apparent in patient a until after fraction 5 (figure 2). the free - breathing superior - inferior stent motion determined from planning for patients a, b, and f were 1.15 cm, 1.87 cm, and 0.53 cm, respectively (table 1). although the sup and inf stent for patient a and f moved similarly during treatment, only the inf stent for patient b showed sustained position changes (figures 2 and 3). the population mean position change (x, y, z) (ie, the mean of all patients ' individual means of position change) for the sup stent was x= 0.44 1.09 cm, y = 0.45 1.26 cm, and z = 0.09 0.26 cm. the population mean position change for the inf stent for all patients was x= 0.33 0.84 cm, y= 0.43 1.65 cm, and z = 0.31 0.95 cm. the standard deviation of position change () exhibited by the inf stent was greater than that exhibited by the sup stent (table 2). for the y component of the stent - l1 distance, six patients (b, c, d, e.1, e.2, and f) had inf stent > sup stent. for the z component of the stent - l1 distance, five patients (a, b, c, e.1, and f) had inf stent > sup stent. this study shows that the position of endobiliary stents can vary greatly over the course of ebrt for lapc in six patients. utilizing endobiliary stents as imaging surrogates for pancreatic tumours should be done cautiously as these stents may lack positional stability during ebrt. in this study, three patients exhibited sustained stent position changes from their planning ct. we saw a greater rate of stent migration than previously published by johanson, which may be evidence of distal stent migration only. the migration rate differential may be related to study population selection as johanson 's sample consisted of a generalized patient population that received endobiliary stents, whereas this study had pancreatic cancer patients that received stents prior to chemoradiotherapy. it is unknown how the presence of malignancy, chemotherapy, or ebrt affected stent migration incidence in this study, but the development of radiotherapy - induced pancreatic fibrosis may play a role. while malignancy and chemotherapy may have influenced stent migration, lofts found that cancer patients on a chemo - regime of epirubicin, cisplatin, and 5-fluorouracil did not have a significant increase in stent blockage or shortening of stent patency duration. significant interfraction deformation of the pancreatic head, duodenum, and stomach can occur during pancreatic head irradiation, and these interfraction anatomic changes may affect stent migration. stent migration could be attributed to pancreatic irradiation ; however, other studies have found that endobiliary stents were stable during radiotherapy [13, 19 ]. zhu found no incidence of stent migration when they treated biliary malignancy with intraluminal radiotherapy, but this may be due to their use of a stent - in - stent configuration. engineer concluded that endobiliary stents were stable tumour surrogates during ebrt after they found population mean position changes of less than 0.4 cm in all directions during ebrt. we found similar population mean position changes when patients were analysed together (< 0.5 cm), but when patients were examined individually, three patients exhibited individual mean interfraction motions of greater than 1 cm in at least one direction, indicating stent migration occurrence. this contradicts the findings of whitfield who reported that their stent patients had individual mean interfraction stent motions of less than 1 cm for all directions during ebrt. previous studies that examined cbct - determined interfraction stent motion during ebrt had small samples (three and five patients). perhaps with larger study sizes, more cases of stent migration would have also presented themselves in these studies. the more inferior stent position during treatment cbcts may be due to the planning cts being captured during exhale breath - hold and the daily cbcts being captured during free - breathing ; the stents appeared inferior on cbcts because inhalation caused inferior pressure on the diaphragm and abdominal contents. however, not all patients showed more inferiorly positioned stents. the influence of variable inhale and exhale lung volumes on stent position as seen on free - breathing cbcts is unknown. the majority of the free - breathing cycle is spent in exhale breath position, which suggests that stent appearance on free - breathing cbct roughly correlates with its appearance on exhale breath - hold ct. the inferior stent position change exhibited by patients a and f was larger than the sup - to - inf breathing motion established during planning. inhalation may not be the only contributing factor to an inferior position change ; distal stent migration may have occurred. this can not be stated conclusively because 4dct - determined pancreatic tumour motion may not have good correlation with the range of motion demonstrated during radiotherapy [21, 22 ]. other nonrespiratory physiological processes may impact on stent position. to distinguish between interfraction motion and intrafraction motion, more detailed treatment imaging such as 4-dimensional cbct could be used to determine stent positions on respiratory - correlated cbct projections. exhale breath - hold cbcts could also be used to minimize the respiratory component of intrafraction motion and facilitate correspondence with the exhale breath - hold planning ct. ideally, stent position would be compared to the pancreas to monitor migration occurrence, but limited soft tissue definition on cbct necessitates the comparison of surrogate motion with a bony match as in previous studies [7, 13 ]. more accurate soft tissue imaging and further study would be required to determine whether change in stent position is representative of position change of the pancreatic head. most patients had greater standard deviation of position change exhibited by the inf stent than the sup stent. greater freedom of movement was associated with the inf stent in the superior - inferior and anterior - posterior planes than the sup stent. sup stent motion was likely restricted by the concentration, size, and rigidity of internal organs in the upper abdomen. the magnitude and direction of position change did not correspond for the sup stent and inf stent. the cause of this is unknown but may be a result of bowel filling changes from planning to treatment or weight loss during treatment. the incidence of stent deformation reinforces the method of selecting two position points on the stent. previous studies selected a single stent position point such as a single end of stent or centre of mass [11, 13 ] to assess interfraction stent motion and did not report on stent deformation. as two points at opposing ends of the stent were used to determine position change for this study, any rotational error was captured as oppositional movement of the two stent ends. endobiliary stents are flexible structures, making the assessment of rotation alone difficult. by using two points of measurement, both potential rotation and potential deformation could be accounted for. because patients were referred from a number of centres, the patients had a variety of both metal and plastic stents in place. although the variation in stent type did not affect visualization of the stent on cbct nor did it affect the results, a greater number of patients with both metal and plastic stents are required to determine this statistically. a limitation of this study was the small sample size, which did not allow for more robust statistical analysis. there was considerable diversity between the patients ' results as shown by the large standard deviation of all patients ' means. without more statistical tests, it is difficult to say whether this diversity would also be found in a larger group of patients. additional study patients would allow for the identification of possible trends with regards to stent migration as well as enable better extrapolation to the wider irradiated lapc population. other limitations of this study were that the method and timing of stent insertion were not controlled nor was the stent type. with patients being referred for radiotherapy after stent insertion, controlling the previously mentioned variables will require forethought and coordination. careful, consistent stent selection and insertion could reduce or eliminate stent migration. shortening the time frame from stent insertion to the start and completion of radiotherapy may also reduce stent migration during treatment. for those receiving short - course radiotherapy for pancreatic cancer [2426 ], the position of the endobiliary stent may be stable during igrt because of the shorter time span of treatment. for those patients who may undergo long - course radiotherapy with a pre - existing stent, it is worth investigating whether these patients may require an additional procedure to exchange for a more stable endobiliary stent or to insert additional fiducials to facilitate image guidance during ebrt. through examination of daily treatment images, large sustained endobiliary stent position changes were observed in a small subset of patients during ebrt for lapc, indicating the occurrence of stent migration and deformation. further study is needed to determine whether change in stent position is representative of position change of the pancreatic head and tumour, and whether stents are appropriate for use as tumour surrogates. | purposeendobiliary stents can be used as surrogates for pancreatic localization when using cone - beam computed tomography (cbct) during external - beam radiotherapy (ebrt). this work reports on interfraction stent position changes during ebrt for locally advanced pancreatic cancer (lapc).materials and methodssix patients with endobiliary stents who underwent ebrt for lapc were assessed. measurements from the most superior aspect of the stent (sup stent) and the most inferior aspect of the stent (inf stent) to the most inferior, posterior aspect of the l1 vertebra central spinous process were determined from daily treatment cbcts and compared with those determined from the planning computed tomography (ct) scan. changes in stent - l1 measurements were interpreted as changes in relative stent position.resultsthree patients showed mean interfraction stent position changes of 1 cm when treatment measurements were compared with planning measurements. the sup stent for patient a moved to the right (2.66 2.77 cm) and inferiorly (3.0 3.12 cm), and the inf stent moved to the right (1.92 2.02 cm) inferiorly (3.23 3.34 cm) and posteriorly (1.41 1.43 cm). the inf stent for patient b moved superiorly (2.23 0.49 cm) and posteriorly (1.72 0.59 cm). the sup and inf stent for patient f moved inferiorly (0.98 0.35 cm and 1.21 0.38 cm, respectively). the remaining three patients c, d, and e showed interfraction position changes of < 1 cm.conclusionendobiliary stent migration and deformation were observed in a small subset of patients. further investigation is required before confirming their use as surrogates for lapc target localization during image - guided ebrt. |
bonding to enamel is been considered satisfactory, but adhesion to dentin is much more complex due to the nature of the substrate. the best way to test the effectiveness of a biomaterial, as well as composite or luting cement or a bonding system, is in vivo experimentation in controlled trials. however, it is quite impossible to distinguish by clinical evaluation and adhesion potential of a dentin - bonding system from many other parameters that influence either the quality or the longevity of a restoration. theoretically, in vitro bonding tests would allow for measurement of the true strength of bonding of a given system to a dentinal substrate. unfortunately, in vitro dentin adhesion testing has not been very successful in predicting in vivo effectiveness, probably because of the number of factors influencing test results and clinical relevance.[13 ] several authors have already studied the influence of some of these factors, such as the origin of the substrate, substrate storage, dentin depth, type of composite, testing mode, etc. some previous reviews have listed most of the variables influencing dentin bond strength, but none has quantified their importance or defined their statistical significance. the purpose of this study is to evaluate the true factor that affects the bond strength of one - step and two - step self - etch adhesive. this quantitative overview could be considered as a meta - analysis study performed under non - experimental conditions to assess the effects of certain clinical parameters on bond strength of adhesives. potential papers were selected from articles published in 13 peer - reviewed journals using pubmed data base. american journal of dentistry, journal of adhesive dentistry, british dental journal, dental materials, european journal of oral science, international dental journal, journal of dentistry, journal of dental research, journal of oral rehabilitation, journal of prosthetic dentistry, operative dentistry, quintessence international and journal of conservative dentistry. study with original data.paper published in english or translated to english.only studies measuring the bond strength of one - step and two - step self - etch dentin - bonding agents to be included.the bonding agents that are commercially available and tested by shear or tensile mode on sound dentin from permanent teeth.dentin-bonding agents involved in the analysis should have been tested in at least five different studies.the outcome measure had to be the dentin bond strength expressed in mpa / psi.light curing unit with intensity of 400 - 500 mw / cm.among the selected studies, some exclusion criteria applied.paper displaying only graphic data.dentin-bonding agents not used according to manufacturer 's instructions.dentin samples, which were contaminated or not treated under clinical conditions before application of the dentin - bonding agents, were excluded from the analysisstudy without data analysis. paper published in english or translated to english. only studies measuring the bond strength of one - step and two - step the bonding agents that are commercially available and tested by shear or tensile mode on sound dentin from permanent teeth. dentin - bonding agents involved in the analysis should have been tested in at least five different studies. the outcome measure had to be the dentin bond strength expressed in mpa / psi. light curing unit with intensity of 400 - 500 mw / cm. among the selected studies, some exclusion criteria applied. dentin samples, which were contaminated or not treated under clinical conditions before application of the dentin - bonding agents, were excluded from the analysis study without data analysis. we classified the parameters into four groups : group a - factors related to the dentin substrate ; group b - type of composite and dentin - bonding agents ; group c - bonding area and ; group d - thermocycling and testing mode. selected studies were included in the meta - analysis the problem of bond - strength testing is that the materials were seldom compared with a standard. rather, different materials were tested under various conditions and bond strengths are reported as point estimates and expressed in mpa. it is difficult to compute a standard effect size for each study, as required in a classic meta - analysis. thus, we decided to analyze directly the measures of bond strength from reports published in literature. this quantitative overview was performed in non - experimental conditions since materials and conditions are not allocated randomly. from each report means and standard deviations of bond strengths were extracted and tabulated with corresponding experimental conditions. the statistical analysis software system mix 2.0 professional (biostat, englewood, nj) was used to perform the statistical analysis. potential papers were selected from articles published in 13 peer - reviewed journals using pubmed data base. american journal of dentistry, journal of adhesive dentistry, british dental journal, dental materials, european journal of oral science, international dental journal, journal of dentistry, journal of dental research, journal of oral rehabilitation, journal of prosthetic dentistry, operative dentistry, quintessence international and journal of conservative dentistry. study with original data.paper published in english or translated to english.only studies measuring the bond strength of one - step and two - step self - etch dentin - bonding agents to be included.the bonding agents that are commercially available and tested by shear or tensile mode on sound dentin from permanent teeth.dentin-bonding agents involved in the analysis should have been tested in at least five different studies.the outcome measure had to be the dentin bond strength expressed in mpa / psi.light curing unit with intensity of 400 - 500 mw / cm.among the selected studies, some exclusion criteria applied.paper displaying only graphic data.dentin-bonding agents not used according to manufacturer 's instructions.dentin samples, which were contaminated or not treated under clinical conditions before application of the dentin - bonding agents, were excluded from the analysisstudy without data analysis. paper published in english or translated to english. only studies measuring the bond strength of one - step and two - step the bonding agents that are commercially available and tested by shear or tensile mode on sound dentin from permanent teeth. dentin - bonding agents involved in the analysis should have been tested in at least five different studies. the outcome measure had to be the dentin bond strength expressed in mpa / psi. light curing unit with intensity of 400 - 500 mw / cm. among the selected studies, some exclusion criteria applied. dentin samples, which were contaminated or not treated under clinical conditions before application of the dentin - bonding agents, were excluded from the analysis study without data analysis. we classified the parameters into four groups : group a - factors related to the dentin substrate ; group b - type of composite and dentin - bonding agents ; group c - bonding area and ; group d - thermocycling and testing mode. the problem of bond - strength testing is that the materials were seldom compared with a standard. rather, different materials were tested under various conditions and bond strengths are reported as point estimates and expressed in mpa. it is difficult to compute a standard effect size for each study, as required in a classic meta - analysis. thus, we decided to analyze directly the measures of bond strength from reports published in literature. this quantitative overview was performed in non - experimental conditions since materials and conditions are not allocated randomly. from each report means and standard deviations of bond strengths were extracted and tabulated with corresponding experimental conditions. the statistical analysis software system mix 2.0 professional (biostat, englewood, nj) was used to perform the statistical analysis. differences between means were computed with analysis of variance. tables 2 and 3 show descriptive statistics of different parameters tabulated from the selected studies according to the four groups described in materials and methods section. some parameters, such as tooth storage medium and dentin surface topography, were not included in the statistical analysis, due to their greater variability or lack of information. it was noted that about one - third of the selected articles provided such information and that the distinction between the different failure modes was not always available and clearly identified. one - way anova comparison between one - step and two - step self - etch adhesives is given in table 4 the p - values indicate the potential relation between each parameter and the bond strength. their respective influences were given by the p - value at the significance level of 0.05(). all the studied parameters showed no significant difference, except for dentin origin / site and bonding area. in addition, statistical analysis done with anova showed statistical significance between the one - step and two - step self - etch adhesives. descriptive statistics of the different parameters studied using one - step self - etch adhesives descriptive statistics of the different parameters studied using two - step self - etch adhesives comparison between one - step and two - step self - etch adhesives using anova as quoted by dersimonian and laird (1986) meta - analysis is the statistical analysis of a collection of analytic results for the purpose of integrating the findings. the advantage of meta - analysis over the traditional literature review is the possibility of combining results from many studies and analyzing the factors that affect the bond strength of self - etch adhesives. in the studies selected, the tensile or shear testing was done using a constant crosshead speed and the samples were continuously loaded such that the crack will propagate at increasing speed until separation. in a clinical situation, if these loads are high enough, small extensions of cracks ensure sub - critical crack growth in bonded area as explained by a. a. griffith. according to griffith 's theory of brittle solid it is stated that the low fracture strength observed in experiments, as well as the size - dependence of strength, were due to the presence of microscopic flaws in the bulk material. in this practical situation, the crack speed is much lower than during tensile or shear forces, which is used to determine the adhesion potential of a dentin - bonding system. the location of the initial flaw is unknown, and the stress concentration at the bonded interface is non - uniform and unpredictable. moreover, the stress distribution is highly dependent on the test geometry, on the loading mode and on the material properties.[2729 ] our analysis revealed that the origin of dentin, site of bonding and bonding area had significant influence on bond strength of one - step and two - step self - etch adhesive. the studies have also showed that there is a difference in bond strength between human teeth and bovine teeth. this difference explains the fact that dentinal tubules are large in the coronal portion of bovine dentin, but one study by nakamichi, showed that there is no difference in bond strength between bovine and human dentin when only the superficial layers was used. oilo and olsson showed that the tensile strength is higher on the buccal surface compared to the occlusal surface, which could be explained by the fact that the tubules are lower in number with less area percentage on the buccal surface than on the occlusal surface. the analysis also showed a definite difference between enamel and dentin bonding which might be explained by various reasons. dentin has a heterogeneous structure and different orientation of tubules with root dentin having smaller and more numerous branching of tubules when compared to crown dentin. acidity of monomer also caused change in surface chemistry and morphology of dentin, which in turn can influence bonding. a significantly thicker hybrid layer was noted in areas with perpendicular tubule orientation than in areas with parallel tubule orientation surface area significantly affects the bond strength in one - step and two - step self - etch adhesives. bond strength is calculated as the load at failure divided by the cross - sectional area of the bonded surface. an inverse relationship between bond strength and bonded surface area has been recently shown, confirming previous studies. these authors explain the lower strengths of large bonded samples by the greater number of defects within the adhesive joint. burrow mf, stated that clearfil se bond showed no statistical difference to g - bond. do amaral rc, suggested application of bonding agents with agitation on the dentin surface to improving the resin - dentin bond strength of one - step self - etch adhesives. when bonded to dentin, the adhesives with simplified application procedures (in particular, one - step self - etch adhesives) still underperform as compared to conventional three - step adhesives. mild two - step self - etch adhesives that provide additional chemical bonding, appear to most optimally combine bonding effectiveness with a simplified application protocol our analysis has showed that two - step self - etch adhesive system showed a superior in vitro performance in comparison to one - step self - etch system. these results are in accordance with those of other studies. the bonding ability of the all - in - one adhesives depends on their specific composition. in light of the low in vitro bond strengths and high rate of spontaneous failures of some all - in - one adhesives compared to those of the two - step adhesives, the newest adhesives should be screened more strictly before they are recommended for clinical use. one - step self - etch adhesives showed higher bond strengths on ground enamel and no reductions in resin - enamel bonds were observed after 12 months of water storage. marginal adaptation was significantly better for clearfil s3 bond than for adper prompt l - pop. serious limitation of all - in - one adhesives are as follows : incomplete polymerization and continued demineralization of the adjacent dentin structure in the tubules. for all - in - one adhesives to be acidic, studies have shown that this water acts as a major interfering factor in polymerization which leads to unpolymerized acidic and aggressive monomers to continue etching the dentin, thereby leading to a detrimental impact on the bond. other factor leading to low bond strength and failure of resin - based adhesives in vivo are harsh conditions of the oral environment, such as intraoral temperature, moisture contact, fatigue of bond due to tooth flexure and bacterial enzymes. the shelf life of one - component self - etching adhesives is determined by their chemical composition. in conventional methacrylate - based adhesives (adhese), the inherently acidic environment of such formulations leads to monomer degradation due to hydrolysis. in contrast, methacrylamide - based adhesives are stable to aqueous acid and exhibit much superior storage stability without monomer degradation - related losses in adhesion performance. intermediary strong self - etch adhesives (adhese and xeno iii) exhibited significantly superior shear bond strength values compared to both strong (prompt - l - pop) and weak (clearfil se bond) self - etch systems, which implies that ph of the adhesive has a significant effect on the bond strength. suzuki and finger found a linear relationship between the remaining dentin thickness to the pulp and the shear bond strengths of three different adhesives, an observation subsequently confirmed by other authors the analysis surprisingly showed that type of composite and thermocycling did not significantly affect bond strength. nevertheless, three studies reported a decrease in adherence after thermocycling whereas other studies reported an increase, depending on the adhesive system tested. from this meta - analysis study, we have analyzed that two - step self - etch adhesive showed better bonding ability than one - step self - etch adhesives. thereby, several clinical parameters among which dentin origin, site and area of bonding have shown a significant influence on mean bond strength of adhesive system. thus, our study suggests, that the practitioners can easily control these parameters by using standardized protocol for the clinical success of self - etch adhesive systems. | aim : the purpose of this study is to critically evaluate the factors that affect the bond strength of one - step and two - step self - etch adhesives by using meta-analysis.materials and methods : potential papers that were selected according to inclusion and exclusion criteria from articles were published in 13 peer - reviewed journals using pubmed data base. from each report, means and standard deviations of bond strengths were extracted and tabulated with corresponding experimental conditions.results:all the studied parameters showed no significant difference, except for dentin origin / site and bonding area. in addition, statistical analysis done with anova showed statistical significance between the one - step and two - step self - etch adhesives.conclusions:our analysis has showed that two - step self - etch adhesive system showed a superior in vitro performance in comparison to one - step self - etch system. nevertheless, certain factors such as dentin origin, site and area of bonding affect the bond strength of adhesives. |
the mineral pneumoconioses (lung disease caused by inhalation of inorganic dust) have been an important disease entity for centuries. in the last several decades, the electron microscope has been used to elucidate the distribution and identification of inhaled minerals, to aid in establishing etiologic factors, and less commonly, to determine the basic biologic mechanisms through which inhaled minerals cause lung disease. in this section, i review the instrumentation and tissue preparation currently used to address some modern problems in particle - induced lung disease. for example, human pneumoconioses of undetermined etiology can be clarified by electron microscopy and x - ray energy spectrometry. in addition, the initial deposition patterns of asbestos and silica are demonstrated in animal models, and the contributions of electron microscopy in establishing the initial lesions of asbestosis are described.imagesfigure 1.figure 2.figure 3.figure 4.figure 5.figure 6.figure 7.figure 8.figure 9.figure 9.figure 10.figure 11.figure 12.figure 13.figure 14. |
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hip fractures are a disabling event and an important cause of morbidity and mortality in elderly people (1 - 4). the majority of patients following hip fracture are treated operatively to obtain pain relief, hasten mobility, and minimize complications. the indications of conservative treatment after hip fracture in previous reported studies were patients with impacted femoral neck fracture, minimally symptomatic following late diagnosis of hip fracture, already bed - ridden, and significant medical co - morbidity (5 - 8). in these patients, high mortality rates in cases with conservative treatment were reported because of poor medical comorbidity (7 - 9). however, little information is available concerning the prognosis of elderly patients who need surgical intervention but who are discharged without surgery after hip fracture because of economic burden. the purpose of this study was to determine the causal factors of non - operative treatment following hip fracture and mortality rate and functional recovery according to the causal factors compared to that of patients who underwent surgery. between january 2009 and december 2011, 451 patients were diagnosed as femoral neck or intertrochanteric fractures. of these, we identified 32 elderly patients (patients over 65-yr - of - age, 32 hips) with hip fracture who refused surgical treatment and received non - operative procedure at the author 's hospital. the exclusion criteria were impacted femoral neck fracture, isolated trochanteric fracture, high - energy injury such as traffic accident, and metastatic pathological femur fractures. we excluded four patients from enrollment : two patients with pathological fracture and two patients with traumatic trochanteric fracture of the femur. fifty six age-, gender-, american society of anesthesiologist (asa) score- (10), and diagnosis - matched patients with hip fracture who had undergone surgical treatment at our hospital were used as the control group (group ii) in a two - to - one ratio using the propensity scoring method. demographic data including gender, age, initial diagnosis, asa score, and pre - fracture ambulatory status by koval 's categories (11) were obtained by reviewing medical records (table 1). the causes of operation refusal were obtained by reviewing medical records and interview with patients or family members. change of patient activity and mortality were compared within a minimum of one year between the two groups. activity levels were defined as follows : i, independent community ambulatory ; ii, community ambulatory with cane ; iii, community ambulatory with walker / crutches ; iv, independent household ambulator ; v, household ambulatory with cane ; vi, household ambulatory with walker / crutches, and vii, nonfunctional ambulatory (11). in the analysis, koval 's grade i, ii, and iii cases were also classified as ambulatory outdoors, whereas koval 's grade iv, v, vi, and vii cases were classified as shut - in patients. patients unable to attend follow - up evaluations were interviewed by telephone. during the follow - up evaluations, care was taken to interview the caregiver previously interviewed during the patient 's hospitalization. the propensity score matching method using age, gender, asa score, and diagnosis was used for a comparable control group retrospectively. cumulative crude mortality rate were calculated at 3, 6, 12, and 24, months and compared between the two groups. kaplan - meier survival analysis was performed on both of the groups with a minimum 12 months follow - up using mortality as a primary end point. the correlation of survival rate in both groups was tested by the log - rank test. the student 's t - test was used to analyze age, and the mann - whitney to analyze asa score and koval 's grade. the chi square test was used to analyze gender, diagnosis, and cumulative mortality. the analyses were carried out using spss version 18.0 (chicago, il, usa). the design and protocol of this retrospective study were approved by the institutional review board of our hospital (cauh - irb no c2013039). all patients were informed that their medical data could be used in a scientific study and provided their consent. the design and protocol of this retrospective study were approved by the institutional review board of our hospital (cauh - irb no c2013039). all patients were informed that their medical data could be used in a scientific study and provided their consent. of 28 patients who could not receive operative treatment, the reason in 10 patients (36%) was medical problems with high - risk of surgery. the reason for the remaining 18 (64%) was economic burden, even if surgery is quite possible. the cumulative mortality rate over 3, 6, and 12 months post - fracture was 54%, 61%, and 64% in group i and 9%, 11%, and 14% in group ii, respectively. the cumulative mortality rate was 82% (23/28) at 24 months in group i and 21% (12/56) at 24 months in group ii (table 2). with regard to the cause - specific mortality in group i, the cumulative mortality rate over the 3, 6, and 12 months post - fracture was consistently 80% in 10 patients who refused surgery for medical problems and 39%, 50%, and 56% in 18 patients who refused surgery because of economic burdens. the cumulative mortality rate at the 24-month follow - up was 90% (9/10) and 78% (14/18), respectively (table 3). the cause of death in group i by economic reasons was pneumonia (n=8), congestive heart failure (n=1), chronic liver disease (n=1), and unknown reasons (n=4). kaplan - meier survivorship analysis with mortality as the end point in group i and group ii estimated a 57.1% (95% confidence interval, 38.7% to 75.5%) chance of survival during 12 months and 82.1% (95% confidence interval, 72.1% to 92.1%) chance of survival during 12 months, respectively (fig. i, the survival rate was 20.0% (95% confidence interval, 0% to 44.7%) with mortality as the end point at 12 months in the medical reason group and 38.1% (95% confidence interval, 15.4% to 60.8%) with mortality as the end point at 12 months in the economic reason group, respectively. of the group i patients at final follow - up, five patients were alive and all were in a nonfunctional ambulatory state (koval 's grade vii) (table 2). of the group ii patients, 44 patients were alive, and 16 patients were outdoor ambulatory and 28 patients were shut - in patients. only seven patients were in a nonfunctional ambulatory state (koval 's grade vii). although many studies have examined mortality and morbidity of conservative treatment after hip fracture in elderly patients, the mortality of conservative treatment that has been done because of economic burden (i.e., inability to pay the costs of surgery) after hip fracture is unclear. to the best of our knowledge, this study is the first study of mortality of conservative treatment after hip fracture in patients who refused surgery because of an economic burden. during the 3-yr study period, 18 of 28 patients who were diagnosed with displaced hip fracture and required surgery refused surgery because of an economic burden and instead received conservative treatment. at a minimum 12-month follow - up, mortality was 56% and all patients were bed - ridden. several recent observational studies addressing several different indications of conservative treatment after hip fracture in elderly patients reported favorable results. (8) conducted a case - control study with 25 patients who were treated non - operatively and 22 patients treated surgically over the same time period. the authors reported that non - surgical treatment with early mobilization did not result in a statistically significant difference in functional outcome compared to surgical treatment patients. gregory. (7) evaluated 102 patients diagnosed with a displaced intracapsular femoral neck fracture. eighty of these patients underwent hemiarthroplasty and 22 were managed non - operatively because of an unacceptably high risk of death within the perioperative period despite medical optimization. the authors reported that the 30-day mortality in the hemiarthroplasty group was 4% (3/82) compared with 34% (7/22) in the non - operative group. patients surviving 30 days after fracture had similar mortality rates at 1 yr, regardless of their treatment (27% non - operative vs 25% hemiarthroplasty). raaymakers and marti (12) performed a prospective observation study with 170 patients with impacted femoral neck fractures treated with early mobilization. of these, 16% had died within 1 yr and 86% had achieved union. although the impacted femoral neck fracture has been described as one of the possible indication of non - operative treatment in some articles, it should be treated surgically because of more than 15% of them displaced eventually. in addition, these findings were not consistent with other previous studies (13). ions and stevens (13) performed a prospective observation study with 158 patients who sustained hip fracture (135 in the operation group and 22 in the conservative treatment group). the authors reported that the 6-month mortality rate of the operation group was 9.6% (13 of 135) and that of the non - operated group who had severe medical comorbidity was 60.8% (14 of 23). in this study, the cumulative mortality rate at 6 months follow - up was 61% in group i and 11% in group ii. in addition, subgroup analysis in conservative treatment group revealed 80% mortality in patients who had severe medical comorbidity and 50% mortality in patients who had refused surgery because of economic burden. at the latest follow - up, five patients in group i were all bed - ridden, but more than half of the remaining 44 patients in group ii were restored to the same pre - injury or grade 1 reduced pre - injury activity levels. therefore, conservative treatment after hip fracture might lead to serious dependency. first, the study was retrospective and was performed in a cohort of prospectively followed patients. however, the study could not be prospectively randomized one because of the high mortality of non - operative treatment. second, the study design included a small cohort of patients and so might not have had sufficient statistical power to be conclusive. however, the calculated sample size of only 20 patients was deemed sufficient because of the high mortality rate of 4-times that of the surgical control group. however, to make a matched case - control study by using a propensity scoring method, we used the asa score as the medical status indicating the risk of surgery. white. (10) reported the asa score is a reliable index of predictor of mortality and surgical risk. fourth, the patients who refused surgery because of economic reasons were not universally followed - up. we persuaded the patients and their family and could finished interviews and get information with one orthopedic surgeon and two nurses. in conclusion, this study demonstrates that the conservative treatment following hip fracture due to economic burden is associated with substantially higher mortality and serious functional loss compared with patients who are treated surgically. this finding should be important for health affiliations and health care providers, and health policy decision makers should take action to reduce mortality and maintain functional activity in elderly patients who refuse surgical treatment. | although many studies have assessed mortality and morbidity of conservative treatment after hip fracture in elderly patients, the mortality of conservative treatment done because of economic burden is unclear. among 451 patients diagnosed with displaced hip fracture during 3 yr, 28 patients (group i) were enrolled as conservative treatment. fifty - six patients matched in age, gender, asa score, and diagnosis (group ii) who had undergone surgical treatment were used as the control group. the causal factors of non - operative treatment and mortality rate and functional recovery were evaluated according to the causal factors of patients with surgical procedure. ten patients (36%) in group i involved medical problems and 18 (64%) by economic burdens. the cumulative mortality rate over 3, 6, 12, and 24 months was 54%, 61%, 64%, and 82% in group i and 9%, 11%, 14%, and 21% in group ii, respectively. at the latest follow - up, all five patients in group i displayed a nonfunctional ambulatory state, whereas only seven of 44 patients in group ii were in a nonfunctional ambulatory state. non - surgical treatment following hip fracture that is done because of the economic burden is associated with substantially high mortality and serious functional loss. |
inverted duodenal diverticulum is an infrequent congenital abnormality that usually is not symptomatic but can become, generally in adult life. this anatomical anomaly has been also related to episodes of upper intestinal bleeding or even acute pancreatitis (1,2). we present a case of an inverted duodenal diverticulum in a patient that presented with an upper intestinal obstruction. a 33-year - old woman with a previous history of fertilization treatments, consulted because of a 2 months vague epigastric and right hypocondric discomfort that was especially more frequent after eating. the patient referred a sensation of fullness / pressure, nausea and abundant alimentary vomiting 2 - 3 hours after eating and that relieved the pain. she was also afraid of eating and had lost 9 kg of weight during this period. upper gastrointestinal series showed the presence of an intraluminal duodenal diverticulum of about 8 cm in length and a partially occluded duodenal lumen (figure 1). intraluminal duodenal diverticulum, pear - shaped sac, occluding duodenal lumen (arrows). an upper gastrointestinal endoscopy showed a inverted intraduodenal diverticulum like a giant pseudo - polip that insufflated and deinsufflated spontaneously (figure 2). upper intestinal endoscopy showing the inverted intraduodenal diverticulum the cholangio - mri and the abdominal ct scan also showed the diverticulum (figure 3), as well as signs of upper intestional malrotation, absence of the pancreatic tail, polysplenia and an hepatic artery that emerged from the superior mesenteric artery. a cholecistectomy, a transcystic cannulation of vater s papilla and a longitudinal duodenotomy on the second portion were performed. an intraluminal formation of 10 cm of length, anchored to three quarters of the duodenal circumference, right next to the papilla and covered by normal mucosa on both sides, was revealed. after circumferencial excision of the inverted intraduodenal diverticulum, an approximation of both mucosal edges was done with reabsorbible stitches, controlling vater 's papilla. the patient was discharged on day 8 after surgery with no complications, except for an autolimited episode of upper intestinal bleeding. only a low percentage of gastrointestinal diverticula are inverted or intraluminal diverticula, that is, they have the sac protruding into the duodenal lumen, and less than 150 cases have been previously described in the literature (3). this lesion is usually found in the 2 portion of the duodenum, very often near vater s papilla, as in our patient, covering a variable percentage of the surface of the duodenum. its origin has been related to a defect in the development of the duodenum which takes place between the 5 and the 12 week of gestation (1). during this period the complete obliteration of the lumen, the vacuole formation, and the subsequently coalescence of the vacuoles until the complete and definitive permeabilization of the duodenal lumen takes place. patients with duodenal diverticula are going to present an impaired and incomplete vacuolization (4). duodenal vacuolization occurs at the same time as the biliary and pancreatic ducts develop and explains the relationship between both congenital defects (5,6). there is a 40% incidence of coexistant anatomical abnormalities : choledochocele, annular pancreas, double diverticula, intestinal malrotation, imperforate anus, hirschsprung`s disease, congenital heart diseases, omphalocele, hypoplastic kidneys, bladder exstrophy, situs inversus, ladd s bands, portal vein anomalies, polysplenia and down syndrome (6,7). the distended diverticulum acts as a large foreign body in the lumen which contributes to produce postprandial fullness and, in some patients, signs of upper intestinal obstruction. when the duodenum is empty, the diverticulum has a tendency to collapse ; this explains the asymptomatic periods. regarding diagnosis, barium contrast radiology shows images that could be considered patognomonic, with a bag of retained barium in the 2 duodenal portion, occlusive or subocclusive, surrounded by a double radiolucid image with perisacular mucosa duodenal folds. this image has been described as a windsock blown into the duodenum (8). the endoscopy can have false negatives because of the device being introduced into the lumen of the diverticulum. specific findings are a blind sac lined by normal duodenal mucosa that gives the appearance of a polyp when inverted (5). in the presented case, ct scans and cholangio - mri allow physicians to complete the study and to evaluate concomitant congenital abnormalities. (9) when the patient develops symptoms, conservative management usually has a poor outcome. nevertheless, surgery is considered as the best treatment, consisting in an anterior duodenotomy and the excision of the diverticulum (1,6). procedures like wide kocher 's manouver, or the indentification of vater 's papilla, biliar duct and pancreatic duct are essentials (3). the diverticulum must be extirpated from the mucosa belonging to the real duodenum in a circumferential manoeuvre. if it is necessary to reconstruct the papilla or the opening of the pancreatic duct, a silicone stent can be used. (1) we conclude that inverted duodenal diverticula should be considered in the differential diagnosis of upper intestinal obstruction of unknown cause. physicians should keep in mind the particular considerations related to vater 's papilla and congenital malformations associated to this condition. | inverted intraduodenal diverticulum is a rare congenital abnormality usually arising near the ampulla of vater. we describe a case of an inverted duodenal diverticulum in a patient that presented with an upper recurrent intestinal obstruction that required surgery. recognition of the entity and its anatomic relationships to the ampulla of vater is essential to the prevention of iatrogenic complications. the inverted intraduodenal diverticulum must be considered in the management of upper intestinal obstruction of unclear origin. |
this was a cross - sectional study of obese (bmi, > 95th percentile for age and sex) pubertal adolescents recruited from an obesity clinic in the children s hospital of philadelphia. exclusion criteria included having previously diagnosed diabetes or sleep disorders, genetic syndromes affecting glucose tolerance or sleep, or major organ system illness, or taking medications affecting insulin or glucose metabolism. the protocol was approved by the children s hospital of philadelphia institutional review board ; informed consent was obtained from the parents or guardians, and assent was obtained from the participants. demographic data and medical history were obtained from guardians and participants. physical examination, including pubertal (tanner) staging, was performed by a study investigator. weight was measured using a digital scale (scaletronix, white plains, ny). height was measured using a wall - mounted stadiometer (holtain inc., crymych, u.k.). bmi was calculated as weight (kilograms) divided by height (meters) squared. bmi percentiles and z scores were assessed using age- and sex - specific reference data (13). after a 12-h overnight fast, an oral glucose tolerance test (ogtt) was performed : subjects ingested oral glucose solution (1.75 g / kg, maximum 75 g), and blood samples for glucose and insulin were obtained at 10, 0, 10, 30, 60, 90, 12, 150, and 180 min. hemoglobin a1c (hba1c) was also measured. the following morning, after an overnight fast, subjects underwent a frequently sampled intravenous glucose tolerance test (fsigt) : infusion of 0.25 g / kg of 25% dextrose intravenously over 30 s, infusion of regular human insulin (0.015 units / kg i.v.) over 5 min at t = 20 min, and drawing of blood samples for glucose and insulin at t = 5, 2, 4, 8, 19, 22, 30, 40, 50, 70, 100, and 180 min. plasma glucose levels were measured by the glucose dehydrogenase method (hemocue analyzer ; hemocue inc., cypress, ca). plasma insulin levels were measured by radioimmunoassay (linco, st. charles, mo). the minmod millenium software program (14) homeostasis model assessment of ir (homa - ir) is a validated measure of insulin sensitivity (15):insulinogenic index (igi) is a measure of insulin secretion that has been validated in children against the hyperglycemic clamp (16):whole - body insulin sensitivity index (wbisi) is an insulin sensitivity measure that has been validated in obese children and adolescents (15) : homeostasis model assessment of ir (homa - ir) is a validated measure of insulin sensitivity (15) : insulinogenic index (igi) is a measure of insulin secretion that has been validated in children against the hyperglycemic clamp (16) : whole - body insulin sensitivity index (wbisi) is an insulin sensitivity measure that has been validated in obese children and adolescents (15) : higher wbisi levels indicate greater insulin sensitivity. acute insulin response to glucose (airg) is a parameter of early pancreatic response to glucose, calculated as the mean incremental plasma insulin concentration over baseline in the first 8 min of the fsigt (14).sensitivity to insulin (si) is a parameter calculated from serial insulin and glucose values during the fsigt (14). acute insulin response to glucose (airg) is a parameter of early pancreatic response to glucose, calculated as the mean incremental plasma insulin concentration over baseline in the first 8 min of the fsigt (14). sensitivity to insulin (si) is a parameter calculated from serial insulin and glucose values during the fsigt (14). signals were recorded on a computerized system (rembrandt ; rescare, buffalo, ny). the following parameters were recorded : electroencephalogram (c3/a2, c4/a1, o1/a2, and o2/a1) ; right and left electro - oculograms ; submental electromyogram ; tibial electromyogram ; electrocardiogram ; chest and abdominal wall motion by inductance plethysmography ; oronasal pressure / airflow (nasal pressure cannula with oral thermistor bead ; pro - tech, woodinville, wa) ; end - tidal pco2, measured at the nose by infrared capnometry (nellcor n-1000) ; arterial oxygen saturation (sao2) by pulse oximetry, and oximeter pulse waveform. studies were reviewed by a single sleep board - certified investigator (l.j.b.), who had no knowledge of subjects metabolic status. sleep architecture (n1, n2, n3, and rem sleep stages) and respiratory disturbances (including the apnea - hypopnea index [ahi ], arousal index, and lowest oxyhemoglobin saturation [lowest sao2 ]) were scored using standard pediatric criteria (17). histograms and one - sample kolmogorov - smirnov tests were used to assess normality of distribution of continuous variables. pearson or spearman correlations were used to examine associations between sleep architecture or osa measures and parameters of glucose homeostasis and insulin secretion and sensitivity. hierarchical linear regression procedures were used to evaluate the aforementioned relationships while controlling for potential confounding variables (e.g., degree of obesity and osa). assumptions of linearity were tested by examining plots of the standardized residuals as a function of standardized predicted values. where curve estimation procedures uncovered curvilinear relationships, polynomial regressions were conducted. analysis of covariance (ancova) models were used to examine differences in the outcome variables between sexes and among different pubertal stages, controlling for covariates. as we tested three underlying hypotheses relating to the relationship between sleep architecture and insulin secretion and sensitivity and overall glycemia, we used an adjusted p value of < 0.017 (0.05/3) for statistical significance. demographic data and medical history were obtained from guardians and participants. physical examination, including pubertal (tanner) staging, was performed by a study investigator. weight was measured using a digital scale (scaletronix, white plains, ny). height was measured using a wall - mounted stadiometer (holtain inc., crymych, u.k.). bmi was calculated as weight (kilograms) divided by height (meters) squared. bmi percentiles and z scores were assessed using age- and sex - specific reference data (13). after a 12-h overnight fast, an oral glucose tolerance test (ogtt) was performed : subjects ingested oral glucose solution (1.75 g / kg, maximum 75 g), and blood samples for glucose and insulin were obtained at 10, 0, 10, 30, 60, 90, 12, 150, and 180 min. after an overnight fast, subjects underwent a frequently sampled intravenous glucose tolerance test (fsigt) : infusion of 0.25 g / kg of 25% dextrose intravenously over 30 s, infusion of regular human insulin (0.015 units / kg i.v.) over 5 min at t = 20 min, and drawing of blood samples for glucose and insulin at t = 5, 2, 4, 8, 19, 22, 30, 40, 50, 70, 100, and 180 min. plasma glucose levels were measured by the glucose dehydrogenase method (hemocue analyzer ; hemocue inc., cypress, ca). homeostasis model assessment of ir (homa - ir) is a validated measure of insulin sensitivity (15):insulinogenic index (igi) is a measure of insulin secretion that has been validated in children against the hyperglycemic clamp (16):whole - body insulin sensitivity index (wbisi) is an insulin sensitivity measure that has been validated in obese children and adolescents (15) : homeostasis model assessment of ir (homa - ir) is a validated measure of insulin sensitivity (15) : insulinogenic index (igi) is a measure of insulin secretion that has been validated in children against the hyperglycemic clamp (16) : whole - body insulin sensitivity index (wbisi) is an insulin sensitivity measure that has been validated in obese children and adolescents (15) : higher wbisi levels indicate greater insulin sensitivity. acute insulin response to glucose (airg) is a parameter of early pancreatic response to glucose, calculated as the mean incremental plasma insulin concentration over baseline in the first 8 min of the fsigt (14).sensitivity to insulin (si) is a parameter calculated from serial insulin and glucose values during the fsigt (14). acute insulin response to glucose (airg) is a parameter of early pancreatic response to glucose, calculated as the mean incremental plasma insulin concentration over baseline in the first 8 min of the fsigt (14). sensitivity to insulin (si) is a parameter calculated from serial insulin and glucose values during the fsigt (14). homeostasis model assessment of ir (homa - ir) is a validated measure of insulin sensitivity (15):insulinogenic index (igi) is a measure of insulin secretion that has been validated in children against the hyperglycemic clamp (16):whole - body insulin sensitivity index (wbisi) is an insulin sensitivity measure that has been validated in obese children and adolescents (15) : homeostasis model assessment of ir (homa - ir) is a validated measure of insulin sensitivity (15) : insulinogenic index (igi) is a measure of insulin secretion that has been validated in children against the hyperglycemic clamp (16) : whole - body insulin sensitivity index (wbisi) is an insulin sensitivity measure that has been validated in obese children and adolescents (15) : higher wbisi levels indicate greater insulin sensitivity. acute insulin response to glucose (airg) is a parameter of early pancreatic response to glucose, calculated as the mean incremental plasma insulin concentration over baseline in the first 8 min of the fsigt (14).sensitivity to insulin (si) is a parameter calculated from serial insulin and glucose values during the fsigt (14). acute insulin response to glucose (airg) is a parameter of early pancreatic response to glucose, calculated as the mean incremental plasma insulin concentration over baseline in the first 8 min of the fsigt (14). sensitivity to insulin (si) is a parameter calculated from serial insulin and glucose values during the fsigt (14). signals were recorded on a computerized system (rembrandt ; rescare, buffalo, ny). the following parameters were recorded : electroencephalogram (c3/a2, c4/a1, o1/a2, and o2/a1) ; right and left electro - oculograms ; submental electromyogram ; tibial electromyogram ; electrocardiogram ; chest and abdominal wall motion by inductance plethysmography ; oronasal pressure / airflow (nasal pressure cannula with oral thermistor bead ; pro - tech, woodinville, wa) ; end - tidal pco2, measured at the nose by infrared capnometry (nellcor n-1000) ; arterial oxygen saturation (sao2) by pulse oximetry, and oximeter pulse waveform. studies were reviewed by a single sleep board - certified investigator (l.j.b.), who had no knowledge of subjects metabolic status. sleep architecture (n1, n2, n3, and rem sleep stages) and respiratory disturbances (including the apnea - hypopnea index [ahi ], arousal index, and lowest oxyhemoglobin saturation [lowest sao2 ]) were scored using standard pediatric criteria (17). histograms and one - sample kolmogorov - smirnov tests were used to assess normality of distribution of continuous variables. pearson or spearman correlations were used to examine associations between sleep architecture or osa measures and parameters of glucose homeostasis and insulin secretion and sensitivity. hierarchical linear regression procedures were used to evaluate the aforementioned relationships while controlling for potential confounding variables (e.g., degree of obesity and osa). assumptions of linearity were tested by examining plots of the standardized residuals as a function of standardized predicted values. where curve estimation procedures uncovered curvilinear relationships, polynomial regressions were conducted. analysis of covariance (ancova) models were used to examine differences in the outcome variables between sexes and among different pubertal stages, controlling for covariates. as we tested three underlying hypotheses relating to the relationship between sleep architecture and insulin secretion and sensitivity and overall glycemia, we used an adjusted p value of < 0.017 (0.05/3) for statistical significance. seventy obese adolescents were screened for participation ; seven cancelled prior to the study date and one did not undergo psg, leaving 62 participants for analysis. insulin and glucose values, calculated indices, and psg results are presented in table 2. subject characteristics glucose tolerance testing and psg results sleep architecture (durations are given in minutes ; percentages are denoted as %). % tst in n = percentage of total sleep time spent in a given sleep stage (e.g., % tst in n1=% total sleep time in n1). total sleep time (tst) was significantly or near - significantly associated with both short- and long - term measures of glucose homeostasis (table 3). curve estimation modeling and regression statistics showed that these relationships were u shaped (quadratic) (fig. c). there was no association between any measure of osa and measures of glucose homeostasis (supplementary table 1). glu 1 h, glucose level 1 h after oral glucose ingestion on ogtt ; glu 2 h, glucose level 2 h after oral glucose ingestion on ogtt. numbers in boldface indicate significant association, and numbers in italics indicate near - significant association (p value between 0.017 and 0.05). spearman correlation analysis. sleep duration and glucose homeostasis measures. b : association between 2-h glucose level on ogtt and total sleep duration (minutes) that evening. in all three panels, the u - shaped relationships suggest that a sleep duration of 420510 min (78.5 h) is associated with optimal glucose homeostasis. on regression analysis, individual sleep stages, pubertal stage, and sex were not significant predictors of any glucose homeostasis measure ; bmi z score was a significant contributor, and tanner stage was a marginal contributor, to the overall 2-h glucose model only (not to the overall fasting glucose or hba1c models). for the overall regression models mentioned above, adjusted r and p values were as follows : 0.201 (p = 0.002) for fasting glucose, 0.442 (p < 0.0005) for 2-h glucose, and 0.200 (p = 0.002) for hba1c. n3 sleep, both total duration and the percentage of total sleep time in n3 (% tst in n3), correlated significantly or with marginal significance (p value between 0.017 and 0.05) with several insulin secretory measures (table 4) in bivariate analysis. curve estimation modeling uncovered a cubic relationship between n3 and airg (r = 0.286 ; p = 0.001), with inflection points at approximately 65 and 98 min. osa measures did not associate significantly with any measure of insulin secretion (supplementary table 2). a marginally significant negative association was seen between pubertal stage and both n3 duration (r = 0.282 ; p = 0.028) and % tst in n3 (r = 0.252 ; p = 0.050), but there was no association between pubertal stage and any of the insulin secretory measures examined, or between sex and insulin secretory measures. correlation of sleep architecture with measures of insulin secretion and sensitivity sleep architecture (durations are given in minutes ; percentages are denoted as %). % tst in n = percentage of total sleep time spent in a given sleep stage. numbers in boldface indicate significant association, and numbers in italics indicate near - significant association (p value between 0.017 and 0.05). other sleep stages, tst, osa measures, and sex were not significant predictors of insulin secretory measures in the final regression model. bmi z score contributed significantly to the airg final model but not to the igi model, and pubertal stage contributed significantly to the final igi model but not the airg model. adjusted r and p values for the overall models were 0.161 (p = 0.002) for igi and 0.383 (p < 0.0005) for airg. correlation analysis showed a marginally significant negative association between n2 sleep and several insulin sensitivity measures (table 4). a marginally significant negative correlation was seen between ahi and si (r = 0.338 ; p = 0.025 ; see supplementary table 3). however, on regression analysis, no strong associations were seen between sleep architecture or osa and ogtt - derived insulin sensitivity measures. we found a marginal relationship between n2 duration and homa - ir (overall r = 0.088 ; p = 0.040), and although the relationship between % tst in n2 and si was stronger, bmi z score (i.e., degree of obesity) was the strongest predictor in that model (overall model r = 0.400 ; p < 0.0005). seventy obese adolescents were screened for participation ; seven cancelled prior to the study date and one did not undergo psg, leaving 62 participants for analysis. insulin and glucose values, calculated indices, and psg results are presented in table 2. subject characteristics glucose tolerance testing and psg results sleep architecture (durations are given in minutes ; percentages are denoted as %). % tst in n = percentage of total sleep time spent in a given sleep stage (e.g., % tst in n1=% total sleep time in n1). total sleep time (tst) was significantly or near - significantly associated with both short- and long - term measures of glucose homeostasis (table 3). curve estimation modeling and regression statistics showed that these relationships were u shaped (quadratic) (fig. c). there was no association between any measure of osa and measures of glucose homeostasis (supplementary table 1). glu 1 h, glucose level 1 h after oral glucose ingestion on ogtt ; glu 2 h, glucose level 2 h after oral glucose ingestion on ogtt. numbers in boldface indicate significant association, and numbers in italics indicate near - significant association (p value between 0.017 and 0.05). spearman correlation analysis. sleep duration and glucose homeostasis measures. a : association between sleep duration and fasting plasma glucose levels on ogtt. b : association between 2-h glucose level on ogtt and total sleep duration (minutes) that evening. in all three panels, the u - shaped relationships suggest that a sleep duration of 420510 min (78.5 h) is associated with optimal glucose homeostasis. on regression analysis, tst was the most significant predictor of glucose homeostasis measures. individual sleep stages, pubertal stage, and sex were not significant predictors of any glucose homeostasis measure ; bmi z score was a significant contributor, and tanner stage was a marginal contributor, to the overall 2-h glucose model only (not to the overall fasting glucose or hba1c models). for the overall regression models mentioned above, adjusted r and p values were as follows : 0.201 (p = 0.002) for fasting glucose, 0.442 (p < 0.0005) for 2-h glucose, and 0.200 (p = 0.002) for hba1c. n3 sleep, both total duration and the percentage of total sleep time in n3 (% tst in n3), correlated significantly or with marginal significance (p value between 0.017 and 0.05) with several insulin secretory measures (table 4) in bivariate analysis. curve estimation modeling uncovered a cubic relationship between n3 and airg (r = 0.286 ; p = 0.001), with inflection points at approximately 65 and 98 min. osa measures did not associate significantly with any measure of insulin secretion (supplementary table 2). a marginally significant negative association was seen between pubertal stage and both n3 duration (r = 0.282 ; p = 0.028) and % tst in n3 (r = 0.252 ; p = 0.050), but there was no association between pubertal stage and any of the insulin secretory measures examined, or between sex and insulin secretory measures. correlation of sleep architecture with measures of insulin secretion and sensitivity sleep architecture (durations are given in minutes ; percentages are denoted as %). % tst in n = percentage of total sleep time spent in a given sleep stage. numbers in boldface indicate significant association, and numbers in italics indicate near - significant association (p value between 0.017 and 0.05). spearman correlation analysis. other sleep stages, tst, osa measures, and sex were not significant predictors of insulin secretory measures in the final regression model. bmi z score contributed significantly to the airg final model but not to the igi model, and pubertal stage contributed significantly to the final igi model but not the airg model. adjusted r and p values for the overall models were 0.161 (p = 0.002) for igi and 0.383 (p < 0.0005) for airg. correlation analysis showed a marginally significant negative association between n2 sleep and several insulin sensitivity measures (table 4). a marginally significant negative correlation was seen between ahi and si (r = 0.338 ; p = 0.025 ; see supplementary table 3). however, on regression analysis, no strong associations were seen between sleep architecture or osa and ogtt - derived insulin sensitivity measures. we found a marginal relationship between n2 duration and homa - ir (overall r = 0.088 ; p = 0.040), and although the relationship between % tst in n2 and si was stronger, bmi z score (i.e., degree of obesity) was the strongest predictor in that model (overall model r = 0.400 ; p < 0.0005). in this multiethnic group of obese adolescents, we found strong relationships between sleep, hyperglycemia, and insulin secretion. specifically, we found u - shaped relationships between total sleep duration and measures of both short- and long - term glycemia, and positive or cubic associations between n3 and insulin secretory measures, even after adjusting for potential confounders such as degree of obesity, osa, sex, and pubertal stage. our sleep duration data suggest that glucose metabolism is optimal when 7.58.5 h of sleep is achieved. this is consistent with adult data noting u - shaped associations between self - reported sleep duration and t2 dm risk (5). adults with t2 dm have also been reported to have shorter n3 duration than nondiabetic adults (18). although one postulated mechanism suggests that n3 loss increases ir (19), our results instead demonstrated a relationship between n3 and insulin secretion. this relationship, which appeared to be a function of n3 itself rather than of total sleep duration, suggested that an absolute n3 duration of 1 h may be needed to achieve a stable amount of insulin secretion, and that increasing n3 might greatly improve insulin secretion. our results may help explain the aforementioned association between n3 lack and t2 dm, as a loss in first - phase insulin secretion is both an early marker of t2 dm and part of its pathogenesis (20). parasympathetic activity, which is increased in n3 sleep (21), stimulates glucose - induced insulin secretion (22) ; we speculate that increased parasympathetic activity may be responsible for our observed associations. although growth hormone (gh) secretion occurs largely during the n3 sleep (23), gh is unlikely to be a factor in the associations, as gh increases insulin resistance (24) rather than insulin secretion. in addition, insulin - like growth factor-1 levels in our subjects did not correlate significantly with either sleep duration or any individual sleep stage, further corroborating that gh secretion is unlikely to explain the observed associations. finally, we found a positive association between n2 sleep and insulin sensitivity, with varying contribution from degree of obesity. although n2 duration was also negatively associated with at least one marker of osa, the ahi, indicating possible confounding, the ahi did not consistently associate with insulin sensitivity measures ; thus, the observed association may represent an intrinsic relationship between n2 sleep and insulin sensitivity. a night in the sleep laboratory does not necessarily reflect sleep at home, a possible limitation of our study. however, in our study, the ogtt preceded the psg. thus, any observed associations between sleep parameters and ogtt - derived measures could not have been short - term effects of a night in the laboratory. the association between tst and hba1c, which reflects long - term glycemia, also supported our findings. also, families completed post - psg surveys asking whether sleep in the laboratory was typical of home sleep. only 32% indicated worse sleep in the laboratory ; these subjects had no significant demographic, anthropometric, or glucose / insulin parameter differences compared with the remaining subjects, and of the sleep parameters, the only difference seen was a lower absolute rem duration in the poorer sleep group. our subjects mean tst of 7.1 h was similar to the mean 7.2-h sleep duration reported in the 2006 national sleep foundation adolescent poll (2). a recent study showed an association between short sleep on actigraphy and metabolic dysregulation in school - age children (25). although home - based actigraphy studies could be useful, actigraphy is less precise and can not discriminate between sleep stages. in conclusion, we found significant relationships between sleep duration and sleep architecture and measures of glucose homeostasis and insulin secretion. to our knowledge, this is the first report of an association between n3 sleep and changes in -cell function and of a u - shaped association between sleep duration and glucose levels in a pediatric population. we speculate that inadequate sleep duration and altered sleep architecture (relative suppression of n3 sleep) may play a role in t2 dm development. ensuring adequate sleep might reduce the risk of t2 dm in at - risk obese adolescents. | objectivesleep deprivation is associated with increased risk of adult type 2 diabetes mellitus (t2 dm). it is uncertain whether sleep deprivation and/or altered sleep architecture affects glycemic regulation or insulin sensitivity or secretion. we hypothesized that in obese adolescents, sleep disturbances would associate with altered glucose and insulin homeostasis.research design and methodsthis cross - sectional observational study of 62 obese adolescents took place at the clinical and translational research center and sleep laboratory in a tertiary care children s hospital. subjects underwent oral glucose tolerance test (ogtt), anthropometric measurements, overnight polysomnography, and frequently sampled intravenous glucose tolerance test (fsigt). hemoglobin a1c (hba1c) and serial insulin and glucose levels were obtained, indices of insulin sensitivity and secretion were calculated, and sleep architecture was assessed. correlation and regression analyses were performed to assess the association of total sleep and sleep stages with measures of insulin and glucose homeostasis, adjusted for confounding variables.resultswe found significant u - shaped (quadratic) associations between sleep duration and both hba1c and serial glucose levels on ogtt and positive associations between slow - wave sleep (n3) duration and insulin secretory measures, independent of degree of obesity, pubertal stage, sex, and obstructive sleep apnea measures.conclusionsinsufficient and excessive sleep was associated with short - term and long - term hyperglycemia in our obese adolescents. decreased n3 was associated with decreased insulin secretion. these effects may be related, with reduced insulin secretory capacity leading to hyperglycemia. we speculate that optimizing sleep may stave off the development of t2 dm in obese adolescents. |
our understanding of physical laws and knowledge of material properties allow us to engineer bridges that do not collapse and car engines that convert energy into mechanical motion. even the simplest bacterium comprises a system whose complexity is humbling [1, 2 ]. in recognition of such challenges, the central goal of synthetic biology is to transform biology into a system that can be engineered just as we engineer bridges and mechanical systems today (reviewed in [38 ]). this approach promises to provide an improved understanding of the living world and will enable us, in the coming years, to harness the diverse repertoire of biology for compelling applications. green chemicals and industrial feedstocks, programmable and personalizable biological therapies, materials with novel properties, cheap and deployable diagnostics and therapeutics to promote global health, and technologies enabling environmental stewardship and remediation. the new paradigm of biology by design can be summarized as follows : conceive a desired biological function, design an engineered biological system to perform this function, the system performs as predicted. achieving this ambitious goal will require an improved understanding of the mechanisms by which biological parts function and by which they interact with one another and their environment. the first wave of synthetic biology has focused on the development of simple biological modules, involving anywhere between two and ten well - characterized biological parts (e.g., genes) that are connected to perform defined functions (reviewed in). in principle, these parts should still operate in a predictable manner when transplanted into different biological contexts. this conceptual framework connects synthetic biology with other engineering disciplines. whether the parts are electrical, chemical, physical, or biological, engineers depend on the predictable behavior of connected components to design and construct complex systems. for example, the automotive industry must develop engines that work within the context of an entire automobile. construction of an engine is possible due to an understanding of how the individual components (e.g., pistons, crankshafts, and spark plugs) interact with one another, how these individual functions can be assembled to achieve a desired task (e.g., conversion of energy released by combusting fuel into mechanical motion), and how this assembled system interacts with the broader context of the vehicle (e.g., drawing fuel from the fuel lines and powering the transmission to transmit energy to the wheels). when synthetic biologists design simple biological systems from individual components in order to perform novel functions, these systems often do not behave as expected, particularly when transported from one biological context to another. in effect, a synthetic biologist today must endeavor to design an engine from imperfectly characterized parts and without knowing how the engine will eventually connect to the rest of the vehicle. to overcome the limitations of our incomplete knowledge of biology, synthetic biologists approach the construction of user - defined functions through an iterative design cycle that incorporates both bottom - up and top - down design perspectives (figure 1). bottom - up considerations include the following : what is the desired function of the synthetic system ? what parts could be used to construct this system (e.g., promoters, ribosome binding sites, etc.) ? do the parts exist and do they require additional characterization ? how should the parts be configured ? what is the predicted behavior of the synthetic module ? conceptually, this design perspective focuses on the characteristics of individual modules (which may be parts or subnetworks of parts) and their assembly into novel configurations in isolation from the endogenous cellular context into which they will eventually be placed. top - down considerations include the following : how might the engineered module be decoupled from or insulated from aspects of the broader biological context (e.g., metabolic state, cell cycle progression, and epigenetic modifications) that may complicate the function of the engineered network ? what interactions are necessary to connect a synthetic controller to the endogenous system it will control ? the top - down perspective focuses on potential interactions both desirable and undesirable between the engineered subsystem and its biological context, and on development of strategies for harnessing or compensating for these influences. top - down and bottom - up perspectives guide our development of improved strategies for engineering biology (figure 1), even in the face of incomplete information. here, we discuss these perspectives in the context of three rapidly progressing areas which exemplify the opportunities and challenges encountered at different scales of biological organization : biochemical transformations, cellular devices and therapeutics, and approaches that expand the chemistry of life (figure 2). each of these topics is leading to new applications and advancing frontiers in synthetic biology. in the nearly four decades since the molecular biology revolution, it has become clear that the biological world provides a rich and diverse repertoire that may help to address some of humanity 's most significant challenges. for example, harnessing biology for production of small molecules has led to applications in biofuels, chemical feedstocks, and therapeutics [3, 9 ]. however, to achieve these advances, substantial investments of resources, time, and labor are currently necessary. for example, it has been estimated that engineering microbes for expression of the antimalarial drug artemisinin required 150-person years of work. biochemical transformations comprise a family of applications for which synthetic biology may complement and expand efforts in metabolic and genetic engineering, by providing a robust technological framework for addressing common challenges. these challenges include targeting substrate flux towards a specific product, avoiding the loss of intermediates to competing pathways, reducing the accumulation of toxic intermediates, and preventing saturation of pathway enzymes [11, 12 ]. synthetic biology tools are used to remove rate - limiting steps and increase titers of target biochemicals through an iterative design cycle of analysis, design, and implementation (figure 1). each cycle invokes both bottom - up and top - down considerations (figure 2). bottom - up considerations may include gene selection, promoter selection, and use of regulatory elements. top - down considerations may include selection of the host organism, potential interactions of the synthetic metabolic network with existing cellular components, and competition with native pathways that impact substrate or intermediate availability. host selection must often include making a choice between transferring heterologous pathways to a well - characterized organism or improving the production capacity (or other properties) of organisms for which robust genetic tools are not available. despite the numerous design requirements and constraints, many biochemical transformation projects in biotechnology, we highlight four types : (i) incorporating nonnative genes to extend natural metabolism, (ii) incorporating whole pathways to add function, (iii) creating new pathways that have never existed before in nature, and (iv) organizing pathways to enhance activity (e.g., through compartmentalization or scaffolds). it should be noted that significant engineering of host metabolism is often a necessity to ensure precursor availability and inhibition of competitive pathways. moreover, in many cases the scope of genetic modifications requires multiple approaches to achieve a desired user - defined objective [1315 ]. to extend natural metabolism, network optimization and selection of nonnative enzymes that catalyze the formation of a desired biochemical is required. in one example of this approach, liao and colleagues converted 2-keto acid metabolites into branched alcohols for use as biofuels. this was accomplished in escherichia coli by first engineering the amino acid biosynthetic pathways that supply precursors required for 2-keto acid metabolite production. then, incorporation of 2-keto acid decarboxylases and alcohol dehydrogenases from a variety of organisms yielded the desired alcohols. in later work, atsumi. improved production titers for isobutanol through the use of alcohol dehydrogenases from different organisms, saccharomyces cerevisiae and lactococcus lactis, rather than the native alcohol dehydrogenase of e. coli. in another example, an e. coli strain was engineered to convert simple sugar to biodiesel through modified fatty - acid biosynthesis pathways. here, native host metabolism was reprogrammed by modulating more than 8 enzymes (such as thioesterase, acyl - ligase, and wax ester synthase) to overproduce fatty acids, deregulate fatty acids, and produce fatty acid ethyl and methyl esters (biodiesel). then, incorporation of nonnative hemicellulase genes enabled direct conversion of biomass to biodiesel in a single e. coli strain. other poignant examples of heterologous gene transfer have come from meeting global health needs, namely, the treatment of malaria. in a landmark study, production of the antimalarial drug artemisinin required incorporation of amorphadiene synthase and cytochrome p450 monoxygenase from the plant artemisia annua. these genes were incorporated into a strain of s. cerevisiae modified to produce large quantities of the amorphadiene precursor, farnesyl pyrophosphate, by a complete overhaul of the mevalonate pathway. beyond heterologous gene transfer, protein engineering is being explored to create nonnatural enzymes that can be combined with biosynthetic pathways to create novel biochemical products. new enzymes can be created through either mutagenesis of existing enzymes or rational design of new enzymes. for example, liao and colleagues expanded their work on the biosynthesis of alcohols by utilizing promiscuous enzyme activity. by altering substrate specificity of an enzyme through site - directed mutagenesis, they were able to synthesis enzymes to make nonnatural alcohols with possible value as biofuels. these enzymes were created through random and site - directed mutagenesis to selectively deprotect different monosaccharide substrates for polysaccharide synthesis. in addition, protein engineering has advanced to a point where design of new enzymes from scratch is possible. an enzyme to perform a diels - alder reaction was created through computational design and experimental fine - tuning to achieve substrate specificity and stereoselectivity. the designed enzyme was then altered to demonstrate the ability to perform similar reactions with different substrates. such work demonstrates a powerful new approach to achieving molecular transformations that can not be efficiently performed by either nature or conventional chemistry. as the number of nonnative and unnatural genes transferred into a host increases to include entire pathway branches, a critical consideration is seamless integration with the organism 's regular metabolic functions. one issue with metabolic network transfer is that the global energy resources of the cell must support both synthetic modules and the host cell. despite this challenge, as well as other hurdles associated with constructing, testing, and balancing large networks of molecular pathways (where design complexity can be intimidating), some examples have shown success. two notable applications include production of high butanol titers through the transfer of the clostridium acetobutylicum butanol pathway to a variety of bacterial hosts and production of valencene in yeast by transferring an isoprenoid pathway consisting of 7 e. coli genes. in addition to manipulating and extending native pathways, it is also possible to engineer novel biochemical pathways that have never been observed in biology. for example, moon. designed a synthetic pathway using genes from three sources : myo - inositol-1-phosphate synthase from s. cerevisiae, myo - inositol oxygenase from mice, and uronate dehydrogenase from pseudomonas syringae. the novel combined pathway was inserted into e. coli to convert myo - inositol into glucaric acid, a value - added chemical used as a dietary supplement, therapeutic, and potential feedstock for polymers. similarly, hawkins and smolke synthesized benzylisoquinoline alkaloids in s. cerevisiae through the combination of genes from three different plant species as well human p450 enzyme. these examples illustrate that complex molecules of therapeutic interest may be produced by combining existing enzymes into novel assemblies to create novel biosynthetic pathways. protein scaffolds that hold tagged pathway enzymes in close proximity have also been used to improve biochemical transformations. in this model, scaffolds have been designed for biosynthesis pathways including mevalonate and glucaric acid pathways, and spatial colocalization of pathway enzymes has lead to increased productivity. the use of scaffolds increases the local concentration of pathway intermediates near the desired downstream enzymes, which improves overall pathway kinetics, avoids the accumulation of hazardous intermediates, and minimizes consumption of intermediates by competing pathways [25, 26 ]. such synthetic biology tools provide novel mechanisms for manipulating metabolic pathways and complement traditional pathway optimization. characterizing transcriptional and translational architectures and developing technologies for manipulating these processes for instance, new genetic elements have been created to carry out circuit - like regulation that mimic toggle switches, and gates, and oscillators [2729 ]. inserting such regulatory elements into cellular devices could lead to high - resolution control and novel functional behavior. translation efficiency, for example, can be adjusted by modifying ribosome binding site affinity. to fine - tune imperfect designs, genetic screening and evolutionary tools are also being developed. sommer. screened a metagenomic library of arbitrary environmental dna to identify three novel genes that convey tolerance to the biomass chemicals syringaldehyde and 2-furoic acid. similarly, bayer. used a metagenomic screen of 89 suspected methyl halide transferase enzymes in e. coli to increase the yield of methyl halides from biomass to serve as precursors for chemicals and fuels. another screening technique called scales (scalar analysis of library enrichments) uses libraries consisting of genomic dna inserts of specific sizes to identify regions of a genome responsible for a particular phenotype. this method has been used to identify gene regions conveying tolerance to inhibitors of metabolic pathways involving aspartic acid. such screens demonstrate approaches that can be used to discover genes responsible for other desirable or undesirable phenotypes. new robust evolutionary techniques such as multiplex automated genome engineering (mage) and global transcription machinery engineering (gtme) have also been developed to enable rapid fine - tuning of metabolic networks. mage generates genomic diversity within cells and across populations by using parallel, site - specific modification of dna. using mage, the lycopene synthesis pathway in e. coli was optimized by genetically modifying several targeted chromosome locations associated with genes of the pathway. the gtme approach uses random mutagenesis of transcriptional machinery to alter the transcriptional program of an organism and has been used to develop e. coli and s. cerevisiae strains with desirable phenotypes for biofuel production, such as high ethanol tolerance [36, 37 ]. evolutionary approaches provide a complementary strategy to design - based engineering that may reveal unexpected routes toward higher system performance and provide a better understanding of biological systems that may be incorporated into subsequent design strategies. the next few years will bring increased attempts to construct and program large - scale user - defined pathways, and even whole organisms. recently, researchers at the j. craig venter institute (jcvi) assembled, modified, and implanted a complete mycoplasma mycoides donor genome into a related mycoplasma recipient cell. this technological milestone marks the dawn of synthetic genomics. while this achievement is an important step towards designer organisms, applications of this approach for example, if one knew how to design a cell that efficiently converts sunlight, water, and co2 into fuel, then jcvi 's technology could enable the synthesis and transplantation of that chemically synthesized genome into a recipient cell. however, we do not yet know how to design such a genome from scratch, and simply screening astronomical numbers of synthetic genome variants is impractical or even impossible. in addition, the costs of genome construction currently prohibit most researchers from applying this approach. in the long run, however, there is no doubt that jcvi 's technology will join a suite of other whole - genome engineering techniques to accelerate the development of microbial factories for producing fuels, pharmaceuticals, green biochemicals, and novel materials. for applications in medicine, synthetic biology may also be used to create new cellular functions, such that the engineered cell itself not just a product that it produces serves a therapeutic role. in this context, a cell can be viewed as a device that receives inputs, processes this information, and produces outputs all based upon its genetic programming. therefore, novel cellular functions may be constructed through strategies that create new connections between existing and novel mechanisms for input (e.g., receptors), processing (e.g., intracellular signaling cascades and regulatory interactions), and output (e.g., the production of bioactive molecules or the induction of specialized cellular effector functions). every such strategy must take into account both bottom - up considerations (including the choice of biological parts and their configuration) and top - down considerations (including the choice of cell type to be engineered and the interactions both desirable and undesirable of the engineered components with native cellular functions) (figure 2). each of these design choices is guided by the type of cellular function required for a given application. for some strategies, a cell may be reprogrammed to change the way it relates existing inputs to existing outputs by rewiring intracellular signal processing. in one example of this approach, signaling through the erbb2 receptor tyrosine kinase, which mediates mitogenic or transformative signaling in tumor cells, was redirected into a proapoptotic pathway. this was accomplished by engineering novel intracellular signaling proteins, in which sh2 or ptb phosphotyrosine - recognition domains from grb2 or shca, respectively, (which interact with erbb2), were fused to the death effector domain of fadd. such intracellular signaling proteins can be engineered to modulate multiple functional characteristics, including autoregulation, ligand specificity, and signaling dynamics using modular functional domains [40, 41 ] and various scaffold motifs and configurations. an alternative approach is one often used in basic research, whereby the extracellular ligand - binding domain of one receptor is fused to the intracellular signaling domain of another receptor to generate a chimeric receptor. strategies such as these could be used to generate cellular therapies for situations in which the natural input / output relationship is dysfunctional, such as tumor - mediated conversion of tumor - reactive t cells to an immunosuppressive phenotype. an important consideration when using this approach is that the engineered signaling pathway does not replace native signaling pathways, but rather engineered and native pathways are coupled by the shared use of native receptors or downstream components. in other applications, cells may be engineered to respond to a stimulus to which the cell does not naturally respond. an early example of this approach is the use of chimeric antigen receptors (cars) for cancer immunotherapy. in this model, ligand - binding domains from antibodies specific for tumor antigens are fused to the intracellular portion of t cell receptors in order to generate t cells capable of killing tumor cells that express the targeted antigen. early implementations of this approach are in clinical trials, and while most benefits have been modest, some responses are quite promising. in the most recent version of this strategy, third generation cars are constructed by fusing the ligand - binding domains of tumor - specific antibodies to intracellular domains of cd28, cd137, and the tcr- chain, in sequence. in this construct, the tcr- chain confers tcr signaling upon binding of the antibody domain to its target, while the cd28 and cd137 domains enhance tcr signaling and t cell survival in vivo. in a related approach, xu. constructed tumor - responsive dendritic cells that recognize the tumor antigen, erbb2, and signal through the toll - like receptor 4 (tlr4) pathway to induce an inflammatory immune response against the tumor. this was accomplished by fusing the ligand - binding domain of an anti - erbb2 antibody to various intracellular mediators of tlr4 signaling. while each of these examples used receptors or antibodies that naturally recognize a particular ligand, it is also possible to re - engineer receptors, such as gpcrs, so that they respond to novel small molecule ligands. such an approach may be applied to generate cells whose functions are regulated by an inducible trigger, such as a small molecule drug. as an example of this strategy, t cells were engineered to express interleukin 2 (il-2, a cytokine necessary for t cell survival) under the control of a synthetic ribozyme switch that responds to the inducer theophylline. when these cells were transferred to recipient mice, theophylline treatment induced il-2 production and engineered t cell survival in vivo. similar ribozyme switches may be designed to respond to a variety of small molecule cues. other rna - based approaches can be used to program cells to perform more complicated functions, including the logical evaluation of multiple inputs, such that activation of a target gene (or genes) is conditioned upon the presence or absence of multiple extracellular cues. like mammalian cells, bacteria may also be programmed for therapeutic applications in which the bacteria itself is the therapeutic. for example, bacteria naturally home to tumor environments, so this propensity may be harnessed by engineering bacteria to selectively invade and destroy cancer cells in vivo. in this model, bacteria were engineered to express the invasin protein in response to the hypoxic conditions that typify tumor microenivronments. invasin expression enables these bacteria to invade mammalian cells expressing 1-integrins, which is a common marker on some types of cancer. this approach can be further enhanced to include the hypoxia - inducible release of cytotoxic agents to destroy the tumor cells after invasion. similarly, synthetic biology could be used to integrate more complex functions and regulatory features into other bacteria - based therapeutics. important considerations when manipulating bacteria for therapeutic applications include both interactions with host biology and preventing the engineered bacteria from spreading beyond the patient being treated. for example, lactococcus lactis was engineered to treat inflammatory bowel diseases by programming these microbes to secrete the immunosuppressive cytokine interleukin-10 (il-10) and by deleting their thymidylate synthase gene. this strategy restricted bacterial growth to cultures supplemented with thymidine, which prevents these cells from replicating efficiently in the gut. this treatment has already shown promise in clinical trials. because the engineered bacteria die quickly within the gut, for example, the immunosuppressive bacteria described above could be modified such that they require survival signals from the gut and quickly die outside of this environment, or such that they secrete il-10 only when they detect disease flareups. such modifications could allow for safer and more effective treatments while still restricting these bacteria to their targeted milieu. synthetic biology provides the framework required to design and implement such sophisticated biotherapy strategies. engineering communication between cells already, simple synthetic communication systems have been constructed in bacteria, yeast, and even mammalian cells. in a representative example, a yeast sender population was engineered to secrete the plant hormone, isopentenyladenine, and a receiver yeast population was made responsive to this signal by expressing the receptor protein, atcre1. for example, two populations of e. coli were engineered to communicate bidirectionally using p. aeruginosa quorum sensing components, such that each cell type secretes a unique sender signal and expresses its own reporter gene only when it detects the signal secreted by the other cell type. similar information exchange between sender and receiver cells can also be used to program spatial organization and pattern formation. other applications of intercellular communication are polling strategies, which can be used to coordinate and synchronize dynamic cellular functions and potentially improve performance by reducing sensitivity to variations in environmental conditions. these approaches can also be extended to interkingdom communication, including bacterial, yeast, and mammalian cells. when applied in mammalian cells, such approaches could eventually be used to program communication and spatial organization for the construction of synthetic organs or organ - like devices. these strategies may also provide useful tools for programming the differentiation of stem cells in a manner that does not require recapitulating the complex extracellular cues that guide natural differentiation programs. interkingdom communication platforms could enable engineered symbiotic microbes, which might patrol the colon or skin for signs of disease, to communicate this information to their human hosts. to date, existing intercellular communication systems rely upon simple small molecule - based signaling intermediates, and sophisticated multicellular communication strategies will require the development of novel orthogonal, high - information - content signaling platforms. finally, synthetic biology could lead to new therapeutics through in vitro applications such as screening and diagnostics. current drug screens are typically based upon measurements such as treatment - induced changes in the replication and viability of diseased cells relative to changes in healthy control cells. this limits the type of information about biological activity and safety that can be assessed at the screening stage. for example, engineered cells can be used to identify drugs that target specific pathogen functions. such an approach was used to identify substances that block the m. tuberculosis protein, ethr, which otherwise confers resistance to the prodrug ethionamide. this approach may be especially useful for pathogens that are dangerous or difficult to culture in vitro. similar approaches could be used to construct screens that evaluate the impact of test molecules on human cellular functions that do not naturally produce an easily - assessed phenotype like apoptosis. for example, bacterial bioreporters have been constructed to detect various compounds and toxins. in most cases, this detection relies upon natural sensing mechanisms and involves expressing a reporter gene in response to the activation of an existing bacterial promoter. synthetic biology could be employed to engineer novel sensing mechanisms and build bacterial bioreporters that perform diagnostic assessments such as detecting biochemical markers within human blood samples. these technologies could be designed to function and produce readouts without requiring external equipment, so that they could be readily deployed for mobile operations or in resource - poor areas. as we continue to develop improved technologies for engineering both human cells and the microorganisms with which they interact, synthetic biology promises to transform medicine just as it has already begun to transform biotechnology. to lessen undesired and unpredictable interactions that often hamper design goals, modules of nonnatural components that operate independently of the cell 's natural machinery such orthogonal modules offer tremendous flexibility for optimization without disrupting the cell 's operating system and, as a bonus, could exploit the renewability and evolvability of biology to synthesize nonbiological materials. in nature, complex and diverse chemical tasks are carried out across a variety of length scales using biopolymers of very simple composition. the expansion of the central dogma of biology to include diverse, nonnatural analogs may lead to a set of general solutions featuring orthogonal information coding, orthogonal cellular machinery, orthogonal scaffolds, orthogonal compartments, orthogonal interactions, or orthogonal communication pathways, which could form the basis for entirely artificial living systems. as an example, even minor modifications to natural biopolymers can endow them with altered interactions and enhanced functionalities. peptide nucleic acids (pnas), which have backbones similar to peptides but nucleobase functional groups (as opposed to the twenty natural amino acids), hybridize to short stretches of dna, and exhibit increased resistance to both proteases and endonucleases. to expand the chemistry of life, orthogonal modules may be constructed through evolved catalytic ensembles that enable synthesis, evolution, and organization of nonnatural or hybrid polymers. in this case, bottom - up design considerations must include template recognition, substrate recognition, catalytic activity, error correction, product folding, and transport properties (figure 2). top - down considerations may require that synthesis should occur under physiological conditions (temperature, buffer conditions, etc.) and that monomer building blocks must be available to the host or able to be synthesized by the host. implicit mass transfer limitations arising from cellular structure must also be addressed. increasing the capacity of nucleic acids to store, recall, and early experiments with modified nucleic acids (termed xenonucleic acids, or xnas) were motivated by the desires to understand how dna and rna developed in the prebiotic world and to create antisense molecules that could be easily taken up by cells. more recently, efforts have shifted towards modulating function by exploring diversity in the composition of bases, sugars, and phosphates. backbone modifications of natural nucleic acids generally decrease recognition by endonucleases and provide an alternative strategy for modulating xna binding affinity to complementary strands. the most studied backbone modifications of natural nucleic acids include pnas, locked nucleic acids (lnas), threose nucleic acids (tnas), phosphorothioates, and morpholinos. beyond antisense applications, these xnas have the potential to vastly expand biopolymer information storage. in order for novel biopolymers to become a viable tool for synthetic biologists, sequence - defined synthesis of the polymers enzymatic template - directed synthesis of nonnatural polymers can be performed. in one approach, naturally occurring dna and rna polymerases have been identified that accept nonnatural substrates, thus allowing the templated synthesis of tna from dna. in addition, these polymerases can be evolved in vitro to improve processivity and decrease error rates with nonnatural monomer units. improved understanding of structure - function relationships in polymerases has further resulted in the rational modification of a natural transcriptase to a polymerase that accepts nonstandard nucleotides. advances in enzyme engineering and our understanding of nature 's design rules will speed progress toward engineered polymerases and enable us to probe the feasibility of enzymatic xna synthesis and application. in another approach, the cell 's protein polymerase, the ribosome, could be engineered to selectively accept and polymerize synthetic monomers with high template fidelity. driven by the promise of new therapeutics, evolvable abiological polymers, and biophysical probes, there has long been interest in harnessing natural translation machinery to synthesize polymers of nonnatural building blocks. in concept, ribosome engineering may provide a more general, modular solution to novel biopolymer synthesis due to the use of transfer rnas (trnas) that serve as adapters between the genetic code and the biopolymer product. incorporating nonnatural monomer units on the ribosome depends on (i) reassignment of codons to nonnatural monomer units and (ii) acceptance of these units in the catalytic center. remarkably, dozens of nonnatural amino acids have already been site - specifically incorporated into polypeptides by the ribosome. this was accomplished by engineering synthetic routes toward aminoacylation of trnas [74, 75 ] and orthogonal synthetase - trna pairs and showing that natural ribosomes could accept nonnatural substrates. even nonpeptide backbones, such as n - substituted amino acids and hydroxy acids have been incorporated by the ribosome, although with varying degrees of success. when using orthogonal synthetase - trna pairs, one potential issue is nonspecific incorporation that results from competing natural aminoacyl - trnas. to obtain greater control of homogeneous sequence composition, genetic code reprogramming [79, 80 ] or synthetic genetic systems (based on xnas or modified bases) can be applied. although a variety of nonnatural monomer units have been incorporated by native ribosomes, it is clear that these substrates are inherently suboptimal. therefore, a major design challenge lies not only in the bottom - up aspect of incorporating nonnatural building blocks, but also in reversing the evolutionary optimization of finely tuned machinery and aligning it onto an entirely new path. to address this limitation, the ribosome and its machinery can be modified to enable increased promiscuity and efficiency at polymerizing nonnatural monomer units. in one case, ef - tu was evolved to more efficiently incorporate amino acids containing bulky aromatic groups. in examples that modify the ribosome directly, ribosomal rna has been mutated or chemically modified to enable more efficient nonnatural substrate incorporation [83, 84 ]. although the ribosome has been shown to be tolerant of modifications, there are relatively few examples because constraints imposed by living cells (e.g., dominant lethality) have hindered efforts to engineer ribosomes. specialized ribosomes overcome the dominant lethality constraint and provide an exemplary illustration of orthogonal performance. originally pioneered by de boer and colleagues, anti - shine - dalgarno sequences can be engineered into 16s ribosomal rna to enable a group of ribosomes (orthogonal from the host) to translate specific mrna populations. this has shown advantages for performing logic operations [85, 86 ], synthesizing proteins with nonnatural amino acids, such as p - benzoyl-(l)-phenylalanine, and efficiently incorporating quadruplet codons. orthogonal ribosomes will provide useful tools in programming synthetic function and expanding the genetic code to make nonnatural products for biotechnology. cell - free systems are being used to remove the most stringent top - down design constraints confronted when attempting to expand the chemistry of life [8992 ]. cellular overhead is reduced (e.g., genes of unknown functions, metabolic redundancies, and unintended interactions), transport barriers are removed, cellular viability is not required, and resources are more efficiently directed towards user - defined objectives. an additional advantage is direct access to the system of interest, similar to opening the hood of a car and accessing the engine. cell - free protein synthesis systems [9395 ] are one of the most prominent examples of cell - free biology and can be employed to create large amounts of nonnatural peptide. looking forward, both in vitro and in vivo efforts towards novel catalytic ensembles promise to meld the complexity and diversity of chemistry with the accuracy and reproducibility of biology. the main rewards will be increased understanding of biology and new classes of evolvable molecules, which may find use as therapeutics or novel materials. given the complexity that characterizes living systems developed through eons of evolution, the challenge to transform biology into an engineerable system is formidable. considering biological design from both top - down and bottom - up perspectives provides a useful framework for proceeding towards this goal. at each scale of biological organization, this approach guides both the design and analysis of novel systems, as well as our investigations into the fundamental mechanisms that impact the performance of engineered biological systems. synthetic biology is already changing the way we think about producing useful molecules, engineering our own biology, and expanding the chemistry of life. making progress on these and other fronts will require both interdisciplinary approaches and new pedagogical strategies for training a new cadre of whole - brain thinkers. if successful, these efforts may revolutionize our ability to meet pressing societal needs through a new paradigm of biology by design. | synthetic biology is a nascent technical discipline that seeks to enable the design and construction of novel biological systems to meet pressing societal needs. however, engineering biology still requires much trial and error because we lack effective approaches for connecting basic parts into higher - order networks that behave as predicted. developing strategies for improving the performance and sophistication of our designs is informed by two overarching perspectives : bottom - up and top - down considerations. using this framework, we describe a conceptual model for developing novel biological systems that function and interact with existing biological components in a predictable fashion. we discuss this model in the context of three topical areas : biochemical transformations, cellular devices and therapeutics, and approaches that expand the chemistry of life. ten years after the construction of synthetic biology 's first devices, the drive to look beyond what does exist to what can exist is ushering in an era of biology by design. |
to ascertain the etiologic agent causing an outbreak of febrile illness with symptoms similar to chikungunya fever, we selected 3 sites in chiapas state, mexico, for sampling : tapachula, la libertad, and ciudad hidalgo (figure 1, panel a). after patients informed consent was obtained, blood samples were collected from persons whose condition met the following case definition for possible chikungunya fever : acute onset of fever > 38.5c, accompanied by severe arthralgia not explained by other medical conditions (6). samples from tapachula were collected from patients who sought treatment at the centro regional de investigacion en salud publica, whereas in la libertad and ciudad hidalgo, researchers surveyed houses to identify potential case - patients. in total, 119 blood samples were collected, and serum was isolated by centrifugation. six samples were stored in magnapure lc buffer (roche, nutley, nj, usa), which inactivates virus particles but preserves the genomic rna. map of mexico showing the 3 sites where serum samples were obtained to test for chikungunya virus in chiapas, mexico, 2014 : tapachula, la libertad, and ciudad hidalgo. viral rna was extracted from serum samples using the zr-96 viral rna kit (zymo research, orange, ca, usa) according to the manufacturer s protocol. one - step quantitative reverse transcription pcr (qrt - pcr) (7) was performed by using the taqman rna - to - ct 1-step kit (applied biosystems, san francisco, ca, usa). standard plaque assays were performed for the samples positive by qrt - pcr with vero cells. anti - chikv igm - capture enzyme - linked immunosorbent assays (elisas) (8) were performed by using a chimeric eilat - chikv (9) that contained the nonstructural proteins from eilat virus and structural proteins from chikv, resulting in a structure indistinguishable from that of chikv. a sample was considered to be chikv negative if the sample was not positive by qrt - pcr or igm elisa. viral rna from 5 serum samples was sent for illumina deep sequencing and assembled by using the virus - specific hive - hexagon algorithm (10) and the ngen module in lasergene suite version 10 (bioinformatics pioneer dnastar, inc., madison, wi, usa). single nucleotide polymorphisms were analyzed by using the sequencing profiling tool in the hive suite of programs (10). sequences were aligned in seaview (11) by using translated proteins for the open reading frames and using nucleotides for the untranslated genome regions, and all gaps were removed. bayesian phylogenetic inference was performed by using the general time reversible plus invariant sites plus gamma distribution 4 model in mrbayes (12) with 1.5 million iterations to reach congruence. partial genome sequencing of the e2 and e1 glycoproteins was performed by using traditional sanger methods on pcr amplicons on an additional 8 samples. over 100 serum samples were obtained from persons seeking treatment for chikungunya fever like illness during october december 2014 in 3 locations in chiapas, mexico (tapachula, la libertad, ciudad hidalgo) (figure 1). these samples were analyzed by qrt - pcr and igm - capture elisa to detect viremia and igm, respectively. no overlap occurred between the samples that were positive for chikv by qrt - pcr or those positive by igm, demonstrating the importance of the humoral response to viral clearance. with few exceptions, viremia was detectable up to 3 days after fever onset (figure 2, panel a), after which most samples were igm positive. all age groups were equally likely to be infected, as expected during infectious disease outbreaks involving a naive population (13). a) number of serum samples positive for chikungunya virus (chikv) by reverse transcription quantitative pcr (qrt - pcr), for chikv igm by elisa, and negative for chikv by both methods, arranged according to day after fever onset. b) phylogenetic tree generated by bayesian analysis showing the relationship of the complete genomic sequences of 5 chikungunya virus isolates from mexico and representative sequences from the genbank library. all nodes showed posterior probabilities of > 0.9, except those indicated with a star. plaque assays were performed to determine serum virus titers (table) ; 3 qrt - pcr samples from tapachula could not be assayed because of sample limitations. mean viremia level was similar among the 3 sampled locations and ranged from < 2 log10 to 4.2 log10 pfu / ml. chikv, chikungunya virus ; qrt - pcr, quantitative reverse transcription pcr. six patient samples that were negative for chikv by qrt - pcr were unable to be used for plaque assays or elisas because they were stored in magnapure buffer (roche, nutley, nj, usa). five serum samples from diverse locations and collection dates were selected for illumina sequencing (genbank accession nos. the strains circulating in chiapas were most closely related to asian lineage strains first detected in the caribbean (represented by a british virgin islands isolate) and now presumed to be circulating in much of latin america (figure 2, panel b). curiously, no novel mutations appear to have been fixed in the year since the british virgin islands isolate was collected. although the genomic sequences confirmed that the circulating virus in chiapas belonged to the asian lineage, which is primarily transmitted by a. aegypti mosquitoes (1), we nevertheless examined the sequences for mutations known to adapt chikv for transmission by a. albopictus mosquitoes. because both mosquito species are found in chiapas (14), adaptation of the chikv strain circulating in mexico to a. albopictus mosquitoes could place temperate regions of the eastern united states and millions of naive persons at risk for infection. none of the e2 or e1 substitutions previously reported to increase fitness in a. albopictus mosquitoes was observed in 8 additional samples analyzed by sanger sequencing (genbank accession nos. one sample had a nonsynonymous mutation, in comparison to the january 2014 british virgin islands isolate (genbank accession no. we found that 79% of febrile illness cases with polyarthralgia in chiapas state during late 2014 were caused by chikv. our sequencing of chikv genomes confirmed spread of an asian lineage strain from the caribbean and suggested that although chikv has circulated in the americas since 2013, no adaptive mutations have occurred. however, continued screening for vector - adaptive mutations will be critical, especially now that strains of the ecsa lineage, which gave rise to the indian ocean lineage, have been introduced into brazil (4). | since chikungunya virus (chikv) was introduced into the americas in 2013, its geographic distribution has rapidly expanded. of 119 serum samples collected in 2014 from febrile patients in southern mexico, 79% were positive for chikv or igm against chikv. sequencing results confirmed chikv strains closely related to caribbean isolates. |
compound 1 (3.90 g, 15.7 mmol), triphenylphosphine (ph3p) (7.40 g, 28.3 mmol), and p - nitrobenzoic acid (3.10 g, 18.8 mmol) were dissolved in anhydrous thf (157 ml). diisopropylazodicarboxylate (diad) (6.35 g, 6.20 ml, 31.4 mmol) was added dropwise over 30 min. the solution was removed from the ice bath, allowed to warm to room temperature, and stirred for an additional 6 h. the solvent was removed under reduced pressure. the crude product was dissolved in ethyl acetate (100 ml) and washed with brine (2 200 ml). the solvent was removed, and the crude product was redissolved in ch2cl2 (75 ml). the crude product was purified via column chromatography (ch2cl2) to yield compound 2 (4.7 g, 12.0 mmol) as a colorless oil in 76% yield. compound 2 (2.0 g, 5.0 mmol) was dissolved in acetone (50 ml). sodium azide (0.49 g, 7.5 mmol) was added, and the solution was heated at reflux for 14 h. the solution was allowed to cool to room temperature, and the solvent was removed under reduced pressure. the crude product was dissolved in ethyl acetate (50 ml) and washed with distilled water (2 50 ml). the solvent was removed, and the crude product was redissolved in ch2cl2 for purification. compound 3 (0.80 g, 3.3 mmol) was purified via column chromatography (2% methanol in ch2cl2 v / v) to obtain a colorless oil in 65% yield. compound 3 (2.23 g, 9.10 mmol) and ph3p (2.86 g, 10.9 mmol) were dissolved in toluene (91 ml) under a nitrogen atmosphere. the solution was cooled to 0 c and stirred on ice for 10 min. diad (2.20 g, 2.15 ml, 10.9 mmol) was added dropwise to the solution over 15 min. (4.30 g, 2.54 ml, 18.2 mmol) and dipea (2.35 g, 3.16 ml, 18.2 mmol) were added to the solution, which was then stirred on ice for another 5 min. the solution was removed from the ice bath, warmed to 45 c in an oil bath, and stirred for 24 h. the solvent was removed under reduced pressure, and the crude product was dissolved in ethyl acetate (50 ml). the crude product was washed with brine (2 75 ml) and dried over sodium sulfate. the solvent was removed under reduced pressure, and the crude product was redissolved in ch2cl2 (50 ml). the crude product was purified via column chromatography (07% ethyl acetate in hexanes v / v) to obtain compound 5 (0.790 g, 2.28 mmol) as a colorless oil in 25% yield. h nmr (400 mhz, cdcl3) 4.91 (s, 1h), 4.494.47 (dd, j = 8.6, 6.4 hz, 0.4h, cis), 4.404.36 (dd, j = 7.8, 7.8 hz, 0.6h, trans), 3.843.75 (m, 1h), 3.75 (s, 3h), 3.693.66 (d, j = 12.5 hz, 0.6h, trans), 3.603.58 (d, j = 12.3 hz, 0.4h, cis), 2.472.44 (m, 1h), 2.282.23 (m, 1h), 1.46 (s, 4h, cis), 1.42 (s, 5h, trans). c nmr (150.8 mhz, cdcl3) 171.9, 171.7, 153.1, 152.4, 119.1 (q, j = 293 hz), 79.8, 77.1, 56.5, 56.1, 51.5, 51.4, 51.2, 36.6, 35.6, 27.2. f nmr (376.3 mhz, cdcl3) 70.47 (trans conformation), 70.53 (cis conformation). hrms (lifdi - tof) compound 4 (0.36 g, 0.78 mmol) and lioh (0.22 g, 0.93 mmol) were dissolved in a solution of water (4 ml) and 1,4-dioxane (4 ml). the solution was stirred at room temperature for 14 h. the mixture was acidified to ph 2 with dilute hcl and extracted with ethyl acetate (2 10 ml). the crude product was purified via column chromatography (02% methanol in ch2cl2 v / v) to obtain compound 5 (0.22 g, 0.49 mmol) as an off - white solid in 63% yield. h (600 mhz, cdcl3) 4.92 (s, 1h), 4.534.51 (dd, j = 7.7, 7.0 hz, 0.6h), 4.424.39 (dd, j = 7.4, 7.4 hz, 0.4h), 3.833.80 (m, 0.4h), 3.743.66 (m, 1.6h), 2.512.48 (m, 1h), 2.442.40 (m, 0.6h), 2.352.32 (m, 0.4h), 1.48 (s, 5h), 1.43 (s, 4h). c nmr (150.8 mhz, cdcl3) 177.7, 174.7, 155.9, 153.4, 120.1 (q, j = 293 hz), 82.3, 81.3, 57.4, 52.6, 37.4, 35.7, 28.2. f nmr (564.5 mhz, cdcl3) 70.49 (trans conformation), 70.55 (cis conformation). compound 4 (1.3 g, 2.2 mmol) was dissolved in a solution of 1,4-dioxane (15 ml) and 4 m hcl (15 ml). the solution was allowed to stir at reflux for 6 h. the solvent was removed under reduced pressure. compound 6 (0.78 g, 2.2 mmol) was used as a crude reagent in the next step without purification. alternatively, compound 5 could be subjected to identical conditions to yield compound 6. h nmr (600 mhz, meod - d4) 5.22 (br s, 1h), 4.554.51 (dd, j = 9.6, 8.8 hz, 1h), 3.813.79 (dd, j = 13.5, 5.0 hz, 1h), 3.463.44 (d, j = 13.5 hz, 1h), 2.532.52 (m, 1h), 2.512.50 (m, 1h). c nmr (150.8 mhz, meod - d4) 168.7, 120.0 (j = 293 hz), 79.0, 58.1, 51.8, 35.7. f nmr (376.3 mhz, meod - d4) 71.56. hrms (lifdi - tof) m / z : [m ] calcd for c9h9f9no3 350.0439, found 350.0420. crude compound 6 (1.01 g, 2.90 mmol) was dissolved in a solution of 1,4-dioxane (15 ml) and h2o (15 ml). fmoc - osu (1.17 g, 3.48 mmol) and k2co3 (0.80 g, 5.80 mmol) were added to the solution, and the resultant mixture was stirred for 14 h at room temperature. the 1,4-dioxane was removed under reduced pressure, and the crude product was acidified with 2 m hcl (10 ml). the crude product was extracted with ethyl acetate (2 20 ml). the solvent was removed, and the crude product was redissolved in ch2cl2. the product was purified via column chromatography (04% methanol in ch2cl2 v / v) to obtain the compound 8 as a white solid (0.83 g, 1.45 mmol) in 50% yield. h (600 mhz, cdcl3) 7.777.76 (d, j = 7.5 hz, 1.3h, trans), 7.747.73 (d, j = 7.6 hz, 0.7h, cis) 7.557.52 (dd, j = 12.4, 7.6 hz, 2h), 7.427.38 (m, 2h), 7.327.29 (dd, j = 7.4, 7.3 hz, 2h), 4.92 (s, 0.7h, trans), 4.89 (s, 0.3h, cis), 4.594.57 (dd, j = 7.8, 7.4 hz, 0.7h, trans), 4.524.43 (m, 2h), 4.384.37 (dd, j = 7.4, 7.4 hz, 0.3h, cis), 4.304.27 (dd, j = 7.0, 6.6 hz, 0.7h, trans), 4.174.16 (dd, j = 6.6, 6.6 hz, 0.3h, cis), 3.833.73 (m, 1h), 3.71 (s, 1h), 2.492.45 (m, 1.7h, mixture of cis and trans), 2.322.29 (m, 0.3h, cis). c nmr (150.8 mhz, cdcl3) 173.1, 156.1, 143.5, 143.4, 141.4, 127.9, 127.2, 124.9, 124.8, 120.12 (q, j = 293 hz), 120.09, 77.9, 68.6, 67.6, 57.6, 56.7, 53.0, 52.7, 47.2, 47.0, 37.6, 35.8. f nmr (564.5 mhz, cdcl3) 70.38 (trans conformation), 70.43 (cis conformation). hrms (ci - tof) m / z : [m ] calcd for c24h18f9no5 571.1041, found 571.1027. 3.96 g, 15.1 mmol) were dissolved in toluene (126 ml) under a nitrogen atmosphere. the solution was cooled to 0 c and stirred on ice for 10 min. diad (3.05 g, 2.98 ml, 15.1 mmol) was added dropwise to the solution over 15 min. perfluoro - tert - butanol (5.95 g, 3.52 ml, 25.2 mmol) and dipea (3.18 g, 4.38 ml, 25.2 mmol) were added to the solution, which was stirred on ice for another 5 min. the solution was removed from the ice bath, warmed to 45 c in an oil bath, and stirred for 24 h. the solvent was removed under reduced pressure, and the crude product was dissolved in ethyl acetate (50 ml). the crude product was washed with brine (2 75 ml) and dried over sodium sulfate. the solvent was removed under reduced pressure, and the crude product was redissolved in ch2cl2 (50 ml). the crude product was purified via column chromatography (07% ethyl acetate in hexanes v / v) to obtain compound 5 (2.07 g, 4.47 mmol) as a colorless oil in 36% yield. h nmr (400 mhz, cdcl3) 4.84 (br s, 1h), 4.524.50 (dd, j = 8.3, 3.7 hz, 0.4h, minor), 4.414.38 (dd, j = 9.0, 3.1 hz, 0.6h, major), 3.843.78 (dd, j = 12.5, 5.6 hz, 0.6h, major), 3.763.75 (d, j = 6.5 hz, 0.4h, minor), 3.72 (s, 3h), 3.683.65 (d, j = 13.0 hz, 0.6h, major), 3.583.55 (d, j = 13.0 hz, 0.4h, minor), 2.532.38 (m, 2h), 1.48 (s, 4h, major), 1.43 (s, 5h, minor). c nmr (150.8 mhz, cdcl3) 172.9, 172.8, 154.2, 154.0, 129.4, 129.3, 124.8, 124.6, 124.5, 120.1 (q, j = 293 hz), 105.1, 80.9, 80.8, 66.6, 66.2, 57.5, 57.1, 52.3, 37.6, 36.6, 28.3, 28.2. f nmr (376.3 mhz, cdcl3) 70.42 (minor), 70.44 (major). hrms (lifdi - tof) m / z : [m ] calcd for c15h18f9no5 463.1041, found 463.1042. compound 8 (1.75 g, 3.78 mmol) and lioh (0.1086 g, 4.53 mmol) were dissolved in a solution of water (20 ml) and 1,4-dioxane (20 ml). the solution was stirred at room temperature for 14 h. the reaction mixture was acidified to ph 2 with dilute hcl and extracted with ethyl acetate (2 75 ml). the solvent was removed under reduced pressure, and the crude product was redissolved in ch2cl2. the crude product was purified via column chromatography (02% methanol in ch2cl2 v / v) to obtain compound 9 (1.05 g, 2.34 mmol) as an off - white solid in 62% yield. h (600 mhz, cdcl3) 4.86 (s, 1h), 4.514.50 (d, j = 6.7 hz, 0.4h, minor), 4.444.42 (d, j = 7.8 hz, 0.6h, major), 3.823.76 (m, 1h), 3.723.67 (m, 0.6h, major), 3.563.55 (m, 0.4h, minor), 2.522.44 (m, 2h), 1.49 (s, 3.6h), 1.44 (s, 5.4h). c nmr (150.8 mhz, cdcl3) 177.4, 175.5, 154.7, 153.5, 120.1 (q, j = 293 hz), 80.9, 57.5, 57.1, 52.9, 52.7, 52.1, 37.7, 37.6, 36.1, 28.1. f nmr (564.5 mhz, cdcl3) 70.38 (major), 70.44 (minor). hrms (lifdi - tof) m / z : [m + h ] calcd for c14h17f9no5 450.0963, found 450.0958. compound 9 (2.0 g, 4.3 mmol) was dissolved in 1,4-dioxane (15 ml), and 4 m hcl (15 ml) was added. the solution was allowed to stir at reflux for 6 h. the solvent was removed under reduced pressure. compound 10 (1.5 g, 4.3 mmol) obtained was used as a crude reagent in the next step. alternatively, compound 8 could be subjected to the same conditions to yield compound 10. h nmr (600 mhz, meod - d4) 5.20 (s, 1h), 4.624.60 (dd, j = 9.6, 3.4 hz, 1h), 3.743.71 (dd, j = 13.4, 4.6 hz, 1h), 3.563.53 (d, j = 13.9 hz, 1h), 2.742.68 (ddd, j = 14.6, 10.0, 5.0 hz, 1h), 2.542.51 (d, j = 14.6 hz, 1h). c nmr (150.8 mhz, meod - d4) 169.3, 120.0 (j = 293 hz), 78.9, 58.3, 52.1, 36.1. hrms (lifdi - tof) m / z : [m ] calcd for c9h9f9no3 350.0439, found 350.0413. crude compound 10 (0.50 g, 1.4 mmol) was dissolved in 1,4-dioxane (7 ml). fmoc - osu (0.50 g, 1.4 mmol) and k2co3 (0.39 g, 2.9 mmol) were added, and the resultant solution was stirred for 14 h at room temperature. the 1,4-dioxane was removed under reduced pressure, and the crude product was acidified with 2 m hcl (10 ml). the crude product was extracted with ethyl acetate (2 20 ml). the solvent was removed, and the crude product was redissolved in ch2cl2. the crude mixture (0.40 g, 0.72 mmol) was purified via column chromatography (04% methanol in ch2cl2 v / v) to obtain compound 11 (0.38 g, 0.70 mmol) as a white solid in 50% yield. h (600 mhz, cdcl3) 7.777.69 (d, j = 7.6 hz, 2h), 7.607.51 (m, 2h), 7.427.28 (m, 4h), 4.92 (s, 0.5h), 4.88 (s, 0.5h), 4.614.56 (m, 1h), 4.534.49 (m, 0.5h), 4.434.31 (m, 2h), 4.164.13 (dd, j = 6.3, 6.1 hz, 0.5h), 3.903.86 (m, 1h), 3.763.63 (m, 1h), 3.74 (s, 1h), 2.572.41 (m, 2h). c nmr (150.8 mhz, cdcl3) 176.3, 175.7, 154.7, 154.3, 143.9, 143.8, 143.60, 143.58, 141.4, 141.3, 127.8, 127.74, 127.70, 127.13, 127.10, 127.0, 125.1, 125.0, 124.8, 121.1, 119.97 (j = 293 hz), 119.96, 78.2, 67.9, 67.8, 67.0, 57.3, 57.0, 53.4, 52.9, 47.1, 37.7, 36.5, 21.9. f nmr (564.5 mhz, cdcl3) 70.42, 70.49. hrms (lifdi - tof) m / z : [m ] calcd for c24h18f9no5 571.1041, found 571.1038. peptides were synthesized on novagel peg - polystyrene graft rink amide resin (emd millipore) by standard solid - phase peptide synthesis. amide coupling reactions with fmoc - perfluoro - tert - butyl 4-hydroxyproline amino acids were conducted using 4 equiv each of amino acid and comu, with the reaction allowed to proceed for 36 h. subsequent amide coupling reactions in ac - gppxppgy - nh2 peptides were conducted as double coupling reactions (2 h for the first coupling, 1 h for the second coupling) using hatu as a coupling reagent. all peptides were acetylated on the n - terminus (5% acetic anhydride in pyridine, 3 ml, 3 5 min) and contained a c - terminal amide. peptides were cleaved from the resin and deprotected for 3 h using a 1 ml reaction volume of 90% tfa with 5% h2o and 5% triisopropylsilane. tfa was removed by evaporation, and the peptides were dissolved in 1 ml of 500 mm phosphate buffer (ph 7.2). crude peptide solutions were filtered using a 0.45 m syringe filter before injection on the hplc. peptides were purified by reverse - phase hplc (vydac semipreparative c18, 10 250 mm, 5 m particle size, 300 pore) using a 60 min linear gradient of 100% to 40% buffer a (98% h2o, 2% acetonitrile, 0.06% tfa) in buffer b (20% h2o, 80% acetonitrile, 0.05% tfa). analytical data for peptides : ac - gpp((2s,4r)-hyp(c4f9))ppgy - nh2 [tr, 37.8 min ; expected mass, 1056.4, observed mass, 1078.4 (m + na) ] ; ac - gpp((2s,4s)-hyp(c4f9))ppgy - nh2 [tr, 36.8 min ; expected mass, 1056.4, observed mass, 1078.4 (m + na) ] ; ac-((2s,4r)-hyp(c4f9))kaaaakaaaakaagy - nh2 [tr, 41.9 min ; expected mass, 1706.7, observed mass, 853.5 (m) ] ; ac-((2s,4r)-hyp(c4f9))kaaaakaaaakaagy - nh2 [tr, 40.8 min ; expected mass, 1706.7, observed mass, 853.6 (m) ]. peptides (1050 m final concentrations : ac-((2s,4r)-hyp(c4f9))kaaaakaaaakaagy - nh2 10 m ; ac-((2s,4s)-hyp(c4f9))kaaaakaaaakaagy - nh2 50 m ; ac - gpp((2s,4r)-hyp(c4f9))ppgy - nh2 20 m ; ac - gpp((2s,4s)-hyp(c4f9))ppgy - nh2 50 m) were dissolved in water containing 5 mm phosphate buffer ph 7.4 and 25 mm kf. the temperatures for each experiment were chosen to allow direct comparison to the analogous peptides with proline. experiments were conducted in 90% h2o/10% d2o containing 5 mm phosphate buffer ph 4, 25 mm nacl, and 100 m tsp as a reference for h nmr spectra. f nmr experiments were conducted without decoupling using a 1.5 s relaxation delay on a brker 600 mhz (545.5 mhz for f) nmr spectrometer equipped with a brker smart probe. | (2s,4r)- and (2s,4s)-perfluoro - tert - butyl 4-hydroxyproline were synthesized (as fmoc-, boc-, and free amino acids) in 25 steps. the key step of each synthesis was a mitsunobu reaction with perfluoro - tert - butanol, which incorporated a perfluoro - tert - butyl group, with nine chemically equivalent fluorines. both amino acids were incorporated in model -helical and polyproline helix peptides. each amino acid exhibited distinct conformational preferences, with (2s,4r)-perfluoro - tert - butyl 4-hydroxyproline promoting polyproline helix. peptides containing these amino acids were sensitively detected by 19f nmr, suggesting their use in probes and medicinal chemistry. |
functional weakness of the lower extremity due to stroke is caused not only by muscular weakness, but also by decreases in muscular endurance, and stability of the joint, and loss of proprioceptive sense. clinically, proprioceptive sense is an important factor in the evaluation and treatment of patients with neurologic problems ; and its weakness leads to declines in postural control, protective reflex, joint movement, balance ability, and gait1. due to weakness of dorsiflexors, mass flexor pattern, or mass extensor pattern, instead of reciprocal innervations, stiffness of the ankle joint, and loss of proprioceptive sense, permanent disabilities, such as foot drop, are common in stroke patients2. primary functions of the ankle joint are the provision of balance control against postural disturbance, absorption of shock during gait, and movement of the lower extremity. to provide these, it is necessary to maintain a sufficient range of motion of the ankle joint, muscular strength, and proprioceptive sense3. limitation of ankle dorsiflexion is a common gait problem of hemiparetic stroke patients. due to abnormal increase of muscle tension in the triceps surae, stroke patients can not actively control dorsiflexion, and the foot drop tends to occur4. a normal range of motion of the ankle joint in the standing position is essential for normal gait5. muscle cooperation in the ankle joint strategy puts the center of gravity on the ankle joint in the standing position. it requires a normal range of motion of the ankle joint and muscular strength. if the range of motion of the ankle joint is limited, postural control provided by the ankle joint is also limited6. compared with subjects without proprioceptive sense, subjects with proprioceptive sense appear to show a greater increase of postural sway, and lowering of balance measure scores7, all of which have important roles in gait pattern. re - education of the ankle joint movement for control of balance is an important factor in remedying gait or balance problems caused by abnormal muscular contraction or proprioception deficit8. therefore, this study investigated the effect of a proprioceptive exercise training program for improvement on the muscular strength of the ankle joint, static balance, dynamic balance, and gait of stroke patients., patients met the following criteria : stroke onset of more than six months previously, in order to minimize the effects of natural recovery ; ability to walk without use of a walking aid for a minimum of 15 m ; a mini - mental state examination score greater than 24/30 ; and ability to comprehend and follow simple instructions. subjects participated in a general physical therapy program lasting 30 minutes per session, two sessions per week, for a period of six weeks. in addition, all subjects practiced additional ankle proprioceptive control training, consisting of 30 minutes per session, two sessions per week, for a period of six weeks. subjects ' general characteristics were : male 10, female 3 ; mean sd age 58.46 years 8.53 ; height 161.09 cm 7.28 ; weight 61.83 kg 10.15 ; stroke onset 53.15 months 7.28, and modified ashworth scale (mas) scores of 01 (n=10), 1 + (n=3). subjects performed not only a general physical therapy program but also the ankle proprioceptive control training program. the ankle proprioceptive control training program consisted of a program for improvement of balance ability and gait by reinforcement of ankle proprioceptive control. the program consisted of three stages : mobility of the ankle joint and muscular strength (stage 1) ; weight bearing in a static standing position (stage 2) ; and a weight bearing and assignment training program (stage 3)9 (table 1). each program was provided 2 times per week for 30 min each session for six weeks. the muscular strength of the ankle joint was measured using a bte - primus (bte technology, usa, 2006). this equipment evaluates patient 's range of motion, muscular strength, endurance, activities of daily living, and task performance. we measured the patients ' ankle dorsiflexion and plantar flexion in the sitting position with placement of the foot on a foothold. subjects performed the timed up and go test (tug) before and after six weeks of physical therapy. tug has shown high intra - rater (icc=0.99) and inter - rater (icc=0.99) reliabilities and is an evaluation of dynamic balance ability10. the test was performed three times and the mean was used in the analysis of this study. the standard gaitrite walkway has six sensor pads encapsulated in a rolled - up carpet with an active area of 3.66 m length and 0.61 m width. as a subject walks along the walkway, the sensors capture each footfall as a function of time and transfer the gathered information to a personal computer for processing into footfall patterns. gaitrite was used for the measurement of spatiotemporal parameters, including gait velocity, cadence, step length, and stride length11. the spss 17.0 program (spss inc., the shapiro - wilk test was used to test the distribution of the general characteristics and outcome measures of the subjects. repeated measures anova was used to compare pretest, posttest, and retest results within groups. at the end of the proprioceptive control training program, muscular strength of the ankle dorsiflexor showed a statistically significant increases (p<0.001) from 29.54 n before training to 33.43 n at week 4, and 36.84 n at week 6. tug, a functional balance ability test, showed significant improvements from 20.47 s before training to, 17.94 s (4 wks), and 15.27 s (6 wks) (p<0.001). in temporal gait ability, gait velocity showed significant increases from 50.35 cm / s before training to, 60.45 cm / s (4 wks), and 67.55 cm / s (6 wks). cadence showed significant improvements from 76.78 steps / min before training to 81.42 steps / min (4 wks) and 88.81 steps / min (6 wks). in spatial gait ability, step length on the paretic side significantly increased from 38.88 cm before training to 44.12 cm (4 wks), and 45.16 cm (6 wks) (p<0.001), and significantly increased from 36.34 cm before training to 42.30 cm (4 wks), and 43.79 cm (6 wks) on the non - paretic side (p<0.001). significant increases in stride length from 72.84 cm before training to 86.63 cm (4 wks), and 88.92 cm (6 wks), were observed on the paretic side (p<0.001), and from 75.85 cm before training to 86.70 cm (4 wks), and 88.90 cm (6 wks) on the non - paretic side (p<0.001). weakened muscle strength in post - stroke patients is one of the factors limiting functional recovery. as a psychological factor, in particular, patient 's self - confidence can lead to a fall, due to self - limitation of activities weakened muscle strength. balance maintenance of anteroposterior postural sway is dependent on co - operative activities between the anterior tibial muscle and internal abdominal muscle. in other words, the activity of the internal abdominal muscle begins before the body falls over a vertical line and the anterior tibial muscle activates before the body becomes posturally erect13. andrews and bohannon2 studied the recovery of stroke patients ' lower extremity muscle strengths over a short period of time. they reported a statistically significant improvement in ankle dorsiflexion on the paretic side, from 75.6 n during hospitalization to 102.4 n after intervention. in a study of lower extremity muscle torque in the movement of stroke patients, kim and eng14 reported that ankle dorsiflexion was weaker than ankle plantarflexion. docherty.15 demonstrated there was a statistically significant increase in dorsiflexor and eversion strength after an ankle joint movement program lasting six weeks. in this study, ankle dorsiflexors showed statistically significant increases on the paretic side from 29.54 n before training to, 33.43 n at week 4, and 36.83 n at week 6. however, despite the increase in ankle dorsiflexor strengths : 44.50 n (0 wk), 47.13 n (4 wks), and 49.39 n (6 wks), plantarflexors showed no significant difference. these results suggest that impairment of proprioceptive sense leads to difficulty in maintaining balance and postural control due to loss of cognitive ability of postural position in the environment16, 17 ; however, proprioceptive sense was improved by participation in the proprioceptive ankle movement program. the increased static postural perturbation of stroke patients may be a result of balance loss due to malfunction of proprioceptive sense or proper weight bearing. the timed up and go (tug) test, berg balance scale (bbs), and functional reach test (frt) are standard assessments of stroke patients ' dynamic balance ability. among them, the reliability and the validity of tug has been proven for the measurement of clinical changes in dynamic balance ability and functional movement with time18. walker.19 reported that tug was 50.9 sec in the first stage of stroke and 24.9 sec after three months. in this study, after participation in an ankle proprioceptive control training program, tug scores showed a significant improvements from 20.47 sec (0 wk), to 17.9 sec (4 wk), and 15.27 sec (6 wk). these results can be interpreted as suggesting that dynamic balance ability was improved by increase of ankle sense of position through proprioceptive control, and they are in agreement with the results of previous studies. values are mean (sd). tug : timed up and go test, p : paretic side, np : non - paretic side, p<0.05, p<0.01, p<0.001 improvement of gait ability during a stroke patient 's functional recovery process is the main goal of both the patient and the therapist, because it is an important factor for the accomplishment of functional independence20. in a study of stroke patients ' walking patterns, edwards1 reported that cadence was 89131 steps / min and stride length was 1.081.63 m. in a walking step analysis using gaitrite of 62 healthy subjects, step lengths were 74.3 cm on the right side and 73.9 cm on the left side. stride lengths were 148.6 cm on the right side and 149.1 cm on the left side. walking velocity was 149.5 cm / s21. in this study, during participation in an ankle proprioceptive control program, subjects gait velocities increased from, 50.35 cm / s (0 wk), to 60.45 cm / s (4 wk), and 67.55 cm / s (6 wk), and cadence improved from 76.78 steps / min (0 wk), to 81.42 steps / min (4 wk), and 88.81 steps / min (6 wk). step length increased from 38.88 cm (0 wk), to 44.12 cm (4 wk), and 45.16 cm (6 wk) on the paretic side, and statistically significant increases from 36.34 cm (0 wk), to 42.30 cm (4 wk), and 43.79 cm (6 wk) were also observed on the non - paretic side. stride length increased from 72.84 cm (0 wk), to 86.63 cm (4 wk), and 88.92 cm (6 wk) on the paretic side, and statistically significant increases from 75.85 cm (0 wk), to 86.70 cm (4 wk), and 88.90 cm (6 wk) were also observed on the non - paretic side. in this study, after participation in an ankle proprioceptive control program, subjects ' gait velocities increased. cadence and step length increased on the paretic side, and statistically significant increases were observed on the non - paretic side as well. stride length were increased on the paretic side, and statistically significant increases were also observed on the non - paretic side (table 2). in this study, the ankle proprioceptive control program improved the spatial - temporal walking pattern of patients with stroke and the results agree with those of previous studies. we consider the ankle proprioceptive control program strengthened the ankle dorsiflexors, which led to improvement in gait ability, due to prevention of foot drop in the swing phase and the provision of lower extremity stability at initial contact. in addition, a more stable walking pattern might have been achieved through improvement of static - dynamic balance ability leading to decreased postural perturbation. this study investigated the effect of an ankle proprioceptive control program on the recovery of balance and gait abilities of hemiparetic patients stroke. the ankle proprioceptive control program focusing on somatosensory sense for the recovery of stroke patients ' balance and walking abilities proved effective. this study used an ankle proprioceptive control program that consisted of three stages : mobility of the ankle joint and muscular strength (stage 1) ; weight bearing in a static standing position (stage 2) ; weight bearing and assignment training program (stage 3). however, we did not compare the three stages to determine which is the most effective for balance, gait ability, or muscle strength. a follow - up study should compare them, to determine which stages are most effective at improving patients ' balance, gait ability and muscle strength. | [purpose ] balance and gait ability determine to a large degree the level of independence of daily living which is an important goal of rehabilitation. this study was conducted in order to examine the effectiveness of an ankle proprioceptive control program on ankle muscle strength, balance, and gait of chronic stroke patients. [methods ] thirteen chronic stroke patients more than six months post - stroke were recruited. all subjects received ankle proprioceptive control training for 30 minutes per session, two days per week, over a period of six weeks. outcome measures were ankle strength (bte - primus), the timed up and go test (tug), and spatiotemporal parameters measured by a gaitrite instrument. [results ] significant improvements in ankle dorsiflexor strength, tug, gait speed and cadence, step length, and stride length were observed on the paretic side. [conclusion ] the results of this study provide evidence in support of incorporation of an ankle proprioceptive control program for effective improvement of both balance and gait ability of chronic stroke patients. the findings of this study suggest the feasibility and suitability of an ankle proprioceptive control program for chronic stroke patients. |
cerebral venous thrombosis (cvt) is a distinct disorder with highly variable symptoms and courses. brain mri, magnetic resonance venography, and positive d - dimer assay confirmed the presence of cvt. the work - up for detecting the cause of the cvt revealed occult prostate cancer. occult malignancy including prostate cancer should be strongly suspected in older patients with idiopathic cvt. cerebral venous thrombosis (cvt) is an infrequently occurring type of cerebrovascular disease with a wide spectrum of clinical presentations and is notoriously difficult to diagnose. several disorders can cause or predispose patients to cvt, including all medical, surgical, and gyneco - obstetric causes of deep vein thrombosis in the legs, genetic and acquired prothrombotic disorders, cancer, hematological diseases, vasculitis and inflammatory systemic disorders, pregnancy and puerperium, infections, and several local causes such as brain tumors, arteriovenous malformations, head trauma, cns infections, and ear, sinus, mouth, face, or neck infections.1 diagnostic and therapeutic procedures such as surgery, lumbar puncture, jugular catheter, and some drugs (particularly oral contraceptives, hormonal replacement therapy, steroids, and cancer treatments) can also cause or predispose people to cvt. however, the relative likelihood of these causes varies with the age and country of origin of the patient.2 in the absence of an obvious provoking risk factor, the presence of an underlying malignancy is often considered because some types of cancer appear to be able to initiate or trigger a thrombotic diathesis through various mechanisms. however, cvt as the presenting sign of occult prostate cancer has not been reported in the english literature. three days prior to admission, he experienced difficulty in using a television remote control and writing a letter, he was clumsy with spoons and chopsticks, and he had forgotten the personal identification number of his bank account. he had no history of diabetes, hypertension, smoking, alcoholism, or cardiac disorders. a neurological examination revealed ideational apraxia, finger agnosia, alexia, and right - to - left disorientation. minimal dysarthria, right central type facial nerve palsy, and pronator drifting of the right arm were also noted. 1a). magnetic resonance (mr) angiography showed no arterial abnormality, but mr venography revealed occlusion of the left transverse sinus (fig. the results of the following laboratory tests were normal or negative : electrocardiogram, complete blood cell count, sedimentation rate, blood sugar, lipid battery, homocysteine, fibrinogen degradation products, prothrombin time, partial thromboplastin time, lupus anticoagulant, antithrombin iii, factor viii, antiphospholipid antibody, anticardiolipin antibody, antinuclear antibody, antineutrophil cytoplasmic antibodies, protein c, protein s, c3, c4, ch-50, c - reactive protein, and antistreptolysin o. however, the plasma levels of d - dimer were elevated (1,700 ng / ml). to search for the predisposing factors of cvt, we performed gastrofiberscopy, colonoscopy, chest ct, abdomen ct, and serological tumor marker studies, but no abnormalities were revealed. the level of prostate specific antigen was markedly elevated (11.8 ng / ml), so an aspiration needle biopsy sample of the prostate gland was taken. malignancy is associated with a high risk of developing thromboembolic disease and is related mainly to the generation of an intrinsic hypercoagulable state as a result of tumor induction of platelet aggregation and expression of various procoagulant factors including tissue factor and thrombin. circulating carcinoma mucins can induce platelet - rich microthrombi without thrombin, a process that is inhibited by heparins but not by warfarin. activation of the coagulation system appears to be intricately linked to processes that control tumor growth and angiogenesis.3 expression of tissue factor in tumors leads to an angiogenic phenotype via the up - regulation of vascular endothelial growth factor and down - regulation of the angiogenesis inhibitor thrombospondin.4 we found no similar reports in the literature of cvt as the presenting sign of occult prostate cancer.5 most series of thromboembolism associated with occult cancers are about deep venous thrombosis of the legs or pulmonary embolism.3,5 it is not clear what percentage of patients with unprovoked venous thromboembolism harbor an asymptomatic occult malignancy. several studies have found that approximately 8 - 12% of patients who presented with venous thromboembolism were diagnosed as having cancer after a relatively simple medical examination based upon symptoms and routine laboratory testing.6 - 8 over 5% of men diagnosed with prostate cancer have an antecedent thromboembolic event without any traditional risk factors for venous thromboembolism.5 however, there are no case reports involving prostate cancer patients with antecedent cvt in those series. because of the wide variety and nonspecificity of the presenting symptoms of cvt, it would be of great practical importance to develop an easy - to - perform test for the emergency room that would reliably rule out cvt. the presence of d - dimer, which comprises fragments of cross - linked fibrin digested by plasmin, indicates the activation of both the blood coagulation system and the fibrinolytic system. indeed, d - dimer concentrations are increased in most patients with recent cvt so that a negative d - dimer assay result may make the diagnosis of cvt highly unlikely. the sensitivity, specificity, and positive and negative predictive values of conventional d - dimer levels of > 500 ng / ml for the diagnosis of cvt are all 100%.9 our patient has some features that differ from typical patients with cvt. cvt is a distinct cerebrovascular disorder that, unlike arterial stroke, most often affects young adults and children. the majority (about 75%) of the adult patients are female, but this is not the case for children or the elderly. 8.2% of 624 consecutive adult patients with symptomatic cvt were aged 65 years or older.10 our case also has several implications for practice. cvt must be included in the differential diagnosis of elderly patients presenting with decreased alertness, delirium, or mental changes, because cvt does not usually present in the elderly as headache or isolated intracranial hypertension. the coincidence of these two entities can not be completely ruled out in older patients. however, occult malignancy including prostate cancer should be strongly suspected in older patients with idiopathic cvt. | backgroundcerebral venous thrombosis (cvt) is a distinct disorder with highly variable symptoms and courses. malignancy is known to be one of the predisposing factors of cvt.case reportwe present the case of a 69-year - old man with sudden behavioral changes. brain mri, magnetic resonance venography, and positive d - dimer assay confirmed the presence of cvt. the work - up for detecting the cause of the cvt revealed occult prostate cancer.conclusionsoccult malignancy including prostate cancer should be strongly suspected in older patients with idiopathic cvt. |
women 's health bears priority in health care services because of its directly associated with family and public health.1 nowadays, life standards has increased with the rise in life expectancy. according to 2015 data of turkish statistical institute (tuik), expected life expectancy of women is 80.7 years. in parallel with increasing average life expectancy the elderly population increases and it is extending the time spend in the postmenopausal period. according to the world health organization (who) the definition of menopause is the ending of mensuration permanently as a result of ovarian activity cessation.2 in order to understand menopause life we need to consider psychological, cultural and social factors as well as biological factors.34 traditions, ethnic structure of the society, the value given to the elderly and women, the role of women, women 's sexuality, women 's life philosophy and meanings ascribed to the menopause by the society and the women has a significant effect on the comprehension of menopause, menopausal complaints and menopausal behavior.56 it is mentioned that marriage and relation with one 's spouse plays an important role with women coping with menopausal complaints.789 varma.10 had reported that thoughts are emerging like agedness and uselessness in the menopausal women. yang.11 found that the psychological and somatic symptoms are more widespread than vasomotor symptoms in china. alpay.12 have been detected 65.5% of women have minor anxiety and 23% of women have major anxiety. it is stated that in the menopause the mood state is 73.5%, the physical and emotional exhaustion is 71.3% and irritability is 68%.13 depressive disorders that experienced during menopause is a major public health problem.14 feelings of guilt and embarrassment are feelings which helps manage oneself and they are two emotions that motivates one to meet certain standards and regulates social behavior. if the experiences of these feelings are too low or intense it can affect the psychology of individuals in a negative way as they can lead to problems in development and socialization.15 the concept of guilt and embarrassment are varies from culture to culture as it is advocated by some researchers.16 it is supposed that people have feelings of guilt and embarrassment in the menopause. it is suggested that these feelings are guiding our behavior and affecting the image of ourselves in the eyes of others. both feelings are enhanced by interpersonal and family relationships, and it is believed that they are based on feelings of moral self - awareness.1517 if these feelings are experienced with the appropriate functional density, it can assist in the socialization of individuals, the formation of personal development and the social adaptation to life in a healthy way. inappropriate experiencing of these emotions is negatively effects the psychology of the individual and can cause a number of problems in development and socialization.15 this study conducted to determine the changes that women experience during menopause and the effect of creating guilt and embarrassment in women and its impact on quality of life (qol). this study had been done as a descriptive research in between september 2014 and january 2015 in a town of gaziantep, ehitkamil in irinevler district with 500 women who agreed to participate among the 800 women aged above 40 years. women 's residences determined by the information taken from the family health centers, all residences are visited and the universe tried to be determined by without sampling with face - to - face interviews. the data was collected by socio - demographic oriented personal information form for menopausal women, guilt - embarrassment, whoqol - bref scales that prepared by the researcher. accordance to the results of first data collected from the pilot interviews, deficiencies of the first personal information form was corrected. whoqol - bref scales consists of 26 items from the original turkish version consists of 27 items. answers to these questions are 5 choices. the qol total score of minimum 4, maximum 20. seven scale physical, mental 6, social 3, article 8 contains the environment. field points, it is obtained by multiplying the average of the four items forming field. there are 41 question which first 4 question is to determine demographic profile of the women such as age, education level, marital status, number of children, education and occupations of the spouses, in the questions between 8 and 41 is to determine spouses point of view to the women, difficulties facing in menopause, perception of menopause, receiving information and treatment about menopause and its impact on sexual life. the data collected is assessed by spss 18 statistical analysis program (spss inc., chicago, il, usa) with independent samples t - test, one - way analysis of variance (anova) tests. participants in the study ; 46.6% are 49 to 54 years old, 78.6% have no literacy, all housewife, and 49.2% witnessed their last menstruation in 45 to 49. in the table 1, women stated that they been in menopause, 60.4% of women for 1 to 5 years, 32.8% of women for 6 to 10 years. ninety - five point four percent of women stated they had difficulties in menopause, 87.8% of it is psychological and physical, 6.6% had psychological only and 1% had physical only distress. thirty - two point two percent of women stated they had hot flashes, 23.6% had hot flashes and night sweat, and 14.6% had hot flashes, night sweats and palpitations together. women experienced psychological symptoms as 42.6% with tension, 15.2% with insomnia, and 7.2% wi th both tension and bad temper. in the table 2, 55% of women stated menopause is a bad situation for women, 45.4% stated menopause is a disease, 56% of women stated it is being infertile. seventy - nine point six percent of the women stated its not depended on the individual, 41.4% stated they felt nothing when they enter menopause, 10% stated they had depressed feelings such as sorrow, over - sensitiveness and unwillingness, 26.6% experienced sorrow, 13% experienced anxiety, 10.2% experienced both sorrow and anxiety. women stated that their first mentioned person about their menopause 53.6% to their spouse, 24.2% to their neighbor, 12.8% to their sibling, and 6% to their children. in the table 3, 74.4% of women stated they had received the information about menopause, 16.6% received from their neighbor, 14.6% received from both neighbor and relative, 13% from relative and medical personnel, 9.8% from a relative, 9% from a medical personnel. seventy - nine point four percent of women stated they did not received menopause treatment ; who received treatment 10.6% had drug therapy, 10% had alternative therapies. ninety - four percent of women stated they did not received depression medication, 76.2% did not seek medical aid, who seek medical aid experienced 6% delayed menstruation, 5.2% hot flushes, 2.2% palpitations, 2.2% insomnia, 2% perspirations. in the table 4, 29.6% women stated the menopause affected their sexual life, 46% of women stated their spouse is sexually less satisfied and 9% stated that their spouse not satisfied. eighty - two point eight percent of women is proud of their ability to childbearing, 73.2% of women said you should not bear a child as long as the spouse wants, 92.8% of women says it is not fair for spouse to marry another woman just for having a child. in the table 5, 54% of women stated they are partially participating the familial decisions, 22.6% of women stated they experienced spousal abuse, 87.8% of women stated there are no changes on their importance and prestige in the family during menopause, 10% stated it is decreased and 2.2% stated it is increased. eighty - five point six percent of women stated that their spouse did not influenced by the menopause, 81.2% stated their spouses ' interest and desire did not decreased, 79% stated that their spouses love did not decreased during menopause. sixty - nine point two percent women stated that their husbands said nothing when they told them about the menopause. in the table 6, thinking menopause as a bad situation significantly affects the guilt emotion (p < 0.001). significant relationship is identified between questions, decrease in love between spouses after menopause, maintaining ability to childbearing pride, women should bear child as long as spouse wants, will spouse justified if he marry another women just to bear a child and the thoughts about early / late menopause starting ages individual woman dependence (p < 0.05). in the table 7, it is found that the perceived emotion guilt is significantly affected by the thoughts about reduced sexual life in the menopause (p < 0.05). after the results of tests carried out by independent sample t - test using bonferroni correction (/3 = 0.017) between subgroups, partially group (p < 0.017). the thoughts of making spouse sexually satisfied after menopause is significantly affects perceived guilt (p < 0.001). after the results of tests carried out by independent samples t using bonferroni correction (/4 = 0.00025) between subgroups, the very group and the low group (p < 0.00025 ; p < 0.00025 respectively). quite group and the low group (p < 0.0125 ; p < 0.00025 respectively). it was determined that the group says menopause is being infertile women has showed higher perceived guilt than says variance analysis was carried out between women should bear child as long as spouse wants question and guilt factor, it is observed that women developed higher perceived guilt in the yes group than the in our study, women stated that they had been in menopause, 60.4% of women for 1 to 5 years, and 32.8% of women for 6 to 10 years. kavlak18 stated 64.1% of women for 1 to 3 years, ertem4 stated 48% of women for 1 to 5 years, tun19 stated 49.2% of women for 1 to 4 years been in menopause. our study is consistent with kavlak, ertem, and tun 's studies. in our study, 55% of women stated menopause is a bad situation for women, 45.4% stated menopause is a disease, 56% of women stated it is being infertile. tun19 reported 26.4% of women stated that menopause is a disease, 89.4% of women stated the menopause is a loss of ability to bear a child. seventy - nine point six percent of women stated early / late starting age of the menopause does no depending on the woman, 41.4% of women stated they felt nothing when they enter the menopause, 10% of women stated they experienced depressive emotions as sadness, sorrow, unwillingness, over - sensitiveness, 26.6% experienced sorrow, 13% experienced anxiety, 10.2% experienced sorrow and anxiety. ninety - five point four percent of women stated they had experienced distress while menopause, 87.8% of it both physical and psychological distress, 6.6% of it is only psychological distress while 1% of it is physical distress only. thirty - two point two percent of women experienced hot flashes, 2.6% of women experienced both hot flashes and night sweating, 14.6% of women experienced hot flashes, night sweating and palpitations. the psychological experiences of women 's are, 42.6% tension, 15.2% insomnia, 7.2% tension and bad temper. in alpay 's study12, it is found that 87.8% of women experienced physical and psychological distress. karlidere and zahin21 has found that first place in the frequency of symptoms of menopause are psychological and vasomotor symptoms. ertem4 has found that 85% of women experienced difficulties related to the menopausal period, there difficulties are 60% physical, 40% physical and psychological difficulties. our study is consisted with karlidere and zahin 's study21 and ertem study4. in karlidere and zahin 's study21, the most commonly identified menopause symptoms are ; 97.7% unrest and distress, 94.7% fatigue, hot flushes and fainting, 93.3% cold sweating, 92.8% soreness in the leg and arm joints, 91.9% amnesia. in the fakil 's study22 ; subjects experienced during the menopause, 63% hot flashes, 49% nervousness, 42% palpitations, 44% insomnia, 50% fatigue, 57% night sweats is determined. in kirolu 's study23, women stated 55% had hot flushes frequently, 42.5% had insomnia occasionally, 47.5% had dizziness occasionally, 47.5% had palpitations occasionally, 50% had headaches occasionally. our research has less resemblance with karlidere and zahin 's study21, but has more close resemblance with fakili 's study22 and kirolu 's study23. seventy - nine point six percent of women stated they felt free to tell their spouse about they are in menopause, women 's first person they told about menopause 53.6% their spouse, 24.2% their neighbor, 12.8% their sibling, 6% their children. kirolu23 found that women prefer to tell about entering menopause to 30% to spouse, 30% to parent / sibling / child, 10% to physician, 10% to another person, 7.5% to a friend, 2.5% to their daughter and 10% prefer to tell no one. women who described the menopause as a lost 32.5% of them stated quite loss and 47.5% often experienced nervousness and unrest, 35% often experienced bad feelings. in ko and salam 's study24, 57% of women think that it is bad and 37.2% of women thinks that they are no longer a woman. in the sis 's study25, women perceived the menopausal period as, 60.8% getting old, 19.4% decreasing of/ end of sexuality, 41.8% end of fertility, 26.2% loss of feminine features, 35% natural and normal process, 62.4% shared their menopausal distress with their spouse, 50.6% with their friend / neighbor, 35.7% with their children, 17.5% shared with no one. it can be mentioned that our study has similar findings on women 's menopause perceiving with sis 's study25, tun 's study19, kapdali 's study20, ko and salam 's study24. the end of the fertility can be perceived as the end of womanhood in the societies that women 's socio - economical existence associated with the ability of childbearing. probably, it is about socio - cultural and psychological factors plays a role on development of this idea (table 2). in the study, women 's stated ; 29.6% their sexual life is affected, 46% their spouse is sexually dissatisfied, 82.8% proud of their ability to child bearing, 73.2% of women says you should not bear a child as long as the spouse wants. ninety - two point eight percent of women said it is unfair for their spouse to marry with another women for a child. shame number of children were significantly statistically significant relationship between the social and environmental areas (p < 0.05). varma.10 stated that 69% of women expressed menopause does not affecting their sexual life. according to kirolu23, in menopause 37% of women balik26 stated that almost half of the menopause women experienced severe sexual problems while 29.1% experienced moderate sexual problems during menopause. menopausal women stated that about 35.5% women occasionally, 22.7% women frequently dissent from sexual intercourse with their spouses. our study shows similarities with balik and kirolu 's studies while it does not with varma can be connected to different - similar culture research universe. in shobeiri 's study27, women with more than three children obtained higher scores of qol compared to those who had fewer than four children, this difference was significant in psychosocial domain. women in our study stated ; 54% of them participating familial decisions, 22.6% experiencing spousal abuse, 87.8% of women stated there are no changes on their importance and prestige among the family during menopause, 10% stated it is decreased and 2.2% stated it is increased. eighty - one point two percent stated their spouses ' interest and desire did not decreased, 79% stated that their spouses love did not decreased during menopause. sixty - nine point two percent women stated that their husbands said nothing when they told them about the menopause. ahin28 detected women who experienced physical abuse at least once from their spouse 35% in whole turkey and 40% in eastern anatolia region. ertem4 detected 10% women 's family relations are affected by menopause, ko and salam24 detected 80% of women participating familial decisions, 9.8% of women partially participating familial decisions. according to the findings the high number of women participating familial decisions is a satisfactory and positive finding. tot29 survey states that in societies and families that gives women rights to speak and respectability, it is easy for women to adapt the menopause smoothly (table 5). in our study, thinking menopause as a bad situation significantly affects the guilt emotion (p < 0.001). having the information significantly affects perceived guilt (p < 0.05). significant relationship is identified between questions, decrease in love between spouses after menopause, maintaining ability to childbearing pride, women should bear child as long as spouse wants, will spouse justified if he marry another women just to bear a child and the thoughts about early / late menopause starting ages individual woman dependence (p < 0.05). tortumolu6 found that among the reasons for turkish women to perceive menopause as a bad thing are loss of motherhood, loss of femininity and marital relationship, changes in the posture, loss of physical and sexual power can be counted. as in many cultures, turkish culture is conceives the menopause as the loss of fertility. cirhinolu and gven30 states, because of maintaining social harmony, loving others and getting connected with them also makes women vulnerable to risk of losing the love of others and increase the dependencies on what others thinking, emerges as a factor that enhances shame and guilt tendencies (table 6). thinking menopause as a bad situation significantly affects the guilt emotion (p < 0.001). it is found that as women 's level of shame and guilt increases their life qualities are decreasing and women guilt - shame and qol is above average level. in order to increase the quality of women 's life during menopause which causes significant changes, women should be told that it is not a disease but an inevitable experience and misunderstandings that negatively affecting qol should be explained. not only women but all society should be informed about menopause and its effects on women 's life and the mass media should be mindful to this issue. the education and consulting services of the medical staff has an important place for women to spend healthy menopausal period, to cope with the problems experienced during this period and the ability to improve their qol. | objectivescurrent study was conducted between september 2014 and january 2015 in gaziantep proviency ehitkamil town irinevler district. purpose of the study is to measure changes experienced, point of view on menopause, and life style of women who are older than 40, living in said district, and within post - menopause and how these factors effected their feelings of guilt and shame.methodsout of universe consisting of 800 women, 500 participants have joined the study. data collected by personal information form, guilt - shame scales, and world health organization quality of life (whoqol - bref) scales. spss 18 was used to conduct statistical analysis.resultsanalysis indicated that 78.6% of the participants was not literate, 37.8% of the participants were was subjected to domestic violence. fifty - five percent of the participants were found to see menopause as a predicament, 45.4% as a disease, and 56% as infertility. results of scales are as follows ; guilt factor 48.95 0.37, shame factor 44.89 0.27, body 10.64 0.42, mental 10.96 0.39, social 11.12 0.49, environment 10.84 0.37.conclusionsno significant correlation found between guilt : body, mental, social, and environmental area (p < 0.05). it was found that there is a significant correlation between the thought that menopause is not a bad thing and the perceived guilt (p < 0.001). results of analyses indicate that information about menopause significantly correlates between perceived guilt (p < 0.05). current study found that as perceived shame and guild increases, the life quality of the sample decreases and the average life quality of the sample is below average. |
selection / amplification is a general tool for directed evolution of nucleic acids and proteins, which is much more complicated for reaction promoters (biocatalysts) than for simple binders. selection and identification for the former require some sort of catalyst - product pairing in an isolated compartment. in vivo selection using living cells has been typical choice for such a purpose. meanwhile, in vitro selection, as opposed to in vivo selection, is simple and convenient to carry out, is free from cytotoxicity problems, and allows for starting with a library of great diversity. griffiths and tawfik proposed an in vitro compartmentalization (ivc) technique for the in vitro evolution of biomolecules including biocatalysts, where biocatalysts are transcribed / translated in a compartmentalized water - in - oil emulsion to allow catalyst - product pairing. in practice, the ivc technique has been applied successfully for evolving or improving biocatalysts such as ribozymes and enzymes [312 ]. here, we applied the ivc technique for evolution of trnas (one of catalysts in protein translation systems) [1316 ] and present a promising concept for in vitro selection of suppressor trnas by the combination of read - through ribosome display (rt - rd, vide infra) [17, 18 ]. binding of suppressor trna to an amber stop codon is in competition with that of release factor 1 (rf1) to terminate the translation. the amber codon is also often misread by the exogenous trna for gln. to maximize the suppression efficiency and minimize incorporation of gln at the amber codon, we used a reconstituted prokaryotic cell - free translation system (puresystem classic), in which rf1, and gln and gln - trna synthetase had not been added unless otherwise stated. balance in the amounts of the fused mrna (mrna - trna) (longer) and the trna (shorter) transcribed, we put gcc immediately downstream of the first t7-promoter so as to lower the translation efficiency for the fused mrna (it is known that a g - less sequence, immediately downstream of the t7 promoter, suppresses the transcription efficiency). gel electrophoresis and blotting were carried out on a be-250 electrophoresis apparatus (bio craft) and a trans - blot sd system (bio - rad), respectively. the first pcr was carried out in 20 l of a reaction mixture containing 4 pmol of a forward primer containing a flag domain (bold) and a tag amber stop codon (italic) 5-d(aa gga gat ata cca atg gac tac aag gat gac gat gac aag tag atc agt ctg att gcg gcg tta g)-3, 4 pmol of a reverse primer with the lower t7 promoter (underlined) 5-d(gtt cag ccg ctc cgg cat ctc tcc tat agt gag tcg tat tac cgg gtg act gct gag ga)-3 for the trna - fused template or 5-d(tgg cgg aga gag ggg gat ttg aac cgg gtg act gct gag ga)-3 for nonfused template, 20 ng of pdhfr, 1.25 u of pfu ultra hf dna polymerase (stratagene), 4 nmol each of dntps (toyobo), and 2 l of 10 pfu ultra hf reaction buffer. after the pcr reaction, the product was purified by agarose gel electrophoresis. the second pcr was carried out in 20 l of a reaction mixture containing 4 pmol of a forward primer with the upper t7 promoter (underlined) and the following sequence after modification (vide supra) of the widely - used one 5-d(t aat acg act cac tat agc ccg gcc aca acg gct ggg ctc tag aaa taa ttt tgt tta act tta aga agg aga tat acc a)-3, 4 pmol of a reverse primer with the sequence for e. coli trna for ser (seru) 5-d(tgg cgg aga gag ggg gat ttg aac ccc cgg tag agt tgc ccc tac tcc ggt l1l2x yzl3 l4ac cgg tcc gtt cag ccg ctc cgg cat c)-3 (l1~4 and xyz represent the anticodon - loop and anticodon bases, respectively), ~20 fmol of the purified first pcr product, 1.25 u of pfu ultra hf dna polymerase, 4 nmol each of dntps, and 2 l of 10 pfu ultra hf reaction buffer. in vitro coupled transcription / translation of template dnas in the reverse - phase micelle was carried out as follows. a 50 l portion of a cell - free translation system containing 50 pm of a dna template (2.5 fmol) was added gradually to 950 l of mineral oil (sigma) containing detergents span 85 (nacalai tesque) (4.5% v / v) and tween 20 (sigma) (0.6% v / v) under stirring on ice. the diameter (d) of the micelles (water droplets) in the resulting emulsion was about 2 m, indicating that the number of micelles was n = v / v = 1.2 10, where v is the total volume of the water phase (50 l) and v is the volume of a micelle with d = 2 m. after incubation of the mixture at 37c for 1 h, the emulsion was spun at 2000 g for 10 sec. a 200 l portion of the supernatant was carefully taken from the upper part and mixed with 200 l of an ice - cold selection buffer (phosphate - k, ph 7.3) containing 92.2 mm of k, 300 mm of na, 50 mm of mg, 0.05% of tween 20 (wako), 2% of block ace (dainippon pharmaceutical co.), and 1 ml of ice - cold water - saturated ether. the mixture was inverted twenty times and centrifuged at 16100 g for 10 min at 4c, and then the organic ether phase was removed. the water phase was washed with 1 ml of ether and mixed with 200 l of ice - cold selection buffer. the mixture was applied on a column packed with prewashed anti - flag m2 agarose (sigma) and gently inverted for 2 h at 4c. after washing the gel retaining the ribosome - protein - mrna (prm) complex with 200 l of selection buffer five times, the mrnas were eluted upon collapse of the rpm complexes with 200 l of an elution buffer (phosphate - k, ph 7.2) containing 92.2 mm of k, 300 mm of na, 30 mm of edta, 0.05% of tween 20, and 2% of block ace. the mrnas eluted were purified with an rneasy minelute cleanup kit (qiagen) and amplified with a qiagen one - step rt - pcr kit according to the manufacture 's protocol using a forward primer with t7 promoter (underlined) and a flag domain (bold) followed by a tag amber stop codon (italic) 5-d(t aat acg act cac tat agc ccg gcc aca acg gct ggg ctc tag aaa taa ttt tgt tta act tta aga agg aga tat acc a atg gac tac aag gat gac gat gac aag tag)-3 and a reverse primer 5-d(tgg cgg aga gag ggg gat ttg aac ccc cgg tag agt tgc c)-3. the resulting rt - pcr products were used for the next round of selection and, after three (for anticodon - randomized trna) or five (for anticodon - loop - randomized trna) rounds, were monocloned using a pcr cloning kit (qiagen) and sequenced. single - round coupled transcription / translation under normal (noncompartmentalized) or compartmentalized conditions (referring to figure 1(b), lanes 14 or 5, resp.) was carried out using 400 pm of a template in 25 l of a cell - free translation system under otherwise identical conditions. the first pcr was carried out in 20 l of a reaction mixture containing 4 pmol of a forward primer with a flag domain (bold) with or without a tag amber stop codon (italic) 5-d(aa gga gat ata cca atg gac tac aag gat gac gat gac aag [tag ] atc agt ctg att gcg gcg tta g)-3, 4 pmol of a reverse primer with an ochre stop codon (italic) 5-d(tat tca tta ccg ccg ctc cag aat ct)-3 ', 20 ng of pdhfr, 1.25 u of pfu ultra hf dna polymerase (stratagene), 4 nmol each of dntps (toyobo), and 2 l of 10 pfu ultra hf reaction buffer. the second pcr was carried out in 20 l of a reaction mixture containing 4 pmol of a forward primer with t7 promoter (underlined) 5-d(gaa att aat acg act cac tat agg gag acc aca acg gtt tcc ctc tag aaa taa ttt tgt tta act tta aga agg aga tat acc a)-3, 4 pmol of a reverse primer 5-d(tat tca tta ccg ccg ctc cag aat ct)-3, ~20 fmol of the purified first pcr product, 1.25 u of pfu ultra hf dna polymerase, 4 nmol each of dntps, and 2 l of 10 pfu ultra hf reaction buffer. template mrnas were obtained by run - off transcription of the 5-flag - tag - dhfr or 5-flag - dhfr template dna obtained using a t7-megashortscript kit (ambion). thus, a t7-transcription mixture (10 l) containing 3 l of the pcr solution of template dna was incubated at 37c for 2 h. after addition of 1 u of dnase i, the mixture was incubated for additional 15 min. mrnas were purified with an rneasy minelute cleanup kit (qiagen) and concentrations of the purified specimens were determined by the absorbance at 260 nm. template dna was prepared by pcr in 20 l of a reaction mixture containing 20 pmol of a forward primer with t7 promoter (underlined) 5-d(g taa tac gac tca cta ta gga gag atg ccg gag cgg ctg aac)-3, 20 pmol of a reverse primer 5-d(tgg cgg aga gag ggg gat ttg aac ccc cgg tag agt tgc c)-3, 100 fmol of a template dna (initial, rt - pcr amplified, or monocloned), 1.25 u of pfu ultra hf dna polymerase (stratagene), 10 nmol each of dntps (toyobo), and 2 l of 10 pfu ultra hf reaction buffer. the resulting pcr solution was used for transcription using a t7-megashortscript kit (ambion) for 37c for 20 h. the transcribed trnas were purified by denaturing page (8%), followed by ethanol precipitation and, after dissolution in 500 l of water, further by passing successively through microcon ym-30 (millipore) and g-25 microspin columns (amersham). translation of a template mrna (5-flag - uag - dhfr or 5-flag - dhfr as a uag() positive control) (2 g) was carried out at 37c for 1 h in the presence of an exogenous trna (2 g) in 10 l of a reconstituted cell - free translation system, in which rf1 had not been added, but gln and gln - trna synthetase had. to the reaction mixture were added 165 l of water and 175 l of a sample - loading buffer (125 mm tris - hcl (ph 6.8), 4% (w / v) sds, 20% (w / v) glycerol, 0.002% (w / v) bromophenol blue, and 10% (v / v) 2-mercaptoethanol). the resulting solution was incubated at 95c for 5 min and applied on 15% sds - page. western blotting was performed on a pvdf membrane (hybond - p, amersham). flag - tagged proteins were visualized with anti - flag - hrp conjugate (sigma) and ecl plus western blotting detection reagent (amersham). the suppression efficiencies were evaluated by comparing the band intensities of the flag - tag - dhfr protein, determined by using the image j software (nih), with those of serially diluted (10, 20, 40, 60, 80, 100%) solutions of the stop - free flag - dhfr reference protein translated under otherwise identical conditions. we previously introduced rt - rd, in which expressed protein could be fully displayed upon suppression of the stop codon(s) downstream of the open reading frame by appropriate suppressor trnas. in this paper, we conversely apply the rt - rd technique for the selection of suppressor trnas that are coded at the 3-terminus of the very template that contains the stop codon to be suppressed. we prepared a dsdna template containing, under the control of a t7 promoter, the rbs (ribosome binding site), the atg start codon, a flag domain, the tag (amber) stop codon, and a spacer (dhfr1192) derived from the e. coli dhfr gene covering the amino acids 164, followed by the trna sequence together with its own t7 promoter (figure 1(a)). there are two t7 promoters in this template, transcription of which thus generates two types of rnas ; the one that contains both an rbs and an aug serves as an mrna, and the other, which lacks them, serves only as a trna. when the latter (trna) happens to suppress (read through) the amber (uag) stop codon in the transcribed mrna, a fused protein containing the flag, dhfr1192, and trna regions would result upon translation and remain attached to the mrna with the dhfr1192-trna portion, serving as a spacer to be anchored in the ribosome tunnel to squeeze the flag - tag peptide for full display (rt - rd ; figure 2). we first confirmed that the right amber suppressor (trnacua) generated in this way worked as such. the anticodon - adjusted (cua) trna is known to be aminoacylated with ser by endogenous ser - trna synthetase and hence suppress (read through) the amber codon with concomitant incorporation of ser at that position [18, 20 ]. coupled transcription / translation for 1 h of the template fused with the gene for trnacua (figure 1(a), nnn = cua) in a reconstituted prokaryotic (e. coli) cell - free translation system containing t7 rna polymerase was followed by affinity selection (4c and [mg ] = 50 mm) of the flag - tag peptide domain displayed (rt - rd) in the protein - ribosome - mrna (prm) complex. the mrna template coding the trnacua sequence was recovered upon disruption of the ternary complex with edta, rt - pcr amplified, and identified as such (figure 1(b), lane 1). however, the template was by no means recovered efficiently when it was lacking (nonfused) the trna domain (lane 2) or fused with anticodon - mismatched (cga) natural trna for ser (nnn = cga) (lane 3). (in respect to the weak spots seen in lanes 2 and 3(figure 1(b)), it is known that the stop codons can be suppressed to some extent (up to several %) in an rf1-minus translation system by misreading even in the absence of a suppressor trna.) in these cases, there is no generation of the correct trna for amber suppression and hence translation stops at the amber (uag) codon, giving rise only to the flag peptide with no linkage to the genotype (mrna). conversely, when equal amounts of nonfused (suppression irrelevant) and trnacua - fused (relevant) templates were used, both templates were recovered (lane 4) (in respect to the apparently stronger spot for the nonfused template in lane4(figure 1(b)), it is generally true that shorter templates are more easily amplified by pcr.) this is because suppressor trnacua generated from the latter template can suppress the amber codon of the former. to avoid such a crossover, we needed to compartmentalize the reactions of each template using a water - in - oil emulsion system [312 ]. with this technique (see below, figure 2), we could selectively recover the trnacua - fused template coding the active suppressor trna (lane 5) from the above mixture. the selection cycle is shown in figure 2. an initial pool (a) of fused templates (figure 1(a), nnn = nnn with a diversity of 4 = 64) with genes for anticodon - randomized trna (figure 3(a), left) was subjected to coupled transcription / translation in a water - in - oil emulsion (b) [312 ]. consequently, each compartment (~2 m) generates and contains a pair of template - related sister rnas, a fused mrna (shown as mrna - trna) and a trna (c). the whole protein, susceptible to rt - rd in the form of a prm complex (step iv in d), can be translated (d) only when the trna serves as an amber suppressor (steps i iii in d). the ternary complex thus formed was recovered by affinity selection of the displayed flag tag after breaking the emulsion and treated with edta to afford the active trna - fused template mrnas (e), which were purified and amplified by rt - pcr back to the dsdna templates (a), for use in another selection cycle. the anticodon of approximately one third of the 29 monocloned trnas was cua (figure 3(a), center), which was the one expected based on codon - anticodon complementarity. these results indicate that the sensitive prm complex survives the ivc and workup conditions to allow the rt - rd / ivc method to select suppressor trnas. finally, we applied the rt - rd / ivc method to the engineering of the two - base loop regions adjacent to the anticodon. these regions are variant in various trnas but are believed to be important in stabilizing codon - anticodon interactions. selection of fused templates with genes of anticodon - matched (cua) and anticodon - loop - randomized (nn - cua - nn with a diversity of 4 = 256) trna (figure 3(a), right) was carried out as above. interestingly, ~40% of the 39 monocloned trnas had the same loop sequence (cu - cua - aa) as the natural trna for ser (cu - cga - aa) even in the base modification - free conditions (figure 3(a)). these results indicate that the anticodon - loop sequence itself has played a preserved or sophisticated role in the evolution of trnas that now have many modified bases (since neither anticodon nor anticodon - loop region is recognized by the serenyl - trna synthetase, the selected loop sequence may play a role in stabilizing the interaction between the amber stop codon andthe suppressor anticodon.) [22, 23 ]. suppression efficiencies were evaluated by western blotting analysis using the nonfused template lacking the trna domain in an rf1-minus translation system under noncompartmentalized conditions (figure 3(b)). enrichment of active trna became notably pronounced after three rounds (lanes 1113) (the reason for the high suppression efficiency (~80%) despite the low percentage amounts of the active trnacua (10% in round 3 and 38% in round 5) is that excess amounts of trnacua were used in the experiments for western blotting (figure 3(b))) and the suppression efficiency of the monocloned trnacu - cua - aa showed ~100% activity (lane9) compared with the suppression - free translation using a uag() reference template (lane 7). interestingly, a single mutation in the anticodon - loop domain led to a dramatic loss of activity (lanes 14 and 15). an essential aspect of it is that trna is a part of mrna (mrna - trna fusion), being located downstream of the particular codon to be suppressed (the amber stop codon in this study). therefore, only active trnas that can self - suppress the codon in a water - in - oil compartment are susceptible to amplification by the read - through ribosome - display technique. although the concept was well demonstrated, the present method must be further optimized especially regarding the selection efficiencies. three or five rounds of selection were required for enrichment from the small library sizes (n = 64 and 256). one possible reason for this low selection efficiency is a misreading of stop codon in an rf1-minus translation system, which would result in the recovery of false positive trna - fused mrnas. optimization of extraction conditions or the use of mrna display technique [24, 25 ] might overcome the instability. of course, compartmentalization conditions or mrna / trna ratios are also the factor to be checked. furthermore, it is not easy to understand that 10 clones out of 39 (26%) had the cc - cua - aa anticodon - loop sequence after 5 rounds (figure 3(a)), which turned out to be completely inactive in suppression (figure 3(b), lane 14). taking into account the fact that the cc - cua - aa occupies 25% of pool even in round 3 in contrast to the less percentage amounts of the active cu - cua - aa sequence (10%), it is feasible to speculate that the inactive cc - cua - aa sequence might be already abundant in the initial pool and/or has been more easily amplified regardless of the selection process. after sufficient improvements | here is presented a concept for in vitro selection of suppressor trnas. it uses a pool of dsdna templates in compartmentalized water - in - oil micelles. the template contains a transcription / translation trigger, an amber stop codon, and another transcription trigger for the anticodon- or anticodon loop - randomized gene for trnaser. upon transcription are generated two types of rnas, a trna and a translatable mrna (mrna - trna). when the trna suppresses the stop codon (uag) of the mrna, the full - length protein obtained upon translation remains attached to the mrna (read - through ribosome display) that contains the sequence of the trna. in this way, the active suppressor trnas can be selected (amplified) and their sequences read out. the enriched anticodon (cua) was complementary to the uag stop codon and the enriched anticodon - loop was the same as that in the natural trnaser. |
acute heart failure is still associated with a high hospital mortality, which ranges between 4.0% and 12.7%.1, 2, 3, 4 emergency physicians, cardiologists, intensivists, nurses, and other healthcare providers have to cooperate to provide optimal benefit. however, many treatment decisions are opinionbased, while relatively few are evidencebased. according to the consensus paper on acute heart failure published in 2015, the recommendation for intravenous vasodilator therapy is that it might be administered as an initial therapy for a symptomatic relief in cases when systolic blood pressure (bp) is normal to high (110 mmhg). alternatively, sublingual nitrates may be considered.5 intravenous vasodilators are the second most commonly used agents in ahf patients. their use was shown to be associated with a lower mortality, while a delay in administration was associated with a higher mortality.6 randomized controlled trial (rct) evidence comparing the clinical outcomes of intravenous vasodilators (especially nitrates) in ahf patients is rather limited, and its methodological quality is relatively low. however, there is a consensus that intravenous vasodilators should be indicated in ahf with rather normal to high bood presure7 and should not be indicated in patients with sbp 75 ml / min/1.73m2. mortality data were obtained from a centralized database of the ministry of health of the czech republic : the 30day mortality was 13.7%, and the 1year mortality 32.3%. in order to identify the subgroup of higherrisk patients in the relaxcomparable group, who might potentially benefit from new therapeutic approaches in future, we divided this group of patients according to ahead, a recently published scoring14 system. this score is based on the presence of comorbidities that worsen the patient 's prognosis : the patient obtains one point for the presence of each comorbidity, and the value of ahead score can thus range between 0 and 5. these monitored comorbidities are as follows : a atrial fibrillation, anaemia (h haemoglobin level lower than 130 g / l for men and 120 g / l for women), e elderly (age 70 years), a abnormal renal parameters (creatinine 130 mol / l) and d diabetes mellitus. standard descriptive statistics were applied in the analysis : absolute and relative frequencies were used for categorical variables, median values supplemented with 5th95th percentile range (or mean and standard deviation) were used for continuous variables. the statistical significance of differences between data from the ahead registry and published data from the relaxahf trial was computed using ttest for continuous variables and chisquare test for categorical variables. the analysis was performed with the use of spss 22.0.0.1 (ibm corporation, 2014). the evaluated cohort (n = 5856) of ahf patients was divided according to the relaxahf criteria into two groups : the relaxcomparable group and the relaxnoncomparable group. the baseline characteristics of both groups of patients recorded in the ahead registry compared with the published characteristics of the placebo group of patients from the relaxahf trial are shown in table 1. baseline characteristics of two ahead groups (relaxcomparable and relaxnoncomparable) compared to the relaxahf placebo group ace, angiotension converting enzyme ; arbs, angiotensin2 receptor blockers ; bp, blood pressure ; copd, chronic obstructive pulmonary disease ; pad, peripheral arterial disease. as compared with the relaxnoncomparable group, patients in the relaxcomparable group were older, had higher systolic, and diastolic blood pressure at admission and lower calculated creatinine clearance. they had a higher number of comorbidities in their history, such as hypertension, stroke, or tia, peripheral artery disease, copd, and diabetes mellitus. at admission, they were more frequently treated by angiotensinconverting enzyme inhibitors, angiotensin2 receptor blockers, betablockers, and digoxin. as compared with the placebo group of patients from the relaxahf trial (which was comparable with the seralaxintreated group8), patients in the relaxcomparable group were older, had higher systolic, and diastolic blood pressure, and higher heart rate ; in terms of medical history, they had less frequently hypertension and atrial fibrillation but more frequently stroke or tia, smoking, and copd. at admission, patients from the relaxcomparable group were less frequently treated with betablockers and spironolactone. the kaplan meier survival estimation (as shown in figure 2) demonstrates a significantly higher mortality of the relaxnoncomparable group as compared with the relaxcomparable group. however, the 180day mortality of the relaxcomparable group is significantly higher in comparison with both relaxahf groups (patients treated with seralaxin and placebo). mortality of two groups of patients from acute heart failure database (ahead) registry (relaxcomparable and relaxnoncomparable) compared to mortality in the relaxahf study. according to the systolic blood pressure at admission, patients from both groups of the ahead registry were divided into six groups. in the relaxnoncomparable group, mortality in the relaxnoncomparable group was consistently higher across all systolic blood pressure categories in comparison with the relaxcomparable group (figure 3). systolic blood pressure as a risk predictor for hospital mortality in acute heart failure database (ahead) registry. overall, 33.8% of patients in the relaxcomparable group were treated with intravenous vasodilators (vd), namely nitrates. the hospital mortality of patients without vd therapy and with vd therapy was 3.1% and 2.9%, respectively, the 1month mortality was 7.1% and 6.1%, respectively, and the 1year mortality was 23.9% and 26.1%, respectively (neither of these differences is statistically significant). according to the recently published risk scoring system ahead, patients from the relaxcomparable group were divided into three groups : lowrisk (ahead score 01), intermediaterisk (score 23), and highrisk (score 45). figure 4 demonstrates that this simple stratification tool clearly differentiates lowrisk patients (ahead score 01) from patients with a higher risk of death (ahead score 23 and 45), even within the first year after the patient 's discharge from hospital. baseline characteristics of relaxcomparable group stratified by ahead score ace, angiotension converting enzyme ; arbs, angiotensin2 receptor blockers ; bp, blood pressure ; copd, chronic obstructive pulmonary disease ; pad, peripheral arterial disease. mortality of patients in relaxcomparable group stratified by acute heart failure database (ahead) score. our results highlight several very important points : (i) only 23% of all consecutive patients hospitalized with ahf met criteria of the large relaxahf trial ; (ii) mortality of patients who met criteria of the relaxahf trial (relaxcomparable group) is significantly lower in comparison to the rest of patients recorded in the ahead registry (relaxahfnoncomparable group) ; (iii) mortality of relaxcomparable group of patients from registry of consecutive ahf patients is significantly higher than mortality of patients in rct regardless to study treatment ; (iv) systolic bp at hospital admission significantly affects the prognosis of ahf patients. based on our data, we were not able to confirm a reduction of mortality associated with the treatment by vasodilators, as it was previously demonstrated by results from the alarm registry, where the treatment by combination of diuretics and vasodilators led to a lower mortality in comparison to diuretics alone (7.6% and 14.2%, respectively, p 110 mmhg at admission. similar results have been shown by the published data from the optimize hf registry, where a systolic bp lower than 100 mmhg was associated with three times higher hospital mortality rate in patients with normal (2.6% and 7.5%, respectively) or with decreased renal functions (5.5% and 16.3%, respectively).18 finally, mortality analysis in the subgroup relaxcomparable showed that the ahead score is a simple tool that can easily identify higher risk patients who might potentially benefit from new therapeutic approaches in the future. our results based on the czech registry ahead are fully consistent with the recently published results. miro. demonstrated similar results from the spanish registry eahfe,19 and wang. showed that approximately 2 out of 10 patients hospitalized because of ahf in the usa, latin america, or asiapacific are potentially eligible for the relaxahf criteria. even after multivariate adjustement, relaxahf type of patients had lower mortality rates as compared with nonrelaxahf type of patients.20 two large rcts with vd therapy the relax ii trial is a multicentre, randomized, doubleblind, placebocontrolled phase iii study to evaluate the efficacy, safety, and tolerability of serelaxin when added to standard therapy in ahf patients. the true ahf clinical trial has finished enrolment in spring 2015, while the relax ii trial is supposed to finish enrolment in winter 2015/2016. results of both trials are expected to be available in 2016, expectantly bringing the definitive answer on the role of vasodilators in acute heart failure treatment. an increased value of the natriuretic peptide level was one of the important criteria for the enrolment in the relaxahf trial, as well as for the subsequent relax ii and true hf trials. we could not use the natriuretic peptide levels because of a limited number of patients in which this parameter was known at the time of their admission. however, we can assume that a low value of the natriuretic peptide would exclude lowrisk patients from the relaxcomparable group and that it could lead to an even higher mortality in the relaxcomparable group in comparison to relaxahf patients than we demonstrated in this study. moreover, we were not able to exclude patients with acute infections that could lead to an acute decompensated heart failure. according to results from the ahead registry, we can expect that about only 2025% of patients hospitalized because of ahf meet the criteria for a potential treatment with new vasodilator serelaxin. significantly, lower mortality rates can be expected in participants of ongoing randomized clinical trials in comparison with realworld ahf patients. ahead score is a simple tool that can easily identify higher risk patients who might potentially benefit from new therapeutic approaches in the future. the new treatment of ahf patients with contraindication for vasodilator therapy remains the challenge for future research. the work was supported by a project of conceptual development of research organization 65269705 (university hospital brno) funded by the ministry of health of the czech republic, by the grant muni / a/1362/2015. | abstractaimsthe randomized clinical trial relaxahf demonstrated a positive effect of vasodilator therapy with serelaxin in the treatment of ahf patients. the aim of our study was to compare clinical characteristics and outcomes of patients from the ahead registry who met criteria of the relaxahf trial (relaxcomparable group) with the same characteristics and outcomes of patients from the ahead registry who did not meet those criteria (relaxnoncomparable group), and finally with characteristics and outcomes of patients from the relaxahf trial.methods and resultsa total of 5856 patients from the ahead registry (czech registry of ahf) were divided into two groups according to relaxahf criteria : relaxcomparable (n = 1361) and relaxnoncomparable (n = 4495). as compared with the relaxnoncomparable group, patients in the relaxcomparable group were older, had higher levels of systolic and diastolic blood pressure, lower creatinine clearance, and a higher number of comorbidities. relaxcomparable patients also had significantly lower shortterm as well as longterm mortality rates in comparison to relaxnoncomparable patients, but a significantly higher mortality rate in comparison to the placebo group of patients from the relaxahf trial. using ahead score, we have identified higherrisk patients from relaxcomparable group who might potentially benefit from new therapeutic approaches in the future.conclusionsonly about one in five of all evaluated patients met criteria for the potential treatment with the new vasodilator serelaxin. ahf patients from the real clinical practice had a higher mortality when compared with patients from the randomized clinical trial. |
immuno - analytical systems have typically been developed by capturing and immobilizing the antibody on solid surfaces since the immobilization provides an easy separation of the binding complexes with the antibody from unreacted reagents. the solid - phase capture antibody, however, is very difficult to control at the level of the molecular state or orientation on the surfaces, which is a major factor that determines the degree of the complex formation. indeed, most immobilization methods (e.g., physical adsorption, covalent attachment, and binding via biotin - streptavidin linkage) [2, 3 ] have not yet been very successful in arranging or orientating the antibody molecules in a manner that substantially improves the random nature of antibody immobilization inherent in the current methods. random immobilization results in a low rate (e.g., 5 to 10%) of the active antibody density, that is, those that can participate in the binding reaction, which decreases even further when the substance to be analyzed is large in molecular size. the rate of active antibody density would significantly increase provided the molecules are deposited by mediating the fc region on the solid matrix, such that the antibody binding sites may face to the bulk solution where the analyte is present. as a typical example, protein a or g binds specifically to the fc region of immunoglobulin and, thus, the capture antibody can be immobilized via this binding protein in the bottom - on configuration. it has been demonstrated that such a controlled molecular orientation of the antibody significantly improves the binding rate compared to random immobilization of the antibody by up to about 10 times for the same antigen. the functional binding affinity measured for the oriented antibody could also increase because the molecules are properly posed to orient the binding sites at a position distal from the surfaces. such improved characteristics would consequently enhance the analytical performances of solid - phase immunoassays [3, 6 ]. however, it is worth noting that the use of antibodies immobilized via the binding proteins is limited to only competitive - type assays in which only a single antibody is used. to develop an immobilization scheme that can be used in a wide range of applications, we have explored site - directed biotinylation of antibodies to bind the molecules via a biotin - streptavidin linkage on the solid surfaces. since the hinge region of immunoglobulin g (igg) could be a potential site for coupling to the matrix, a novel method was devised to selectively attach a biotin derivative to the functional groups present within this region. although this product, if used as the capture antibody, led to an enhancement in the performance of the sandwich - type immunoassay, the fc region in the molecule was likely interfere with immobilizing the antibody in an erect position, which is desirable for binding. to address this, we next synthesized a product biotinylated at the same region but using an antibody fragment, f(ab)2, in place of igg. the use of this capture antibody further enhanced the performances of the immunoassay, indicating that the molecules could not only be positioned on the streptavidin layer in an arrangement that efficiently exposed the binding sites to the bulk solution, but also with a high flexibility for access to the antigen. such improvements would be crucial to the analytical systems for, particularly, cardiac troponin i (ctni), as a specific marker of acute myocardial infarction (ami), requiring a high detection capability. in this study, we analyzed the complex formation with the immobilized antibodies to ctni prepared in different schemes using variable biotinylation methods and antibody types. in the previous report, the site - directly biotinylated antibodies were shown to be superior to the conventional random preparation if they were used as the capture antibodies in sandwich immunoassays. as to the molecular types, since the fragmented antibody without the fc had an enhancement effect, the smaller fragment, fab, was also employed in this study to examine a new factor, that is, the number of binding units per molecule. the respective complex formation with such different preparations have been analyzed with respect to two major parameters determining the reaction ; the surface density of active binding sites and binding affinity. based on these analyses, we have attempted to construct binding models that represent the reactions of each antibody with the antigen on the solid surfaces. the models could consequently be used to elucidate the effects of the following factors on the complex formation : biotinylation method, the presence of fc, and the number of binding units per molecule. the stock of ctni - t - c complex (from human cardiac muscle tissue) and mouse monoclonal antibody specific to ctni (clone 19c7) were supplied by hytest (turku, finland). goat anti - mouse igg antibody, two types of goat anti - mouse igg antibodies (specific to whole igg and fc of mouse igg, resp.) conjugated with horseradish peroxidase (hrp), succinimidyl 4-[n - maleimidomethyl]cyclohexane-1-carboxylate (smcc), 1-biotin - amido-4-(4-[maleimidoethylcyclohexane]-carboxamido)butane (biotin - bmcc), succinimidyl-6-[biotinamido]-6-hexanamidohexanoate (nhs - lc - lc - biotin), and immunopure igg1 fab and f(ab)2 preparation kit were purchased from pierce (rockford, il). mercaptoethanol (me), dithiothreitol (dtt), sephadex g-15 gel, 3,3,5,5-tetramethylbenzidine (tmb), casein (sodium salt type, extracted from milk), glutaraldehyde (ga), protein marker (low range), and polyoxyethylene sorbitan monolaurate (tween 20) were obtained from sigma (st. louis, mo). ficin (from a fig tree) protease was purchased from wako (osaka, japan). a protein g affinity column was supplied by bio - rad (hercules, ca). the substrate solution for the microtiter - plate - based immunoassays contained 10 ml of 50 mm sodium acetate (ph 5.1), 100 l of 1% tmb, and 10 l of 3% hydrogen peroxide. a monoclonal antibody specific to ctni (bd clone 12) was produced by following a standard protocol as described elsewhere. the antibody was fragmented using the immunopure igg1 fab and f(ab)2 preparation kit according to the manufacturer 's instructions. briefly, the ficin column (gel volume : 2 ml) was equilibrated with activation buffer containing 10 mm cysteine to eventually yield fab as the product. the antibody solution, diluted to 1 mg / ml in phosphate buffered saline (pbs), was added to the equilibrated ficin column, and incubated at 37c for 5 hours. the digested antibody solution eluted from the column was immediately transferred to the protein a column (gel volume : 2.5 ml). after collection of the unbound fractions, the bound antibody components were collected with the elution buffer supplied in the kit. each fraction obtained from the protein a column was then analyzed by means of a solid - phase immunoassay with an immobilized anti - mouse igg antibody. the goat anti - mouse igg antibody (1 g / ml, 100 l) dissolved in pbs was added to microwells and incubated within a container maintained at 100% relative humidity and 37c for 1 hour. these incubation conditions were also applied to all subsequent reaction steps. after washing the microwells three times with deionized water, the residual surfaces were treated with 100 mm tris buffer, ph 7.6, containing 0.5% casein (casein - tris ; 200 l). after washing, each eluted fraction from the protein a column were diluted 100 times with casein - tris containing 0.1% tween (casein - tris - tw) and added (100 l) to duplicate wells. after washing, two types of anti - mouse igg antibodies (specific to whole igg and fc region of mouse antibody, resp.), which were labeled with hrp, were diluted 5000 times with casein - tris - tw and subsequently added (100 l) to each sample. after washing, the substrate solution for hrp (200 l) containing soluble tmb was dispensed into each well and reacted at room temperature for 15 minutes. sulfuric acid (2 m, 50 l) was added, and the absorbance was then measured at 450 nm with a microtiter plate reader (versamax, molecular device, chicago, il). the eluted fractions were also characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds - page) analysis (12% gel) under non - reducing conditions, as described elsewhere. the same monoclonal antibody described in the production of fab section was also fragmented by ficin protease dissolved in digestion buffer (50 mm tris - hcl containing 2 mm edta, ph 7.0). the antibody (1 mg) was mixed with the dissolved ficin solution (molar ratio of ficin to antibody was 1 to 30) containing 1 mm cysteine as an activator and the mixture was then incubated at 37c for 2 hours. after the enzymatic digestion, the mixed solution was immediately transferred to the protein g column (gel volume : 1 ml), which had been pre - equilibrated with 10 mm pbs (ph 7.4). after collecting the unbound fractions, each fraction from the column was then analyzed by of the bradford assay at 595 nm. to characterize the unbound and bound fractions, the conditions used for the sds - page analysis (12% gel) are described elsewhere. in the immunoassay, the goat anti - mouse igg antibody (1 g / ml, 100 l) dissolved in pbs was added to microwells and incubated within a container maintained at 100% relative humidity and 37c for 1 hour. in this immunoassay, the incubation and washing procedure of each reaction step were the same as the conditions described in the production of fab section. after blocking the residual surface with casein - tris, each unbound fraction, bound fraction and f(ab)2 fragment, pre - made with a commercial kit as a control, was serially diluted (0 to 1000 ng / ml) with casein - tris - tw and then added to microwells. following the reaction, anti - mouse igg labeled with hrp was added and signal generation was performed by the same procedures described above. the antibody molecule can be biotinylated with a few commercially available cross - linkers, which are reactive to some chemical functions that exist on the outer surface of the antibody. we prepared three types of antibody species (i.e., igg, f(ab)2 and fab) and then produced the biotinylated antibodies using not only an amine - reactive biotin (nhs - lc - lc - biotin) linker for random biotinylation of igg type but also a sulfhydryl - reactive biotin (biotin - bmcc) linker for site - directed biotinylation of igg, f(ab)2 and fab types in this study. for random biotinylation of igg and site - directed biotinylation of igg and f(ab)2, in addition, unlike the two antibodies from the site - directed biotinylation method, that is, igg and f(ab)2, synthesis of site - directed biotinylation of fab was performed by a simple biotinylation process (five - fold molar excess of biotin - bmcc) without inducing the competitive reaction among maleimide derivatives because fab fragment molecule contains just one disulfide bond that can be reduced by reducing agents at the interchain between ch1 and cl domain. after site - directed biotinylation of fab, excess biotin - bmcc reagent was removed on a sephadex g-15 gel column (gel volume : 10 ml). the monoclonal antibody, clone 19c7, was coupled to the enzyme, that is, hrp, via a cross - linking protocol that was similar to a protocol previously described elsewhere. briefly, the antibody was first reduced using 10 mm dtt at 37c for 1 hour, and the excess reagent was removed on sephadex g-15 gel column (10 ml volume). hrp was also activated with smcc in a 50 molar excess, and the excess reagents were also separated on the sephadex g-15 gel column. the reduced antibody was then combined with the activated hrp in a 5 molar excess and the conjugation was carried out overnight at 4c. the conjugates synthesized were stored as aliquots after snap freezing. to analyze the performance of the four different biotinylated antibodies, that is, random igg, site - directed igg, site - directed f(ab)2, and site - directed fab, as capture agents, antibody immobilization on the inner surfaces of microwells were performed by a streptavidin - biotin linkage [11, 12 ]. a streptavidin solution (10 g / ml, 100 l) dissolved in pbs was added to the microwells pre - treated with 0.5% glutaraldehyde and reacted within a humidified container at 37c for 1 hour. these incubation conditions were used in all subsequent steps, and the wells were washed with deionized water three times after completing each step. after treating the well surfaces with casein - tris, the four types of biotinylated antibodies dissolved in casein - tris - tw were serially diluted (0.5, 5, 25, and 75 pmol / ml) and added to each microwell. antigen - antibody complexes were then formed by sequentially adding variable analyte concentrations of ctni - t - c (0, 0.01, 0.1, 1, 10, 100, 1000, and 2000 ng / ml), and a constant amount of mouse anti - ctni antibody - hrp conjugates (1 g / ml). the rest procedure for signal generation and measurement were performed by the same procedures described above. to investigate the antigen binding patterns of the four types of biotinylated antibody participants immobilized on streptavidin layer, we analyzed the data obtained from the two - site immunoassay as described above. the colorimetric signal data obtained from the each analyte concentration were quantified as molar concentration using a linearized hrp standard curve. the surface density of the reactive binding site and the equilibrium binding constant were determined from the x - intercept and slope of the regression curve. to improve detection ability of ctni, immunoassay systems employing different capture antibodies were investigated in this study by immobilizing them, via a biotin - streptavidin linkage, in various configurations. it has been reported that the adoption of the site - directly biotinylated antibody was superior in its analytical performance over the use of the randomly prepared biotinylated antibody. furthermore, the presence of the fc region in the site - directly biotinylated antibody molecule may result in a steric effect that was significantly alleviated provided an antibody fragment, f(ab)2, was employed. the aim of this study was to continue this work by characterizing not only the surface density of immobilized captured antibodies that were actively participating in the reaction but also the binding affinity, which primarily determines analytical performance. it is worth noting that the fab fragment, which was used as the capture antibody and could determine the minimal size of the binder, was newly synthesized to more fully determine the controlling factors. the smaller antibody fragment was produced by enzymatically digesting the igg1 mouse antibody using a protease, ficin, immobilized on a gel column. to derive the reaction for mainly synthesizing fab, the antibody was incubated with 10 mm cysteine, which acted as an activator, within the column for an extended time period (e.g., 5 hours). after collecting the reaction mixture from the ficin column, the product was sequentially purified by affinity chromatography on a protein a column. the unbound portion was retrieved as fractions and the bound portion was eluted by shifting the medium to a low ph buffer. each fraction was analyzed for total antibody with a sandwich - type immunoassay using a secondary antibody, as the capture binder, raised against mouse whole igg and the same antibody but chemically conjugated to an enzyme, hrp, as the detection molecule. to monitor fab, the same immunoassay system was employed except with the use of a detection antibody specific only to the fc region. the immunoassay results, as shown in the chromatogram in figure 1(a), revealed that the major component in the unbound fractions in the first peak was fab and the components in the bound fractions in the second peak contained fc, including the non - digested igg. to verify that the major component in the unbound fraction was fab, the fractions were pooled and analyzed by means of sds - page under non - reducing conditions (figure 1(b)). this analysis showed that the major protein band had a molecular weight between 66 and 45 kda. impurities were also present although they were relatively minor. since the molecular size of fab is approximately 50 kda, we can conclude that this major protein band is fab cleaved from igg by ficin. it should be noted that the yield of fab fragmentation by enzymes was approximately 50% provided the areas under each antibody fragment peak were compared with each other (refer to the curve marked with open square in figure 1(a)). unlike the above enzymatic digestion in the solid phase, f(ab)2 was synthesized by carrying out the same cleavage reaction in the solution phase containing 1 mm cysteine as an activator. this method provided an improved yield of the reaction when compared to the previous method although the enzyme could not be reused. the antibody fragments were then purified by affinity chromatography on a protein g column, which was essential for separating f(ab)2 in a high purity from the reaction mixture containing the enzyme. after eluting the unbound fractions, the bound material was dissociated from the column by switching the medium to a low ph buffer. to obtain the chromatogram the protein concentration in each elution fraction was analyzed by the bradford assay (figure 2(a)). after pooling the fractions in each peak, the components were analyzed by sds - page under non - reducing conditions (the inset, figure 2(a)). this analysis showed that the unbound fractions included mainly the smaller fragment, fc, which had a molecular weight of about 50 kda, and the bound fraction contained the larger fraction, f(ab)2, which had a molecular weight of about 100 kda. these results indicate that this method resulted in a complete isolation of the product. the gel data, however, was in conflict with the preconception that protein g specifically binds to the fc region of the antibody molecule, which would have resulted in the opposite order of the eluted components. to settle this contradiction, each fragment was reacted with the antigen, ctni, immobilized on the solid matrix, and the complex was traced using a secondary antibody, raised against mouse igg, conjugated to an enzyme, hrp (figure 2(b)). the fragment that came from the bound fractions showed a binding pattern to the antigen similarly to that obtained with the f(ab)2 control. these results were supported by a previous study where f(ab)2 produced by the enzymatic digestion of the same subtype antibody using ficin was found to strongly bind to protein g whereas the cleaved fc poorly reacted with the binding protein under physiological conditions. the liquid phase cleavage reaction had several advantages relative to the solid phase cleavage employed in the earlier investigation. first, as shown in the sds - page data, only the two fc and f(ab)2 components were contained in the unbound and bound peaks, respectively. this indicated that the yield of fragmentation under the conditions used was close to 100%, which was a significant improvement over the solid phase reaction (about 20 to 30%). secondly, the antibody cleavage was completed via enzymatic digestion within 2 hours, which was about one - tenth the reaction time required for the previous protocol (approximately 20 hours). finally, the reproducibility of the cleavage reaction was remarkable as shown in the quadruplicate chromatograms of figure 2(a). all of these did not result from only the enzymatic cleavage in the liquid phase, but also from the use of ficin without limitation in the quantity. using the biotinylated preparations as the capture antibody, immuno - analytical systems were constructed to obtain the respective dose - response curve for evaluating the analytical performance of the antibody, in particular, the detection capability (also expressed as sensitivity). the antibodies used in this study were highly specific in measuring analyte, that is, ctni as a specific ami marker, present in serum as a medium for clinical tests [16, 17 ]. each antibody were immobilized on the inner surfaces of microwells in low and high densities to establish the binding models, and sandwich - type enzyme immunoassays were carried out to plot the signal against the ctni concentration (figure 3). from such prepared dose - response curves, the detection limits for each antibody preparation were determined as described previously. with the antibodies immobilized at a low density (figure 3(a)), the conventional, randomly biotinylated whole antibody showed the poorest sensitivity (detection limit = 1.826 ng / ml). when the site - directly biotinylated whole antibody was employed, the detection capability increased 6.1 times (detection limit = 0.297 ng / ml). adoption of the antibody fragments, f(ab)2 and fab, further enhanced the sensitivity (0.205 ng / ml for the both). when the capture antibodies were immobilized at a high density (figure 3(b)), the assay sensitivity with the random preparation remarkably increased by 6.6 times (detection limit = 0.275 ng / ml) compared to immobilization at a low density. the site - directed products also showed somewhat improvements (detection limits = 0.246 ng / ml for igg, 0.178 ng / ml for f(ab)2, and 0.162 ng / ml for fab), but the increments were relatively small. this may result from a change of the performance - controlling factor when the antibody density increased, which will be cleared in the rest of this paper. although the site - directly biotinylated fragments gave the best performances regardless of the surface density, a high density of the random igg immobilization significantly enhanced the sensitivity. the fragmented products may have been immobilized in a controlled orientation as well as having flexible steric freedom and, consequently, had better accessibility to the antigen. this will be discussed in more detail below. as the surface density increases in the random preparation, the antibodies may change their molecular conformation into a more accessible orientation via self - assembly. however, the use of a large quantity of antibodies as the capture may cause unwanted problems, for example, an increase in non - specific binding of the enzyme - labeled detection antibody to the immobilized layer or negatively influencing the system [2, 3 ]. the site - directly prepared fragments immobilized at a 10 times lower density than the random igg gave approximately an identical sensitivity. in this context, fragments with such performance would be preferable in most immunoassays that require a high signal - to - noise ratio as a precondition for enhanced detection capability. the complex formation of antigen with the immobilized antibody is affected mainly by three factors : method of immobilization, surface density of the antibody, and size of antigen. in general, these alter not only the density of reactive binding sites on the surfaces, but also the binding affinity of the antigen to the immobilized antibody. the number of reactive binding sites represents the status of the molecular orientation of the layer, and the affinity represents the steric freedom of the antibody accessible to the antigen. therefore, these two parameters were used to analyze the improved performances of the site - directly biotinylated and suitably fragmented antibodies. to determine the reactive binding sites of the immobilized antibody, scatchard analysis can be carried out with a sandwich - type enzyme immunoassay using a microtiter plate for relatively easy measurements. the use of the detection antibody labeled with an enzyme (e.g., hrp), however, may sterically hinder binding between the antigen (e.g., ctni) and the capture antibody. we performed these experiments and analysis based on the following four assumptions : (1) the binding sites on the surfaces are uniform for reaction with antigen ; (2) each antigen - antibody binding event is independent of one another ; (3) dissociation of the complex during incubation and washing is negligible ; and (4) the detection antibody labeled with the enzyme covers all of the binding complexes such that one enzyme molecule represents a paired complex. while the first three assumptions are usually applicable to any solid phase immuoassay, the fourth may be dependent on the quality of labeled detection antibody. the detection antibody used in this study was chemically coupled to hrp, which was analyzed by sds - page. from this analysis we found that the major portion (over 80%) of protein was indeed positioned at the 200 kda band (data not shown). since the molecular weights of the antibody and hrp are 155 and 44 kda, respectively, the conjugation ratio on average should be very close to one. in addition, the three different types of antibodies, that is, whole igg, f(ab)2, and fab, employed in this study had approximately same association / dissociation rate constants when measured using a surface plasmon resonance sensor system under the identical conditions. this indicated that the fragmentation of the antibody using protease did not alter the binding kinetic properties. under these assumptions, the surface density of the binding sites participating in the antigen - antibody reaction was determined by means of the scatchard analysis for four different preparations of antibody : randomly or site - directly biotinylated whole igg, site - directly prepared f(ab)2, and site - directly synthesized fab. the quantity of complex formation was estimated on the basis of the hrp activity coupled to the detection antibody, and the bound over the unbound antigen was plotted against the bound antigen concentration. the surface density of the binding sites and affinity was determined from the x - intercept and slope, respectively, of the scatchard plot. it is worth noting that the scatchard plot was linear, indicating that the four assumptions proposed above were valid. the density of binding sites available for the complex formation was determined at a constant concentration of each antibody, which was then replicated at different concentrations (figure 4). at a low antibody concentration (e.g., 0.5 pmol / ml), the randomly biotinylated igg showed a significantly low reactivity comparing to the site - directly prepared antibody products, although the total number of immobilized antibody was higher (data not shown). however, this gap decreased as the antibody concentration increased, which indicates that there was a gradual formation of a self - assembled molecular layer on the surfaces. for the site - directed preparations, their relative densities were in an order of fab > f(ab)2 > igg at a low concentration, and became approximately the same but still greater than igg as the concentration increased. these results indicate that the fragmented preparations essentially oriented the binding sites to face the bulk solution, but it may be very difficult for igg to bind to the solid surfaces without impairing the binding sites for the reaction. there, however, could be subtle discrepancies between the fragmented species with regard to accessibility to the antigen present in solution, which will be further analyzed in terms of the binding affinity below. as described above, the binding affinity of the immobilized antibody can also be determined concurrently with the reactive density of antibody binding sites from the scatchard analysis. the binding affinities of four different types of the biotinylated antibody species were plotted against the antibody concentration added (figure 5). compared with the randomly immobilized igg, the site - directly prepared antibodies (igg, f(ab)2, and fab) showed high affinities over the entire antibody concentration range tested. among the site - directed preparations, the relative affinities were consistent (fab > f(ab)2 > igg) regardless of the concentration, suggesting formation of unique conformations to each antibody species upon insolubilization onto the matrix. conformations of each antibody preparation at the solid surfaces could be distinctive and determined mainly according to differences in the molecular shape (shown as igg, f(ab)2, and fab) and biotinylation method (figure 6). among the antibodies tested, both of the native igg type biotinylated using either the random figure (6(a)) or site - directed method figure (6(b)) showed the lowest binding affinities (refer to figure 5), which may be linked to the presence of fc. although the fc region has definite biological roles in vivo, this can hinder the attainment of a molecular orientation suitable for antigen - antibody binding when immobilized under an artificial environment. the igg preparation did indeed seem to be present on the solid surface layer in a configuration, that is, side - on, with multiple contacts, which brought the binding sites of the molecules into the proximity of the surface. thus, the surface can partially restrain the steric freedom of immobilized molecules required for antigen - antibody binding, resulting in a reduction of the binding affinity (known as surface effect). between the biotinylation methods, the random protocol resulted in the worse configuration (e.g., top - on) and was the major factor that reduced the number of reactive binding sites, particularly, when the surface density was low (refer to figure 4). the other two site - directly synthesized fragments without fc, f(ab)2 and fab, which relieved the surface effect when used as the capture antibody (refer to figure 5), are also different in their shape with respect to the number of binding units. the f(ab)2 were immobilized via the hinge and sat on the surfaces in an orientation that turned the binding sites toward the bulk solution (figure 6(c)). since the molecules consisted of two binding sites located on the opposite ends of the left and right arms, the antigens in solution had to diffuse in a lateral direction to approach the sites to form the binding complex. in contrast, since the fab fragment consisted of a single binding unit, it could be immobilized in an erect position where the binding site faced the bulk solution even though the fab fragment was biotinylated on the same region (figure 6(d)). in this case, the antigens can access the binding sites in the vertical direction, which may be more free of diffusion limitations caused by the surface layers present on the solid matrix. the analyses done in this study show that factors controlling the performance of the capture antibody seem to be different according to the antibody density present on the solid surfaces. when the antibody density was low, orientational arrangement of the molecules mainly limited the number of binding sites for antigen in solution. particularly, in case of randomly biotinylated igg, the antibody may be immobilized mostly in top - on and side - on configurations, resulting in an inaccessible binding site. side - on conformation due to the presence of the fc region, which could decrease the accessibility of the antigen in solution. when the density increased, a molecular layer organization called self - assembly occurred and under such crowding conditions accessibility of the antigen to the binding sites could alter the binding affinity. in the case of antibody molecules with two associated binding units, this process could be regulated by the molecular layer formed on the surfaces, which was more pronounced provided the steric freedom of the antibody was restricted by, for instances, the presence of fc and immobilization via multiple biotin - streptavidin linkages. various antibody fragments, f(ab)2 and fab, biotinylated at the hinge region were produced and when used as the capture antibody in a sandwich immunoassay for ctni displayed an enhanced analytical sensitivity that was about 6 times higher than the randomly biotinylated whole igg under a low antibody density condition. this performance was comparable to that of the randomly prepared igg immobilized at a 10 times higher density on the solid surfaces. such a high detection capability with the scanty fragments could result mainly from a controlled conformation of the capture antibody and also from the flexibility due to the absence of fc, which offer conditions suitable for the binding complex formation. these features would be beneficial in immuno - analytical systems for the reduction of non - specific binding caused by protein - to - protein interactions or even by cross - reaction between antibody species employed as reagents. | the enhanced analytical performances of immunoassays that employed site - directly immobilized antibodies as the capture binders have been functionally characterized in terms of antigen - antibody complex formation on solid surfaces. three antibody species specific to cardiac troponin i, immunoglobulin g (igg), fab, and f(ab)2 were site - directly biotinylated within the hinge region and then immobilized via a streptavidin - biotin linkage. the new binders were more efficient capture antibodies in the immunoassays compared to randomly bound igg, particularly, in the low antibody density range. the observed improvements could have resulted from controlled molecular orientation and also from flexibility, offering conditions suitable for binding complex formations. |
segmental absence of intestinal musculature (saim) was first reported as a new entity in 1947 by emanuel. as a cause of intestinal obstruction. the cases are now increasingly reported in the medical literature, so far more than 25 cases have been reported in the english literature with additional cases in european and asian literature. however, occasionally the cases have been reported in adults. beside small intestine, musculature defect has been reported in stomach and colon. these cases of saim have been divided into primary and secondary groups. in primary group etiology is unknown, the onset of symptoms is acute and there is no pathologic finding in the remaining layers of the small intestine except for superimposed perforation or intussusception. in secondary group histologically there may be ischemic necrosis of the remaining layers, fibrosis, calcification, chronic inflammation and presence of macrophages. these findings indicate secondary destruction of muscle coat due to ischemia and/or infarction, interruption of blood supply or trauma. most of the authors agree that this is a congenital defect and some believe ischemia to be only etiological factor in cases of perforation. here, we present a rare case of saim in an adult of 48 years. a 48-year - old male, previously operated in some other hospital for pain in abdomen and vomiting for 2 days. resection anastomosis of the bowel was done nearly 20 days back, and the surgical pathology report mentioned perforation peritonitis with the gangrenous bowel. he was admitted in our hospital for the complaints of abdominal distension and vomiting and treated conservatively. he was discharged from the hospital and was readmitted after a week for abdominal distension, pain and vomiting for 1-day. he was reoperated, and resection anastomosis of the bowel was done where perforation peritonitis was present. in surgical pathology department, two pieces of small intestine together measuring 45 cm were received. the pieces were markedly congested with rough peritoneal surface and thin wall with a perforation of 1 cm. the sections near the perforation showed necrosis of mucosa with inflammatory cell infiltration, marked vascular congestion, sub - mucosal fibrosis and peritonitis [figure 1a ] with gangrenous changes. the section from the mesentery showed inflammation and a thrombosed artery [figure 1b ]. the sections taken from the normal looking parts of intestine revealed focal absence of muscularis propria [figure 2 ]. (a) photomicrograph of small intestine showing focal ulceration of the mucosa with marked inflammatory cell infiltration and vascular congestion (h and e, 40). (b) photomicrograph of mesentery showing thrombus (arrow) in the mesenteric artery (h and e, 40) photomicrograph of small intestine showing absence (arrow) of muscularis propria (h and e, 40) the patient subsequently developed abdominal fistula and was subjected to computed tomography angio based on the histopathology report of thrombosed artery. it revealed dilated small bowel loops in mid and left side of lower abdomen, proximal mesenteric artery was normal in caliber, distal superior mesenteric artery wall was irregularly thickened causing 60% narrowing of the lumen with thrombosis of few branched vessels [figure 3 ]. the patient was advised re - exploration that he refused and left against medical advice. photographs of computed tomography abdomen angiography sagittal (a) and axial (b) : calcified plaques in the abdominal aorta with focal narrowing of the celiac trunk at origin approximately 50% seen. there is normal caliber and flow in proximal superior mesenteric artery (sma) with irregular slight thickening of sma wall causing narrowing of sma approximately 60%, mesenteric fat stranding in lower abdomen segmental absence of the intestinal musculature or segmental congenital defect of intestinal musculature is a rare clinical entity. most of the patients are from neonatal age group and present as intestinal obstruction along with other associated congenital anomalies like patent ductus arteriosus, biliary atresia, intestinal atresia and ventricular septal defect. the preoperative diagnosis is not possible in these cases, and the diagnosis is only possible on postoperative pathologic specimen provided the pathologists are aware of the condition. however, it is suggested that transillumination of the intestinal wall may be helpful to identify the defect during the surgical procedure. so in clinical set up of the cases of intestinal obstruction and/or perforation not only in neonates but also in higher age group, saims may be one of the differential diagnosis if the patient has repeated recurrences of the symptoms as is seen in our case. there have been several proposed theories of pathogenesis of saim such as : a normal embryogenesis leading to incomplete or dicontinuous myogenesisan ischaemic event during fetal life or postnatally leading to injury of mucosa or muscularis propria, and differences in regenerative capacity between mucosa and muscle, in which mucosa rather than muscle regenerates, resulting in exclusive absence of the musculature. a noteworthy fact is that animal studies have not yet been successful in producing such lesion. ligation of the mesenteric vessel causes intestinal atresia involving all the three layers of intestinal wallgenetic and familial factors have been postulated in some cases, however, some investigators do not support this theory as there is not enough evidencesome authors believe that this malformation could be a relic of developmental diverticula in the embryonic small bowel. others believe that it could also result from resorption of ileal muscle at the time of regression of omphalomesenteric duct, considering the frequency of proximity to the site of meckel 's diverticula. a normal embryogenesis leading to incomplete or dicontinuous myogenesis an ischaemic event during fetal life or postnatally leading to injury of mucosa or muscularis propria, and differences in regenerative capacity between mucosa and muscle, in which mucosa rather than muscle regenerates, resulting in exclusive absence of the musculature. a noteworthy fact is that animal studies have not yet been successful in producing such lesion. ligation of the mesenteric vessel causes intestinal atresia involving all the three layers of intestinal wall genetic and familial factors have been postulated in some cases, however, some investigators do not support this theory as there is not enough evidence some authors believe that this malformation could be a relic of developmental diverticula in the embryonic small bowel. others believe that it could also result from resorption of ileal muscle at the time of regression of omphalomesenteric duct, considering the frequency of proximity to the site of meckel 's diverticula. the present case is 48-year - old and did not have history of any other ailment. he was nondiabetic, nonhypertensive and developed intestinal perforation at the age of 48 years. the thrombosis in the vessels suggests that it may be a benign congenital anomaly and ischemia may be the cause of perforation as agreed upon by majority of the authors but still the etiology is controversial. in summary, saim remain puzzling and challenging disease with variable clinical presentation not only in neonates but also in higher age group and adults and the pathologists as well as clinicians should be aware of the condition, especially, when repeated complications are developing in a patient of intestinal obstruction and/or perforation. | segmental absence of intestinal musculature (saim) is a rare cause of intestinal obstruction and/or perforation seen in neonates, and rarely in adults. we present a case of saim in a 48-year - old male, who presented with acute abdominal symptoms and was repeatedly explored because of recurrence of abdominal symptoms. |
austere renal replacement therapy (rrt) describes the provision of renal replacement therapy in any setting in which traditional, first - world therapies and resources are limited, incapacitated, or nonexistent. the provision of rrt in an austere environment is very different from that in a routine situation in a first - world country. in the latter case, the following apply (1) the environment is secure from violence and physical risk to the providers and patients ; (2) the transportation infrastructure is functioning ; (3) there are plentiful and stable sources of electricity, rrt supplies, and potable water ; (4) engineering systems are in place for the production of pure water ; (5) sophisticated equipment is available ; (6) adequate equipment maintenance and nursing / technician staff support exist ; (7) patient acuity and numbers are predictable and stable. in an austere situation, some or all of these components may be inadequate or completely absent. if austere environment rrt is to be successful, the provider must identify the components that are lacking and attempt to offer reasonably safe and effective substitutes for them if they can not be controlled or repaired. this requires flexibility, the ability to triage, and a thorough understanding of the engineering and physiologic principals of rrt. moreover, specific advance planning is necessary, especially in an environment or geographic area where certain disasters are likely to occur (particularly true for storms and earthquakes). moreover, it is important during planning and implementation not to allow (as in voltaire 's aphorism) the perfect to become the enemy of the good. ultimately, the optimal scenario for rrt provision after a disaster, or in an austere situation, is for rrt to be unnecessary or able to be delayed. others have provided expert opinion regarding the appropriate response to certain likely disasters (especially earthquakes and storms) for both providers and patients requiring both acute and chronic rrt [17 ]. many recent reviews focus on the epidemiology and management of patients with acute kidney injury (aki) due to crush injury sustained after earthquakes [2, 3, 7 ]. the renal disaster relief task force (rdrtf) and european renal best practice (erbp) are currently developing comprehensive guidelines for the management of crush syndrome. therefore, in this paper, although we will discuss the situation of aki due to crush injury as the paradigm for austere rrt, we will focus more on general practical aspects of managing patients who require rrt in austere settings, regardless of the cause of renal failure. many such patients are likely to be encountered where chronic dialysis units have been incapacitated or resources are otherwise severely limited due to unanticipated disaster events. rrt situation may exist where there are only a few patients to manage, and no disaster has occurred, but rrt provision is limited by logistical and equipment considerations alone. although crush syndrome with resultant myoglobulinuria and aki due to acute tubular necrosis (atn) is not the only type of renal failure requiring rrt seen under austere circumstances, it has received the most attention. although most associated with earthquake events, it also may be seen in the setting of entrapment after building collapse due to any cause and was first described in 1941 in patients removed from beneath collapsed buildings during the aerial bombardment of london. there was no rrt infrastructure in 1941 (dialysis had not yet been developed), and death due to hyperkalemia was the outcome for the patients described in this series. subsequently, much of the disaster nephrology literature has focused on preparing for and treating the influx of patients with atn due to crush - related muscle injury after an earthquake event [7, 10, 11 ]. rrt management of such patients can be very resource intensive because of the associated muscle damage with resultant accelerated hyperkalemia, hypocalcemia, and acidosis. in addition, in situations where intravenous fluid resuscitation is available and employed, severe symptomatic rebound volume overload can occur in those who develop oliguria. even with efficient, single - pass hemodialysis, rrt may be necessary more than daily, and dialysis dependence may last for weeks. less efficient forms of rrt, such as peritoneal dialysis and continuous therapies, may not provide enough clearance to control the metabolic abnormalities (particularly hyperkalemia). although patients with myoglobinuric atn require very resource - intensive rrt, the effects of the earthquake itself may significantly limit rrt delivery. after an earthquake, there may be prolonged interruptions of electricity and water delivery, transportation infrastructure and medical building may be severely damaged, and medical personnel themselves may be casualties. it is also difficult to predict the number and severity of casualties that may develop atn and require rrt. the type of buildings in the area, the rapidity of rescue, the provision of intravenous (iv) fluid prophylaxis at the injury site, and the strength and timing of the earthquake can substantially affect the number of casualties with crush injury and subsequent atn. for example, after the large california earthquakes of 1971, 1983, and 1989, crush injuries were small in number, but in situations where there are collapses of multistory stone or reinforced buildings (as seen in the armenian earthquake in 1988), there may be many [12, 13 ]. the damage can be so great that there are paradoxically few crush injuries and cases of atn, because few persons are rescued or are able to access medical care. they simply die at the scene, as observed after the haitian earthquake, as well as after the collapse of the world trade center in new york in 2001 [8, 14 ]. in another scenario, where buildings are small and constructed of relatively light materials, such as brick, wood, or adobe, there are few crush injuries because rescues are very rapid, and crush syndrome does not develop [15, 16 ]. despite these observations, it is important to recognize that crush syndrome remains the second most likely cause of death in earthquake disasters after direct trauma and, unlike the latter, may be medically prevented. the pathophysiology of crush syndrome and rhabdomyolysis - associated atn is complex and beyond the scope of this paper. direct tubular toxicity due to heme iron released from myoglobin, other toxins released from injured muscle (to include uric acid), formation of obstructing myoglobin casts, volume depletion, free radical activation, reperfusion injury, cytokine release, and acidosis all appear to contribute to renal injury and development of atn [1719 ]. it has been shown in animal models as well as in humans that volume repletion, with increased glomerular filtration and tubular flow, prevents atn, along with (and perhaps to a lesser extent) alkalinization and administration of free radical scavengers (such as mannitol). this has been translated into the clinical practice of prophylactic isotonic iv fluid resuscitation to victims of crush injury in the field, in many cases while still entrapped the rtrtf of the international society of nephrology (isn) has developed a disaster plan and organized a response team to assist in the management of rrt for victims presenting with atn after earthquakes. they have reported extensively on their experiences with crush syndrome and management of aki, after numerous large earthquakes. it is noteworthy that crush syndrome casualties are proportionately few relative to the overall injured population, and even fewer require rrt. of those who do require rrt, mortality appears relatively low, and the majority who survive will regain renal function (table 1). that being said, the burden of aki requiring resource - intensive management will vary widely from one earthquake event to another, depending on a multitude of factors, only some of which are predictable prior the disaster event. there are many other situations, besides earthquakes, when nephrologists may be called upon to manage an influx of patients requiring rrt, in an environment in which optimal physical and personnel resources are not available, or are severely compromised (table 2). first, it is useful to summarize, in an orderly manner, the situations in which rrt may be required in an austere situation taking into consideration the capacity and demand for rrt. (1) the most commonly described, and planned - for event is a disaster that results in an increased incidence of aki, requiring an increased demand for rrt services. in this situation present previously and now severely damaged. in this scenario, not only is there an influx of patients requiring acute rrt, but also there is a population of patients with pre - existing esrd who are receiving either chronic hemodialysis or peritoneal dialysis. examples of events that could cause such a situation include, but are not limited to, earthquakes and urban battlefields. damage to infrastructure may vary considerably depending on the age, design, and location of the buildings housing dialysis units, and the size and intensity of the damaging event. this would be the situation predicted for the recent chilean earthquake. however, there appear to have been very few cases of aki after this event, and the major impact was on the patients with esrd who were unable to receive care because of severe damage to their dialysis units.previously nonexistant or negligible, although other medical services may exist. in this scenario, there are few patients with pre - existing esrd receiving chronic rrt, and the patients who require rrt largely have aki. events that could cause this situation include earthquakes, urban battlefields, infectious disease outbreaks associated with aki (e.g., hantavirus - associated hemorrhagic fever, gastroenteritis - associated hus), or wide population exposure to renal - toxins (e.g., melamine - contaminated infant formula) [3840 ]. earthquake events in third - world countries, such as the haitian earthquake of 2010, are examples of this scenario. one would presume, in this situation, that dialysis resources would have to be brought to the area, but this is not clear - cut. after the haitian earthquake of 2010, although limited dialysis resources were brought to the area aboard the usns comfort, which had 2 standard hemodialysis machines and provided 15 treatments within the first 9 days, the isn rdrtf repaired the existing infrastructure of the university hospital dialysis unit in port au prince to support the care of both patients with aki due to crush injury and 30 of the 100 haitian chronic dialysis patients [8, 34].present previously and now undamaged, but insufficient to handle the influx (demand) of patients with aki (and/or esrd). this scenario is most likely to be seen in a refugee situation in areas adjacent to, but not affected by, an earthquake or war, after an isolated building collapse, or in the setting of large case numbers of aki after an infectious outbreak or toxic release. a special case of this is when refugees with esrd travel from a disaster site to an adjacent area with intact rrt infrastructure, as could be seen after a devastating earthquake, storm, flood, or in the aftermath of a battle or terrorist attack. excellent examples were the aftermath of hurricane katrina in 2005 and after the recent chilean earthquake [1, 8 ]. present previously and now severely damaged. in this scenario, not only is there an influx of patients requiring acute rrt, but also there is a population of patients with pre - existing esrd who are receiving either chronic hemodialysis or peritoneal dialysis. examples of events that could cause such a situation include, but are not limited to, earthquakes and urban battlefields. damage to infrastructure may vary considerably depending on the age, design, and location of the buildings housing dialysis units, and the size and intensity of the damaging event. however, there appear to have been very few cases of aki after this event, and the major impact was on the patients with esrd who were unable to receive care because of severe damage to their dialysis units. previously nonexistant or negligible, although other medical services may exist. in this scenario, there are few patients with pre - existing esrd receiving chronic rrt, and the patients who require rrt largely have aki. events that could cause this situation include earthquakes, urban battlefields, infectious disease outbreaks associated with aki (e.g., hantavirus - associated hemorrhagic fever, gastroenteritis - associated hus), or wide population exposure to renal - toxins (e.g., melamine - contaminated infant formula) [3840 ]. earthquake events in third - world countries, such as the haitian earthquake of 2010, are examples of this scenario. one would presume, in this situation, that dialysis resources would have to be brought to the area, but this is not clear - cut. after the haitian earthquake of 2010, although limited dialysis resources were brought to the area aboard the usns comfort, which had 2 standard hemodialysis machines and provided 15 treatments within the first 9 days, the isn rdrtf repaired the existing infrastructure of the university hospital dialysis unit in port au prince to support the care of both patients with aki due to crush injury and 30 of the 100 haitian chronic dialysis patients [8, 34 ]. present previously and now undamaged, but insufficient to handle the influx (demand) of patients with aki (and/or esrd). this scenario is most likely to be seen in a refugee situation in areas adjacent to, but not affected by, an earthquake or war, after an isolated building collapse, or in the setting of large case numbers of aki after an infectious outbreak or toxic release. a special case of this is when refugees with esrd travel from a disaster site to an adjacent area with intact rrt infrastructure, as could be seen after a devastating earthquake, storm, flood, or in the aftermath of a battle or terrorist attack. excellent examples were the aftermath of hurricane katrina in 2005 and after the recent chilean earthquake [1, 8 ]. (2) a less commonly discussed event is a disaster that does not produce an increased incidence of aki or an influx of patients with esrd, but results in inability of existing local esrd patients to access dialysis due to disruption of transportation, damage to the dialysis infrastructure, or both. the demand for rrt is unchanged, but the capacity to provide it is degraded. the most common cause of such an event would be a weather emergency (such as a hurricane, tornado, flood, or blizzard), but civil unrest, war, and terrorist attacks could also be causes. this was the local scenario after hurricane katrina, but occurs to some extent quite commonly after local flooding or blizzards (as seen after the blizzards on the east coast of the us in early 2010) [1, 42 ]. if one considers the numbers of victims with aki needing acute rrt after a disaster in the last several decades (tables 1 and 2), it is striking how proportionately few were identified as having aki, and then the fewer number who required dialysis. for instance, after the january 2010, haitian earthquake in which over 200,000 people died, the dialysis response team dialyzed 19 patients with aki, but also managed 30 patients with esrd who were dialysis dependent before the earthquake. in chile, after the earthquake of february 2010, most fatalities were associated with the tsunami, and only 2 patients are reported as requiring rrt due to crush injury - induced aki. however, there were over 2400 patients with esrd on chronic dialysis and their management after the destruction of many chronic dialysis units was the primary challenge facing the local nephrology community. evacuation to areas unaffected by the earthquake and adjustment of dialysis schedules at units within the earthquake zone that had survived were used to accommodate those patients whose dialysis units were nonfunctional. in the aftermath of hurricane katrina, the population of patients requiring chronic dialysis in baton rouge, la increased by approximately 700 patients (the usual population was about 1000) due to the influx of refugees from areas of louisiana affected by the hurricane. although there were no immediate deaths reported due to unavailability of dialysis services in this population, cms data indicated that there was an increase in deaths among esrd patients in the area within the first 180 days after the hurricane, although later data suggests that there was no significant change in mortality rate in the 6 months following the disaster, when compared to the 6 previous months [1, 43 ]. thus, as esrd services become more commonly available, even in countries which have relatively poor medical infrastructure such as haiti, the management of patients with esrd who are unable to access chronic dialysis after disaster may become the primary concern of local nephrologists and renal response teams, rather than rrt for aki, even in the setting of very severe earthquakes. how then does an individual nephrologist or chronic dialysis unit prepare for and respond to a disaster that may result in an austere rrt environment ? in the united states, the center for medicare and medicaid services (cms) requires that dialysis facilities must develop written policies and procedures for emergencies. the kidney community emergency response coalition (kcerc), formed in the us after hurricane katrina, has published a set of guidelines for emergency planning. although many of the recommendations apply to federal, state, and local emergency providers, effective strategies are available for patients and providers. the kcerc time - line to safety is a helpful, general resource for patients, local dialysis units, and providers in developing a disaster plan, especially for disasters that are predictable (such as weather emergencies). the copy of the cms publication preparing for emergencies : a guide for people on dialysis should be available to all patients. the first step is to identify the disasters most likely to occur. regardless of the type of disaster event, there are several planning recommendations that nephrologists, dialysis directors, nursing staff, and local policy makers should consider in localities with an existing dialysis infrastructure and many dialysis - dependent patients with esrd. (1) assess and implement measures that will keep the dialysis facility functional and safe during and after a disaster event. simple measures are important, such as knowing where utility shut - off valves are located. (2) educate patients about modifications to chronic diet and fluid intake in the event of a disaster. volume overload and hyperkalemia are the most likely complications to force rrt treatments. the cms publication preparing for emergencies : a guide for people on dialysis contains a detailed 3-day diet plan and food supply list. (3) provide medications which may delay the need for dialysis to patients before a predictable disaster (such as a hurricane or snowstorm). high - dose loop diuretics may increase urine output and kaliuresis in patients with residual renal function. explicit instructions should be given to the patient on when to begin such medications, and these instructions should be reviewed at regular intervals with all chronic dialysis patients [5, 6 ]. (4) ask patients to maintain updated lists of medications, allergies, health problems, and contact information for his / her providers and local dialysis unit, and to carry these records with them in the event of travel out of a disaster area. in fact, it should be assumed that communication systems will be nonfunctional for a period of time, and advance planning should focus on optimizing self - sufficiency for each patient as much as possible [1, 5 ]. (5) develop emergency evacuation plans at both the unit and individual esrd patient levels that provide for efficient, practical, and safe egress for both patients and staff. it is crucial that this planning incorporate both on - site and from - home scenarios. these plans should be routinely practiced by both staff and patients during scheduled and unscheduled drills. home dialysis patients, especially pd patients, should not be forgotten, and specific guidelines exist for them with regard to infection control management [5, 43 ]. these plans require frequent review and training for both patients and staff. at the local and regional levels, policy leaders need to incorporate planning for the orderly and timely evacuation of the chronic dialysis population to areas unaffected by a disaster event. nurses and physicians themselves may be injured, be unable to travel to the facility, and may have personal responsibilities that are equal to their professional ones. development of a defined and flexible coverage plan for staff during the first days of a disaster should be in place. any successful plan for managing rrt in the event of large - scale resource incapacitation will need to incorporate several key elements, regardless of the type and number of patients being managed. (1) conserve resources (e.g., supplies, transportation, purified water, and staff). a dialyzer reuse plan, plans for limitation of water use (decrease of dialysate flow rate), shortening of dialysis times, and reduction of supply consumption all should be considered. available supplies (especially dialyzers) may not be the type used before the disaster, or may be in short supply. dialyzer reuse may not be feasible or safe in many situations, but in scenarios where patient numbers are low, and resupply is totally disrupted, reuse should be considered. (2) determine thresholds for rrt initiation and frequency. at the height of the disaster, with its attendant difficulties of transportation and resource access, the presence of acute indications for dialysis and the catabolic state may determine which patients receive dialysis treatments, and standard treatment schedules may need to be abandoned. as the situation improves, accepted standards of rrt adequacy should guide treatment decisions regarding both esrd and aki patients, although this may be constrained by available resources. single - pass hemodialysis with dialysate water delivered by a state - of - the - art portable or fixed water treatment plant may not be possible. alternate rrt modalities must be considered, and each hemodialysis center should plan for the provision of an alternative means of renal replacement therapy should the pre - disaster, existing infrastructure be rendered nonfunctional. many of the same resource constraints are likely to apply to these as to conventional dialysis. realistically, the modality that can be made to work in the existing environment is the one preferred ! peritoneal dialysis : peritoneal dialysis is an attractive alternative in settings where the electrical supply and the water plant are disrupted. drawbacks include the need for peritoneal catheter placement, the risk of peritonitis, the need to obtain (or make) large volumes of appropriate sterile dialysate, and difficulty of metabolic control in the severely hypercatabolic patient. cavh / d and cvvh / d using replacement fluid / dialysate from readily available commercial iv crystalloid solutions : cavh / d and cvvh / d have the disadvantage of requiring large volumes of replacement fluid / dialysate and may also be resource intensive from the standpoint of personnel. cavh requires arterial access, and use of upper extremity av fistulae / grafts may be difficult with cvvh. a distinct advantage is the limited electrical power requirements (cavh requires none), and there is no need for water purification. isolated ultrafiltration for volume control, which does not require dialysate or a functioning water treatment plant and may even be achieved with dedicated slow continuous ultrafiltration devices used for management of congestive heart failure (aquadex ref). although this approach can control volume, there is no solute clearance [6, 50 ]. alternative devices developed for home hemodialysis based on either sorbent or cvvh technology [51, 52 ] : because these devices have been developed for the home market, they are simple to use and quite robust. existing chronic dialysis access can be used, and water treatment capacity is not necessary, as they either use a sorbent column or premixed replacement fluid. knowledge of water preparation and monitoring for dialysis not only among nursing and technical staff, but among physicians is essential. because chloramines may be increased in potable water after a disaster to prevent water - borne illness, they may need to be monitored with greater frequency. if possible, product water should be carefully monitored, especially if preparation is by mixed bed deionizer. product water may need to be stored in tanks, rather than continuously made [8, 34, 48 ]. (5) provide for electrical back - up systems / generators, which should be considered and in place before the disaster, with a plan for fueling them. (6) include an infection control plan, especially for patients who may be infected with tuberculosis or hepatitis b [1, 34 ]. the united states military medical services have well - described protocols for the provision of rrt to casualties in theater [46, 48 ]. augmentation team consisting of two dialysis technicians, a nephrologist, and an icu nurse. the dialysis machine specified for use, until recently, was the redy 2000, a sorbent - based system, which required 6 - 7 liters of potable water to manufacture dialysate for a 3 - 4-hour dialysis treatment. however, because of the success of aeromedical evacuation systems in rapidly removing casualties with aki from theater, deployment of this augmentation team was never required, and the redy 2000 is no longer manufactured. there have been occasions when alternative, short - term solutions, including peritoneal dialysis, cvvh, and cavh, have been used in austere conditions to manage individual patients presenting acutely who could not be evacuated in a timely manner. the us navy maintains a state - of - the art dialysis facility on the usns comfort, which uses single pass dialysis machines that one might encounter in a tertiary care medical center. this ship assisted with the dialysis needs of patients in haiti after the earthquake in 2010. the management of rrt for aki / esrd under austere conditions can be conveniently divided into management of crush syndrome patients (and, more generally, any patient in a hypercatabolic state) versus those patients with aki or esrd who are not hypercatabolic and may require less intense dialysis. for hypercatabolic patients, single - pass hemodialysis is the most efficient method of managing the hyperkalemia and acidosis which are immediately life - threatening, and other modalities (such as crrt and pd) may not be adequate to prevent life - threatening hyperkalemia. however, such modalities may be tried in settings where single - pass hemodialysis is not feasible, and they may be effective [4448, 53 ]. in patients with aki who are not hypercatabolic and in patients with esrd, in settings where resources are limited, an effort should be made in the early period after a disaster to triage patients on the basis of their acute need for rrt [1, 48 ]. this approach to provision of rrt is supported by experience with aki at the very beginning of the dialysis era. as early as the 1950s, it was well recognized that patients with atn more - or - less followed a defined course, and that nonoliguric patients had better outcomes than oliguric patients. if an acute event could be avoided (i.e., fatal hyperkalemia, acidosis, volume overload, and life - threatening uremia), patients could be expected to recover, and acute dialysis (which was technically very complex and difficult) was reserved only for these potentially fatal events. using this approach, mortality in aki (even trauma - associated aki) was reduced to approximately 5060% from the previously near - universal fatality rates which accompanied the most severe aki [54, 55 ]. studies of dialysis withdrawal in well - dialyzed esrd patients have shown that with aggressive volume restriction and judicious use of kaliuretics and potassium - binding resins, routine dialysis may be delayed for several days before the classic signs and symptoms of uremia develop or a life - threatening electrolyte imbalance occurs [6, 56 ]. isolated ultrafiltration, which does not require the use of dialysate, may be helpful to those in whom volume overload is the only indication for rrt and is resource friendly in conditions where supplies are constrained. screening for the need for acute dialysis can be done simply and requires little in the way of laboratory support. a physical examination and history assessing for symptoms and signs of severe uremia (pericardial friction rub, asterixis, vomiting / severe nausea, neurologic instability) can be done. solid - state, hand - held blood analyzers may be invaluable in assessing for hyperkalemia and acidosis and may also be used to check the electrolyte content of dialysate., dialysis treatments may be reserved for patients who are in acute need of them, thus directing scarce resources to those most likely to benefit. resources may be limited, and optimal rrt therapy, as defined in a nondisaster setting, may be impossible to deliver. however, difficult to recognize, it is important for patients and direct providers to remember that in large - scale disaster events, there are likely to be many more victims / refugees who will not require rrt services than those who will, and that there are likely to be other resource - intensive injuries present. emergency providers must therefore focus on the principals of triage, a systematic patient - prioritization technique whereby decisions regarding care, medical evacuation, or any other resource - intensive intervention of limited supply are made based on a combination of factors to include illness or disease severity, likelihood of survival within the constraints of the resources available, and the number of casualties relative to the resources at hand., this can be difficult, as in rare cases decisions to withhold available care to the most severely injured may be necessary in order to save others. applying the concepts of triage to decisions regarding maintenance hemodialysis therapies to chronic esrd patients in a disaster setting may require that the nephrologist set aside his or her standard approaches and adherence to dosing and management guidelines in order to maximize outcomes for the greatest number of individuals. the underlying chronic illness burden and the age of the esrd patient must be taken into account, as well as adherence to diet and volume restriction, overall dialysis adequacy prior to the disaster, and the likelihood of evacuation. in a situation of fixed and inadequate / barely adequate rrt capacity, it may not be possible to intensify rrt for a particularly fragile or non - dietary - compliant esrd patient at the expense of dangerously decreasing rrt therapy intensity for others overall. in more extreme situations, especially those involving a mix of esrd, aki, and highly catabolic aki patients, even more difficult rrt triage decisions may be required. helpful reviews and guidelines are available for those medical personnel involved with disaster planning who may be less familiar with these important concepts [5860 ]. the experience of others would support the notion that triage concepts can be applied successfully to the management of esrd patients after natural disasters : sever. have reported on the successful management of esrd patients using a reduced dialysis schedule at seven dialysis centers in turkey after the marmara earthquake. in their report, an approximate 50% reduction in functional capacity contributed to a 3-fold increase in the number of once - weekly dialysis treatments. despite this, interdialytic weight gain and blood pressures remained relatively stable, likely due to successful self - management of fluid intake and dietary restriction. the views expressed in this paper are those of the authors and do not reflect the official policy of the department of army, the department of defense, or the us government. | myoglobinuric renal failure is the classically described acute renal event occurring in disaster environments commonly after an earthquake which most tests the ingenuity and flexibility of local and regional nephrology resources. in recent decades, several nephrology organizations have developed response teams and planning protocols to address disaster events, largely focusing on patients at risk for, or with, acute kidney injury (aki). in this paper we briefly review the epidemiology and outcomes of patients with dialysis - requiring aki after such events, while providing greater focus on the management of the end - stage renal disease population after a disaster which incapacitates a pre - existing nephrologic infrastructure (if it existed at all). austere dialysis, as such, is defined as the provision of renal replacement therapy in any setting in which traditional, first - world therapies and resources are limited, incapacitated, or nonexistent. |
the prevalence of depression in diabetes is well documented, with a raft of research over the past two decades focusing on links between the two conditions. people with diabetes are between 1.4 and 3 times more likely to develop depression compared to the general population (3), with depression affecting 1520% of diabetes patients overall (4). when subclinical depression is included, prevalence estimates are greater, with at least two - thirds of a large sample of type 2 diabetes patients experiencing depressive symptoms (5). comorbid depression in diabetes patients is associated with poorer metabolic control, an outcome that predicts hyperglycemia and an increased risk of diabetes - related complications and mortality (6). these complications are, in turn, thought to be at least partly mediated by depression s association with poor adherence to medication and lifestyle recommendations, both of which predict reduced quality of life and increased health care costs (7). if anything, these figures likely underestimate both the prevalence of affective disturbances in diabetes and the associated costs of disease management, suggesting that the problem of depression may be far larger than is currently accepted. commensurately, the negative effects of these conditions on crucial self - care regimens and the resulting consequences in terms of increased disability, health care utilization, and mortality also may be far more widespread. given the scale of the problem of depression in diabetes, evidence to support the effectiveness of current antidepressant therapies is surprisingly sparse, and overall outcomes are still unclear. a recent systematic review and meta - analysis (8), including 14 randomized, controlled trials (rcts) evaluating psychotherapy, pharmacotherapy, and collaborative care in 1,724 diabetes patients with comorbid depression, showed moderate effects for treatment overall. it should be noted, however, that four of the five rcts (n = 310) involving a psychotherapeutic intervention such as cognitive behavioral therapy (cbt) also included other supportive treatment such as diabetes education alongside the psychotherapy, making it difficult to assess the merits of the psychological intervention in isolation. additionally, most studies were small (10 investigated samples ranging from 13 to 60 participants) and mainly involved type 2 diabetes patients, meaning results may not be generalizable to insulin - dependent type 1 diabetes patients. furthermore, although depression in diabetes patients is linked to glycemic indices, it is not yet clear if successfully treating low mood is associated with improved metabolic control. one review (7) found that improvements in depression were linked to improvements in glycemic control and overall perceptions of well - being. contradicting these findings, however, are results of a meta - analysis (11 studies in adults and 10 studies in children and adolescents) showing that, although both cbt and antidepressant medications were associated with an improvement in blood glucose readings in some studies, overall, there was no significant effect of either of these treatments on glycemic control in adults and only weak evidence of an effect in children and adolescents (9). successfully treating depression in diabetes is further complicated by the difficulty of detecting the presence of low mood, with evidence that only a small fraction of depressed patients receive any form of treatment, likely because of an overlap between symptoms of both conditions, such as pain and fatigue (7,10,11). furthermore, patients with diabetes may find depression difficult to acknowledge because it represents another area for them to be dissatisfied with themselves, perhaps further evidence of personal failure beyond their daily struggle with self - management targets (12), and a focus, therefore, for continued negative self - judgment. in summary, evidence for the treatment of depression in diabetes patients is limited, with existing studies suggesting modest effects in some groups, at best. further investigation is essential in light of the devastating downstream effects of depression on glycemic control and overall quality of life. emerging research into the construct of self - compassion, with its specific focus on reducing self - criticism and treating oneself kindly, suggests its potential utility for this population. self - compassion is defined as the practice of treating oneself with kindness, care, and concern in the face of negative events (13,14). for diabetes patients, negative events may include receiving less - than - optimal blood glucose readings together with other self - management failures involving nonadherence to medication, diet, and exercise prescriptions (15). in this context, self - criticism, a common consequence of self - care failure among diabetes patients, neff (13) conceptualizes self - compassion as being composed of three components, all of which may be relevant to the experience of living with diabetes and managing its daily demands. first, self - kindness refers to the tendency to be caring and understanding of oneself rather than being harshly critical or judgmental. second, common humanity recognizes that all humans are imperfect, fail, and make mistakes, framing difficulties and painful experiences in light of the shared human experience. finally, mindfulness, the third component of self - compassion, involves being aware of the present - moment experience so that one neither ignores nor ruminates on disliked aspects of oneself or one s behavior (17). taken together, developing these capacities may not only enable individuals to reflect concern and compassion toward others, but also may improve their capacity to direct this same concern and compassion toward themselves (13). in doing so, self - compassion may offer not only a gentler way of self - relating, but also one that may have the potential to reduce the depression and psychological suffering often associated with diabetes and its management. in the following section, the direct and indirect pathways by which both self - compassion and depression may relate to diabetes self - management are briefly reviewed, suggesting the utility of self - compassion for improving both mood and overall diabetes outcomes (figure 1). interlocking cogs illustrate the hypothesized processes linking self - compassion to improved outcomes in diabetes patients. increasing self - compassion may reduce depression, leading to improved outcomes via mechanisms including increased motivation and reduced self - criticism. although the exact mechanisms by which depression is associated with markers of diabetes remain uncertain, there is evidence to suggest both direct (physiological) and indirect (behavioral) pathways. interestingly, in terms of the current discussion, emerging evidence suggests that self - compassion may operate through similar processes. early studies indicate that depression may complicate, or possibly contribute to, the cause of diabetes due to direct, reciprocal effects on endocrine and other physiological processes. these effects include abnormalities of the hypothalamic - pituitary - adrenal axis, changes in sympathetic nervous system functioning as measured by decreased heart rate variability (hrv), and increased release of inflammatory cytokines (11,18,19). support for a possible biological link between depression and diabetes was demonstrated in a recent meta - analysis (20) involving 24 studies that found patients with depression had significantly higher concentrations of tumor necrosis factor- and interleukin-6 (il-6) levels compared to nondepressed subjects. in turn, markers of inflammation such as il-6 have been proposed to be involved in diabetes disease onset (21). interestingly, emerging evidence suggests that self - compassion might be linked to improved mood through similar or related metabolic and autonomic processes, including inflammatory and sympathetic nervous system responses to stress. cross - sectional reports show that self - compassion is associated with lower blood plasma levels of il-6 (22), as well as improved autonomic nervous system responding to stress (23), as measured by increased hrv (24). such data are consistent with the theoretical proposition that self - compassion may calm the threat system, which is associated with defensiveness and autonomic arousal, and activate the self - soothing system (14). a series of experimental studies suggests that quantifiable physiological and neurological processes underlie the experience of self - compassion. (25) used functional magnetic resonance imaging to demonstrate that the specific neuronal networks associated with love and affiliation were activated after the experimental stimulation of compassion. participants also demonstrated an increase in positive affect ; compared with a memory control, participants who reacted with negative affect when witnessing others in distress before compassion training subsequently showed increased positive affective experiences after training. (26) also found that compassion training increased both altruistic behavior and neural responses to suffering, including activation of the inferior parietal cortex and dorsolateral prefrontal cortex. taken together, these early fin - dings suggest that activation of attachment and affiliation circuitry through self - compassion may be linked to particular direct processes involving autonomic and metabolic pathways that may be common to both depression and diabetes. although depression and self - compassion may be associated with processes directly linked to physical health, a number of cross - sectional and experimental studies in both patient and nonpatient populations suggest that compassion may alter psychological and behavioral processes leading to reductions in depressed affect and better self - regulation. first, consistent evidence suggests that self - compassion is related to physical and psychological health as a result of reduced negative affect (17,2729). for example, in a study in patients with obesity and pain problems (30), self - compassion predicted lower negative affect, higher positive affect, more adaptive pain coping, higher pain self - efficacy, and lower pain catastrophizing. self - compassion also predicted more adaptive reactions to having hiv, including better adjustment and lower levels of stress, anxiety, and shame (31). such studies suggest that more self - compassionate people may treat themselves more kindly, recognize that their problems are common aspects of human experience, and, as a consequence, be less ruminative and self - judgmental when confronting negative or difficult feelings (32,33). on the other hand, the opposites of self - compassion, including self - criticism, self - hate, self - judgment, and negative perfectionism, have been linked to greater psychological distress, including depression (16). one recent study in chronically ill patients found that self - criticism predicted depression, illness - related stress, and diminished quality of life (34). other nonpatient (college student) data likewise suggest a key role for self - criticism ; self - reported physical symptoms increased as self - judgment increased and self - kindness decreased (35). self - judgment may be particularly important to investigate in relation to diabetes patients given the daily demands of managing the illness and the difficulty in achieving ideal metabolic control, which seems a likely trigger for self - criticism (15). a second indirect pathway by which self - compassion may improve physical health among diabetes patients is as a consequence of greater self - care (36). evidence for this possibility can be seen in research, indicating that people reporting greater self - compassion may take greater responsibility for their problems and be less overwhelmed by difficulties, suggesting that they are more likely to take care of themselves when ill or injured (37). for diabetes patients, it may be that taking care of oneself includes seeking medical treatment, maintaining regular physical activity, maintaining appropriate dietary behavior, having regular foot checks, and self - monitoring blood glucose. this suggestion is consistent with data from a study with type 2 diabetes patients showing that depression predicted a decline in the self - care routines that protect against poor glucose control and the development of complications (12). provocatively, egede and osborn (12) suggest that the link between depression and poor self - care is mediated by a decline in the motivation to maintain self - care. a series of experimental studies in nonpatient groups (38) adds weight to this suggestion, showing that self - compassion leads to increased self - improvement motivations, possibly because it provides a nonjudgmental context in which to appraise one s strengths and weaknesses and to strive to improve without the threat of unhelpful self - criticism. in the context of diabetes, enhancing self - compassion thus may enable patients to address evidence of less - than - ideal control without paralyzing self - criticism and help them reappraise and adjust their goals in support of good control. the idea that self - compassionate individuals may be more motivated to take care of their health out of a desire to maximize well - being is also consistent with studies of several health behaviors that are directly related to outcomes among patients with diabetes. for example, interventions to maintain diet (39) and exercise (40) and to quit smoking (41) have demonstrated the positive influence of self - compassion on affect and behavior. (41) found that smokers who were highly self - critical were helped to achieve their goals with an intervention that stimulated warmth and understanding while they attempted to quit. these authors suggest that for highly self - critical people in particular, self - compassion might inhibit a reflex toward rumination and self - judgment when faced with setbacks, helping them tolerate the distress they experience while trying to quit and enabling self - regulation via the soothing system rather than the threat system (14). a further intervention study (39) found that a self - compassion induction helped reduce negative self - evaluation, distress, and subsequent food consumption among highly restrictive eaters compared to dieters in a control condition. participants in the self - compassion condition were able to hold eating goals in mind without ruminating or allowing negative evaluation to interfere with their eating goals. alterations in self - management when confronting failures may be highly relevant to diabetes patients, who are generally required to monitor food intake as part of controlling blood glucose and can be assumed to experience setbacks as part of daily life. finally, another study (40) found that self - compassion predicted superior exercise - related outcomes among women. specifically, self - compassion was related to greater intrinsic motivation (behaviors initiated and regulated through choice as an expression of oneself) and lower external motivation (behaviors pressured by environmental forces) (42). in sum, these initial studies are encouraging in that they illuminate the potential utility of self - compassion based interventions in improving some of the health behaviors that routinely challenge diabetes patients. finally, a theoretical model (43) suggests a third indirect pathway by which self - compassion may be linked to improved health as a consequence of broad improvements in motivational management and self - regulation (44). successful self - regulation involves selecting goals, engaging in behaviors that support achievement of those goals, monitoring goal progress, and adjusting those goals when sufficient progress is not being made all of which are highly relevant to diabetes self - care. it seems likely that more self - compassionate people may therefore attend to their own self - care out of a desire to treat themselves kindly and well by engaging in behaviors that support their ultimate good. they may, for example, set more specific, achievable, and appropriate goals related to diet, exercise, and blood glucose targets ; prioritize attendance to these goals perhaps through regular medical appointments ; adhere to regular medication and testing regimens ; disengage from diet and exercise goals that are not working ; and establish new behavioral targets associated with better control. evidence of this can be seen in a study (37) with a large sample of participants (n = 241) with a range of serious medical conditions that found that self - compassionate people were more likely to take action with regard to their medical problems, including promptly visiting health care professionals when needed. further analyses suggest that the benefits of self - compassion on outcomes results from the combined influence of benevolent self - talk, a motivation to treat oneself kindly, and a tendency to be proactive with regard to one s health. early studies indicate that depression may complicate, or possibly contribute to, the cause of diabetes due to direct, reciprocal effects on endocrine and other physiological processes. these effects include abnormalities of the hypothalamic - pituitary - adrenal axis, changes in sympathetic nervous system functioning as measured by decreased heart rate variability (hrv), and increased release of inflammatory cytokines (11,18,19). support for a possible biological link between depression and diabetes was demonstrated in a recent meta - analysis (20) involving 24 studies that found patients with depression had significantly higher concentrations of tumor necrosis factor- and interleukin-6 (il-6) levels compared to nondepressed subjects. in turn, markers of inflammation such as il-6 have been proposed to be involved in diabetes disease onset (21). interestingly, emerging evidence suggests that self - compassion might be linked to improved mood through similar or related metabolic and autonomic processes, including inflammatory and sympathetic nervous system responses to stress. cross - sectional reports show that self - compassion is associated with lower blood plasma levels of il-6 (22), as well as improved autonomic nervous system responding to stress (23), as measured by increased hrv (24). such data are consistent with the theoretical proposition that self - compassion may calm the threat system, which is associated with defensiveness and autonomic arousal, and activate the self - soothing system (14). a series of experimental studies suggests that quantifiable physiological and neurological processes underlie the experience of self - compassion. (25) used functional magnetic resonance imaging to demonstrate that the specific neuronal networks associated with love and affiliation were activated after the experimental stimulation of compassion. participants also demonstrated an increase in positive affect ; compared with a memory control, participants who reacted with negative affect when witnessing others in distress before compassion training subsequently showed increased positive affective experiences after training. (26) also found that compassion training increased both altruistic behavior and neural responses to suffering, including activation of the inferior parietal cortex and dorsolateral prefrontal cortex. taken together, these early fin - dings suggest that activation of attachment and affiliation circuitry through self - compassion may be linked to particular direct processes involving autonomic and metabolic pathways that may be common to both depression and diabetes. although depression and self - compassion may be associated with processes directly linked to physical health, a number of cross - sectional and experimental studies in both patient and nonpatient populations suggest that compassion may alter psychological and behavioral processes leading to reductions in depressed affect and better self - regulation. first, consistent evidence suggests that self - compassion is related to physical and psychological health as a result of reduced negative affect (17,2729). for example, in a study in patients with obesity and pain problems (30), self - compassion predicted lower negative affect, higher positive affect, more adaptive pain coping, higher pain self - efficacy, and lower pain catastrophizing. self - compassion also predicted more adaptive reactions to having hiv, including better adjustment and lower levels of stress, anxiety, and shame (31). such studies suggest that more self - compassionate people may treat themselves more kindly, recognize that their problems are common aspects of human experience, and, as a consequence, be less ruminative and self - judgmental when confronting negative or difficult feelings (32,33). on the other hand, the opposites of self - compassion, including self - criticism, self - hate, self - judgment, and negative perfectionism, have been linked to greater psychological distress, including depression (16). one recent study in chronically ill patients found that self - criticism predicted depression, illness - related stress, and diminished quality of life (34). other nonpatient (college student) data likewise suggest a key role for self - criticism ; self - reported physical symptoms increased as self - judgment increased and self - kindness decreased (35). self - judgment may be particularly important to investigate in relation to diabetes patients given the daily demands of managing the illness and the difficulty in achieving ideal metabolic control, which seems a likely trigger for self - criticism (15). a second indirect pathway by which self - compassion may improve physical health among diabetes patients is as a consequence of greater self - care (36). evidence for this possibility can be seen in research, indicating that people reporting greater self - compassion may take greater responsibility for their problems and be less overwhelmed by difficulties, suggesting that they are more likely to take care of themselves when ill or injured (37). for diabetes patients, it may be that taking care of oneself includes seeking medical treatment, maintaining regular physical activity, maintaining appropriate dietary behavior, having regular foot checks, and self - monitoring blood glucose. this suggestion is consistent with data from a study with type 2 diabetes patients showing that depression predicted a decline in the self - care routines that protect against poor glucose control and the development of complications (12). provocatively, egede and osborn (12) suggest that the link between depression and poor self - care is mediated by a decline in the motivation to maintain self - care. a series of experimental studies in nonpatient groups (38) adds weight to this suggestion, showing that self - compassion leads to increased self - improvement motivations, possibly because it provides a nonjudgmental context in which to appraise one s strengths and weaknesses and to strive to improve without the threat of unhelpful self - criticism. in the context of diabetes, enhancing self - compassion thus may enable patients to address evidence of less - than - ideal control without paralyzing self - criticism and help them reappraise and adjust their goals in support of good control. the idea that self - compassionate individuals may be more motivated to take care of their health out of a desire to maximize well - being is also consistent with studies of several health behaviors that are directly related to outcomes among patients with diabetes. for example, interventions to maintain diet (39) and exercise (40) and to quit smoking (41) have demonstrated the positive influence of self - compassion on affect and behavior. (41) found that smokers who were highly self - critical were helped to achieve their goals with an intervention that stimulated warmth and understanding while they attempted to quit. these authors suggest that for highly self - critical people in particular, self - compassion might inhibit a reflex toward rumination and self - judgment when faced with setbacks, helping them tolerate the distress they experience while trying to quit and enabling self - regulation via the soothing system rather than the threat system (14). a further intervention study (39) found that a self - compassion induction helped reduce negative self - evaluation, distress, and subsequent food consumption among highly restrictive eaters compared to dieters in a control condition. participants in the self - compassion condition were able to hold eating goals in mind without ruminating or allowing negative evaluation to interfere with their eating goals. alterations in self - management when confronting failures may be highly relevant to diabetes patients, who are generally required to monitor food intake as part of controlling blood glucose and can be assumed to experience setbacks as part of daily life. finally, another study (40) found that self - compassion predicted superior exercise - related outcomes among women. specifically, self - compassion was related to greater intrinsic motivation (behaviors initiated and regulated through choice as an expression of oneself) and lower external motivation (behaviors pressured by environmental forces) (42). in sum, these initial studies are encouraging in that they illuminate the potential utility of self - compassion based interventions in improving some of the health behaviors that routinely challenge diabetes patients. finally, a theoretical model (43) suggests a third indirect pathway by which self - compassion may be linked to improved health as a consequence of broad improvements in motivational management and self - regulation (44). successful self - regulation involves selecting goals, engaging in behaviors that support achievement of those goals, monitoring goal progress, and adjusting those goals when sufficient progress is not being made all of which are highly relevant to diabetes self - care. it seems likely that more self - compassionate people may therefore attend to their own self - care out of a desire to treat themselves kindly and well by engaging in behaviors that support their ultimate good. they may, for example, set more specific, achievable, and appropriate goals related to diet, exercise, and blood glucose targets ; prioritize attendance to these goals perhaps through regular medical appointments ; adhere to regular medication and testing regimens ; disengage from diet and exercise goals that are not working ; and establish new behavioral targets associated with better control. evidence of this can be seen in a study (37) with a large sample of participants (n = 241) with a range of serious medical conditions that found that self - compassionate people were more likely to take action with regard to their medical problems, including promptly visiting health care professionals when needed. further analyses suggest that the benefits of self - compassion on outcomes results from the combined influence of benevolent self - talk, a motivation to treat oneself kindly, and a tendency to be proactive with regard to one s health. although research to date on self - compassion has not specifically examined its possible utility in diabetes patients, several considerations suggest that findings linking self - compassion to improved psychological and physical health, through both direct and indirect processes, could be of particular relevance to this group. first, the daily struggle of diabetes patients to maintain adequate control is frequently characterized by negative feelings of stress, anxiety, guilt, and shame (15), particularly when patients are continually reminded that their long - term health is dependent on their ability to self - manage. second, maintaining good control involves ongoing adherence to a relentless daily self - management regimen to maintain optimal health and reduce the risk of long - term complications. self - compassion may support adherence by reducing distressing and demotivating cognitive and emotional responses to medical problems such as self - blame, nonacceptance, and anger (43,45). negative emotions often accompany a diagnosis of a chronic, incurable illness such as diabetes (15), and these emotions in themselves may be linked to self - regulatory failure (31). third, self - compassion may be linked to improved adherence through increased conscientiousness (32). it may be that the emotional stability provided by self - compassion helps engender more responsible behavior, even though taking care of one s health by maintaining a good diet and exercising frequently might initially involve a certain amount of displeasure. overall, the relationships between diabetes, psychological distress, depression, and self - care are complex, and the pathways by which they are related are not yet fully understood. what is clear, however, is that depression and distress play an important role in increasing the suffering and complications associated with managing this chronic condition (10,18) and that current approaches are having mixed success. given the links between self - compassion and improved psychological and physical health, self - compassion based interventions may be a supplementary approach with the potential to reduce suffering and its negative effects on physical health outcomes among diabetes patients. nascent research suggests that intervening to increase self - compassion, a construct that appears to tap into brain structures adapted for self - soothing and calming, may have quantifiable physiological, psychological, and behavioral effects that may help diabetes patients cope better with their condition and enjoy improved quality of life. in particular, the bulk of research to date suggests that processes involving a reduction in negative self - evaluation and improved motivations for self - care may underpin the beneficial effects of self - compassion on physical and psychological health. thus, the utility of self - compassion as an intervention to reduce self - criticism, improve mood, and increase motivation to maintain self - care among diabetes patients may be worthy of substantial further investigation. | depression and severe psychological distress are frequently comorbid with diabetes and are associated with reduced adherence to medication and healthy lifestyle regimens, poorer glycemic control, and increased complications. the mixed success of existing treatments for depression in diabetes patients suggests a need for supplementary approaches to this common problem. this article reviews recent evidence for the benefits of self - compassion in chronically ill patients, suggesting its utility as a clinical tool for improving self - care, depression, and glycemic control in diabetes. possible physical and psychological pathways by which self - compassion may promote better outcomes in diabetes patients are considered, with particular attention given to reductions in negative self - judgment and improved motivation to undertake self - care. |
leptin is a hormone secreted from the white adipocytes and plays a role in the maintenance of energy homeostasis. although the role of ghrelin in energy homeostasis is still unclear, its secretion depends largely on nutritional state, with ghrelin concentrations increasing prior to a meal and decreasing after eating. both chronic and acute sleep restriction cause a reduction in serum concentrations of leptin and an increase in serum ghrelin concentrations [27 ]. subjects who had a 4-hour sleep opportunity for 6 consecutive nights had 19% lower mean and 26% lower maximum concentrations of leptin.. showed that those subjects who habitually slept 5 hours compared to 8 hours per night had 15.5% lower leptin concentrations. sleep restriction is also associated with an increased sense of hunger [3, 7 ]. leptin concentrations exhibit a strong circadian pattern, with the highest concentrations occurring at night and the lowest concentrations in the middle of the day. it has been shown that neither feeding time nor adrenalectomy affected the rhythmicity of leptin release. ablation of the suprachiasmatic nuclei (scn) in rats, where the circadian pacemaker is located, however, eliminated leptin circadian rhythmicity in rodents, suggesting that the central circadian clock regulates leptin expression. ghrelin concentrations also exhibit a strong circadian pattern, with the highest concentrations occurring during the daytime hours and the lowest concentrations occurring at night. light / dark patterns, conveyed from the retina to the scn via the retinohypothalamic (rht) tract, are the major synchronizer of the scn to the 24-hour solar day. the scn controls the timing of production of a series of biomarkers, such as the timing of melatonin synthesis. melatonin, a hormone produced at night and in darkness, is believed to be a primary timing messenger for the entire body, providing seasonal and daily photoperiod information to every neural, physiological, and cellular system. nighttime exposures to certain light levels and spectra will cease or diminish the production of melatonin. in humans, nocturnal melatonin suppression is maximally sensitive to short - wavelength (blue) light peaking close to 460 nanometers (nm). light can also impact hormones and brain activity without affecting nocturnal melatonin production [10, 11 ]. figueiro and rea showed that exposures to both 470-nm (blue) and 630-nm (red) lights increased cortisol concentrations at night, while only blue light suppressed melatonin. in another study, figueiro. demonstrated that blue and red light exposures at night increased alertness, measured via electroencephalogram, showing that melatonin suppression is not required for light - induced nighttime alertness. fluctuations in body weight have been associated with changes in day lengths, suggesting a role of the biological clock in regulating metabolism. it has been shown that in middle - aged rats, daily melatonin administration (liquid diet containing 0.2 mcg / ml melatonin) decreased weight gain in response to a high - fat diet and decreased nighttime plasma leptin concentrations within 3 weeks of the treatment. thus, it seems that the circadian clock may play an important role in determining body weight, likely by influencing the expression and secretion of hormones, such as melatonin. the present study was an investigation of how different light spectra might counteract the impact of sleep restriction on concentrations of leptin and ghrelin in subjects who maintained a 5-hour sleep schedule for 5 consecutive days. since sleep restriction decreases leptin and increases ghrelin concentrations and since light has a strong influence on hormone production, such as melatonin production, it was conceivable to hypothesize that light modulate concentrations of leptin and ghrelin after sleep restriction. specifically, as in previous studies, it was hypothesized that, after sleep restriction, morning leptin concentrations collected in dim light would be lower and ghrelin concentrations would be higher than after the baseline week (8-hour sleep opportunity). it was further hypothesized that when subjects underwent a sleep - restricted schedule (5-hour sleep opportunity per night for 5 consecutive nights), exposure to narrowband long - wavelength (red), middle - wavelength (green), and short - wavelength (blue) lights would counteract the effect of sleep deprivation on these biomarkers by increasing concentrations of plasma leptin and decreasing concentrations of plasma ghrelin. if it could be shown that narrowband lights modulate concentrations of leptin and ghrelin, a secondary goal of the study was to determine whether their spectral sensitivities were similar to that of acute melatonin suppression, which is maximally sensitive to short - wavelength light. the mean standard deviation (sd) age of participants was 27.4 8.7 years and the mean sd body mass index (bmi) was 25.2 4.2 all subjects included in the study were screened for medical illness and previous gastrointestinal surgery, and all reported being nonsmokers and medication - free. shift workers or subjects who had traveled across time zones four weeks prior to the experiment were not accepted into the study. the institutional review board at rensselaer polytechnic institute approved the study and participants provided informed consent and were paid for their participation. subjects experienced four morning light - exposure conditions for two hours upon wakening : (1) 60 lux of red light (0.325 w / m, peak = 633 nm, full - width - half - maximum [fwhm ] = 14 nm) ; (2) 60 lux of green light (0.105 w / m, peak = 532 nm, fwhm = 34 nm) ; (3) 60 lux of blue light (0.585 w / m, peak = 475 nm, fwhm = 21 nm) ; (4) dim light conditions (< 0.5 lux at the eye of a 630-nm, red light). two light emitting diodes (leds) were mounted to each of the goggle lenses. prior to every experiment, the light goggles were calibrated in the laboratory using a spectroradiometer (model 2300i, action research, ottobrunn, germany and spectra - sense version 4.3.0, advanced fiber sensors inc., ann arbor, mi, usa) with a uv - vis optical fiber ending in a lambertian diffuser. the experiment was conducted over 9 weeks ; data were collected in the laboratory on alternate weeks starting at the end of week 1 (thursday evening). participants were required to maintain an 8-hour sleep schedule during the first baseline week. during weeks 3, 5, 7, and 9, participants were asked to maintain a 5-hour sleep schedule from sunday through thursday prior to data collection. during the sleep restricted weeks, subjects were asked to maintain the same wake - up times as the baseline week and they were required to go to bed 3 hours later than their bedtimes during the baseline week. six subjects (group 1) were asked go to bed at 10:00 p.m. and wake up at 6:00 a.m. and five subjects (group 2) were asked to go to bed at 11:00 p.m. and wake up at 7:00 a.m. during the 8-hour schedule weeks. the two groups were selected based on their typical wake - up times, which were always kept the same during the experiment. during the sleep - restricted schedule weeks, the same six subjects who had a wake - up time at 6:00 a.m. were required to stay awake until 1:00 a.m. and the other five subjects who had a wake - up time at 7:00 a.m. were required to go to bed at 2:00 a.m. during weeks 1, 3, 5, 7, and 9 subjects were asked to sleep at the laboratory on thursday evenings, and data collection occurred on friday mornings. during weeks 2, 4, 6, and 8, participants were required to maintain a regular 8-hour sleep schedule (similar to the baseline week) so that they could return to baseline ; they were not required to come to the lab at the end of those weeks. compliance was assured from data obtained from actigraphy and checked against their sleep log reports. the dimesimeter is a calibrated light meter that contains accelerometers and three sensors which record and store data in flash memory. light data from the photosensor channels were downloaded to a host computer and converted into photopic illuminance, circadian light (cla), and circadian stimulus (cs) levels. concisely, photopic illuminance is irradiance transformed by the photopic luminous efficiency function (v()), providing an orthodox measure of the spectral sensitivity of the human fovea. cla, based on nocturnal melatonin suppression, is irradiance weighted by the spectral sensitivity of the retinal phototransduction mechanisms stimulating the scn. cs is a transform of cla into relative units from 0, the threshold for circadian system activation, to 0.7, response saturation, and corresponds to relative suppression of nocturnal melatonin after one hour of light exposure for a 2.3 mm diameter pupil during the midpoint of melatonin production. participants reported for the first, baseline overnight session at 9:00 p.m. (group 1) and at 10:00 p.m. (group 2) and were given an 8-hour sleep opportunity from 10:00 p.m. to 6:00 a.m. (group 1) and from 11:00 p.m. to 7:00 a.m. (group 2). during weeks 3, 5, 7, and 9, participants reported to the laboratory at 11:00 p.m. (group 1) and midnight (group 2) and were kept awake until their scheduled bedtimes, after which they were given a 5-hour sleep opportunity in complete darkness. again, rest / activity patterns were analyzed and confirmed for compliance to the schedule. participants were not allowed to eat or drink from 12 hours prior to awakening until the last blood draw was completed on the following morning. caffeine and alcohol use was voluntarily restricted starting 24 hours prior to the overnight session in the laboratory. for the baseline, 8-hour sleep condition (week 1) and for the first 5-hour sleep - restricted condition (week 3), participants were in dim red light (peak = 630 nm ; less than 0.5 lux at the eye) for 120 minutes after morning awakening in the laboratory. for weeks 5, 7, and 9 participants were exposed to the red, green, or blue lights for 120 minutes upon awakening ; light spectrum (red, green, and blue lights) presentation was counterbalanced across subjects. for every morning session, the first blood sample was collected 60 minutes after awakening and subsequent samples were collected 90 and 120 minutes after awakening. blood was drawn into two 4 ml vacutainer tubes containing k2 edta 7.2 mg, and immediately centrifuged for 10 minutes at 3500 rpm at 4c. the plasma was separated and frozen at 20c until assayed. this protocol was followed for every laboratory session. plasma leptin and total ghrelin concentrations were measured in duplicate by radioimmunoassay using commercially available kits from millipore (billerica, ma, usa). the lower limits of detection were 0.75 ng / ml for leptin and 93 pg / ml for ghrelin. intra- and inter - assay precisions were 5% and 7% for leptin and 6% and 16% for ghrelin, respectively. five (sleep - duration / light - exposure combinations) by 3 (sample times) analyses of variance (anovas) were performed on both plasma leptin and ghrelin concentrations. post hoc, two - tailed student 's t - tests were performed to further investigate the significant main effects and interactions from the anovas using the unnormalized data. in addition, plasma leptin and ghrelin concentrations following the 5-hour sleep restriction in the dim - light condition were taken as the reference values for post hoc inferential statistical analyses. plasma leptin and ghrelin concentrations at 60, 90, and 120 minutes after 8-hour sleep / dim - light condition and concentrations after light exposures (red, green, or blue light) were normalized (ratio) to concentrations at 60, 90, and 120 minutes after the 5-hour sleep / dim - light condition. one - sample, two - tailed t - tests using the data collected at the three sample times were used to determine whether the changes in leptin and ghrelin concentrations for each of the other four experimental conditions were significantly different than those obtained under the 5-hour sleep / dim - light condition. an a priori criterion probability for a type i error was set at 0.05 for all post hoc statistical analyses. the anova using leptin concentrations revealed significant main effects of sleep - duration / light - exposure combinations (f4,40 = 3.3 ; p = 0.02) and of sample times (f2,20 = 7.8 ; p = 0.003). post hoc, two - tailed student 's t - tests revealed that leptin concentrations after the morning red light exposure were significantly greater than after the 8-hour sleep / dim - light condition (p = 0.009) and after the 5-hour sleep / dim - light condition (p = 0.001). the mean standard error of the mean (sem) and the median concentrations are presented in table 1. the anova using ghrelin concentrations revealed a significant main effect of sample times (f2,20 = 17.5 ; p < 0.0001) and an almost significant interaction between sleep - duration / light - exposure combinations and sample times (f8,80 = 1.8 ; p = 0.09). the mean sem ghrelin concentrations were 927 69 pg / ml after 60 minutes, 947 73 pg / ml after 90 minutes, and 961 72 pg / ml after 120 minutes. although the main effect of sleep - duration / light - exposure combinations was not statistically significant (f4,40 = 1.6 ; p = 0.2), ghrelin concentrations were the highest after the 5-hour sleep / dim - light condition and all three morning light exposure spectra reduced ghrelin concentrations. the mean sem and median ghrelin concentrations are listed in table 1. in order to determine the changes in leptin and ghrelin concentrations by light relative to the reference condition (5-hour sleep / dim - light condition), data collected at 60, 90, and 120 minutes for the other four experimental conditions (8-hour sleep / dim - light, 5-hour sleep / red - light condition, 5-hour sleep / green - light condition, and 5-hour sleep / blue - light condition) were normalized to the data collected at 60, 90, and 120 minutes during the 5-hour sleep / dim - light condition. one - sample two - tailed t - tests revealed that, with respect to the 5-hour sleep / dim - light condition, normalized leptin concentrations (average of the three samples collected at 60, 90, and 120 minutes) were significantly higher for the 8-hour sleep / dim - light condition (t1,32 = 2.9 ; p = 0.007) and for the (sleep - restricted) red - light (t1,32 = 5.7 ; p < 0.0001), green - light (t1,32 = 3.6 ; p = 0.001), and blue - light conditions (t1,32 = 3.0 ; p = 0.005). leptin concentrations following the 8-hour sleep condition were nearly 20% higher than those following the 5-hour sleep condition, both of which were obtained under dim light. exposure to the three morning light spectra increased leptin concentrations by more than 50% relative to the reference sleep - restricted / dim - light condition. one - sample two - tailed t - tests revealed that compared to the 5-hour sleep / dim - light condition, the normalized ghrelin concentrations (average of the three sample concentrations collected at 60, 90, and 120 minutes) were significantly lower after exposure to morning red (t1,32 = 3.3 ; p < 0.003) and green (t1,32 = 2.2 ; p = 0.04) lights, but not following the blue - light exposure (t1,32 = 1.0 ; p = 0.3) and not after the 8-hour sleep / dim - light condition (t1,32 = 0.6 ; p = 0.6). the present study is the first to demonstrate that narrowband, morning light exposures can modulate leptin and ghrelin concentrations, biomarkers associated with hunger, following sleep restriction. previous studies have shown that sleep deprivation decreased overall leptin concentrations by 18% and increased ghrelin concentrations by 28% [2, 3, 7 ]. the present results are consistent with the literature in that they reveal a decrease in leptin concentrations of 19% when subjects were sleep restricted. although ghrelin concentrations following sleep restriction were lower than following the 8-hour sleep / dim - light condition in the present study, these reductions were not statistically significant. the present results extend those in the literature by showing that light exposure in the morning can increase leptin and decrease ghrelin concentrations in sleep - restricted individuals although, again, the impact of morning light on leptin concentrations was more robust than on ghrelin concentrations. it is possible, however, that still higher light levels (e.g., morning daylight) are needed to reduce ghrelin concentrations. an experimental protocol similar to the one used here should be conducted to examine this possibility. it seems unlikely that the increases in leptin concentrations observed in the present study were the result of a circadian phase shift due to subjects ' delayed bedtimes. peak concentrations of leptin occur during the middle of the night, but leptin concentrations following the 5-hour sleep / dim - light condition were significantly lower than those in the 8-hour sleep / dim - light condition (and for all of the morning light exposure conditions). if a light - induced phase shift of the leptin rhythm had occurred before sleep during the later bedtimes, leptin concentrations in the morning would have been higher, not lower as observed. moreover, the light measurements from the wrist actigraph showed that the median light levels between 10:00 p.m. and the participants ' bedtimes were below 12 lux of white light, which would result in a predicted melatonin suppression of less than 1%, suggesting that these evening light levels were too low to delay their circadian clock [15, 16 ]. since it has been shown that rats on a high - fat diet and treated with melatonin had a decrease in leptin concentrations compared to controls, the impact of light on leptin concentrations could have been mediated by light 's ability to suppress morning melatonin. in the present study, melatonin concentrations were not measured, so it is not possible to make a direct link between the observed effects of light on leptin and ghrelin and melatonin concentrations. although it can not be ruled out that light - induced melatonin suppression also may play a role in mediating these effects as shown in animal models, the present study logically suggests that light - induced increases in leptin concentrations and decreases in ghrelin concentrations were not dependent upon acute melatonin suppression. the level of morning red light exposure used in this study was not high enough to suppress melatonin, but, like the blue- (and green-) light exposure, it was sufficient to modulate leptin concentrations. the same, or a parallel, effect may have been observed by figueiro and colleagues [10, 11 ]. they showed that the same level of red light used in the present study increased cortisol concentrations and alertness, as measured by electroencephalogram [10, 11 ]. possibly the same neural mechanisms mediating the observed light - induced alertness and observed increase in cortisol concentrations are also mediating the effects obtained in this study. although we can not determine the spectral sensitivities of light - induced leptin and ghrelin modulation in this experiment, the fact that the morning red light had a significant impact on both leptin and ghrelin concentrations strongly suggests that the long - wavelength cones contribute to these acute responses ; there is no other known photoreceptor in the retina that would have sufficient sensitivity to red light to evoke the observed effects. a more extensive study should be performed to determine the spectral sensitivities of the light - induced increases in leptin and decreases in ghrelin before we can conclude that this is a cone response. moreover, it is not known if color information relayed to the brain, rather than retinal irradiance levels, per se, is mediating these effects. studies suggest that colors seen as warm (red, orange, yellow) evoke feelings of arousal while colors seen as cool (violet, blue, green) are associated with calming feelings. the color red, for example, has been associated with feelings of danger, love, rage, and excitement as well as negative feelings such as anxiety, anger, and annoyance [1722 ]. it has been suggested that the color red increases human receptiveness to external stimuli and increases excitation, therefore affecting a person 's emotional and motor responses [1722 ]. it is conceivable that these subjective interpretations of color may be mediating the physiological changes in leptin and ghrelin concentrations. the present results need to be replicated in a larger group of subjects, but they suggest that light can have a significant impact on reducing hunger that accompanies sleep deprivation. it has been shown that obese individuals have higher leptin concentrations than lean individuals and are more resistant, or have a greater tolerance, to the effects of leptin. animal studies should be performed to determine the pathways via which light is modulating these biomarkers and if light can change leptin resistance in obese animals. whether light can be used as a nonpharmacological tool to clinically impact obesity should be the subject of future research. | acute and chronic sleep restrictions cause a reduction in leptin and an increase in ghrelin, both of which are associated with hunger. given that light / dark patterns are closely tied to sleep / wake patterns, we compared, in a within - subjects study, the impact of morning light exposures (60 lux of 633-nm [red ], 532-nm [green ], or 475-nm [blue ] lights) to dim light exposures on leptin and ghrelin concentrations after subjects experienced 5 consecutive days of both an 8-hour (baseline) and a 5-hour sleep - restricted schedule. in morning dim light, 5-hour sleep restriction significantly reduced leptin concentrations compared to the baseline, 8-hour sleep / dim - light condition (t1,32 = 2.9 ; p = 0.007). compared to the 5-hour sleep / dim - light condition, the red, green, and blue morning light exposures significantly increased leptin concentrations (t1,32 = 5.7 ; p < 0.0001, t1,32 = 3.6 ; p = 0.001, and t1,32 = 3.0 ; p = 0.005, resp.). morning red light and green light exposures significantly decreased ghrelin concentrations (t1,32 = 3.3 ; p < 0.003 and t1,32 = 2.2 ; p = 0.04, resp.), but morning blue light exposures did not. this study is the first to demonstrate that morning light can modulate leptin and ghrelin concentrations, which could have an impact on reducing hunger that accompanies sleep deprivation. |
in the following chapter we summarize patterns of medication use for adhd and describe what is known about factors influencing adherence to adhd medication. choices about using medication are complex, with more recent work focusing on beliefs and attitudes that shape patient preferences. in addition, effectiveness and adverse effects are important factors and we review what is known about the differences among commonly used adhd medications. we end with a discussion of the challenges and related opportunities facing clinicians who work with young people with adhd. the field remains under - researched with much more to learn about how to assist greater numbers of young people with adhd to maximize their potential.table 1factors associated with adhd medication adherencefactorpredictors of increased adherencepredictors of decreased adherenceparent / familytwo - parent families older parents higher socioeconomic status increased parent - child conflict [1, 40]belief that medication is safe [33]belief that symptoms are not a disorder [12, 38]belief that adhd is neuro - biological disorder distrust of the medical system [12, 38]stigma [12, 38]burden of medication regimen concerns about medication safety [33]healthcare system/ professionalinsurance coverage cost of medication specialty care lack of providers in the community [24, 46]prior history of medication treatment [12, 38]good relationship with doctor childcaucasian racial background [20, 21]older child age at diagnosis increased symptom severity [12, 38]family history of adhd combined subtype severe behavior problems at home [12, 40]comorbidities present (oppositional defiant disorder, depression, social skills, anxiety, developmental delay, learning disabilities) [12, 38]child unwilling [12, 40]adolescentacademic benefits [33]negative attitudes toward medication [33]few adverse effects [33, 41]stigma [12, 33, 42, 44]concerns about treatment dependence experience of social withdrawal [16, 42]medicationlong - acting formulations [20, 25, 26]medication ineffectiveness adverse effects (physiological / psychological) [1, 42]multiple daily doses difficulties adjusting the dose regimen the evidence regarding direction of influence is not consistent factors associated with adhd medication adherence the evidence regarding direction of influence is not consistent attention deficit hyperactivity disorder (adhd) is a neurobiological disorder, characterized by symptoms of inattention, overactivity and impulsivity. adhd is estimated to affect 5 % children worldwide and boys are classified with adhd approximately twice as frequently as girls and primary school age children approximately twice as frequently as adolescents. throughout childhood and adolescence, clinically significant adhd is often associated with concurrent oppositional and aggressive behaviors, also anxiety, low self - esteem, and learning disabilities. while adhd begins before children enter school, it is most commonly identified and treated in primary school, ages 7 to 9 years old. in the preschool age group, adhd is characterized not only by impairment in attention span, excessive impulsivity, but also is frequently accompanied by additional disruptive behavior symptoms, including severe temper tantrums, demanding, uncooperative behavior and aggressiveness. while levels of symptoms decrease with age, the majority of children with adhd continue to show impairment relative to same - age peers throughout adolescence and into adulthood. many studies have documented increased risk for youth with adhd for leaving school early, increased contact with the law, early onset substance use, and associated conduct, mood and anxiety disorders. more recently studies have also documented higher risk for dangerous driving, suicidal behavior, problem gambling, eating disorders and early parenthood [26 ]. over the years, ` adhd interventions have largely targeted children in the primary school age group `, with the hope and expectation that early treatment will diminish such poor psychosocial and mental health outcomes in adolescence. however few studies exist that examine long - term effectiveness of interventions with follow up beyond 12 months. overall outcomes in adolescence remain less than optimal with many youth continuing to show functional impairment even where stimulant medications have been combined with intensive behavioral interventions. a few studies suggest that continued medication use during adolescence improves academic outcomes and postpones substance initiation [810 ]. however it is difficult to distinguish between medication treatment and other family, socioeconomic and psychosocial factors that may support increased use of healthcare services. interestingly, a recent population based study from sweden among persons older than 16 years, shows that periods of medication use are associated with diminished contact with the law, and non - use is associated with a return to illegal activities. this study demonstrates effectiveness of medication interventions for an important functional outcome, but also underlines that the benefits of medication are not maintained when treatment is discontinued. there are primarily two types of pharmacological agent with proven efficacy over 12 months for adhd : psychostimulants, (e.g., methylphenidate, or amphetamine derivatives such as dextroamphetamine and mixed amphetamine salts) and a norepinephrine reuptake inhibitor, atomoxetine. psychostimulants continue to provide control of adhd symptoms and are generally well tolerated for months to years at a time, although adverse effects, most commonly decreased appetite, sleep disturbances, headaches and stomachaches, may continue to be present. methylphenidate improved adhd symptoms and overall functioning alone or in combination with psychosocial / behavioral interventions for 14 months and up to 24 months [7, 14 ]. the benefits and safety of methylphenidate for symptom control and general functioning are most clearly documented, for boys, ages 7 to 9 years at initiation, with adhd combined type. however the benefits of psychostimulants wear off when the medication is not taken as the pharmacological half life is 3 to 5 hours in immediate release medication. best practice guidelines recommend that for full benefit both in school and at home, children with adhd should use these agents all day every day. children who use psychostimulants, often continue treatment for three years or more, although many discontinue use once they reach adolescence [16, 17 ]. rates of clinical identification and medication treatment for adhd have increased over time from the early 1990s to 2005 or later. rates of medication use have increased due to increased duration of use as well as increased use among girls, preschool children, adolescents and adults. rates vary by geography, provider type and patient characteristics as well as formulation of pharmacological agent. diagnosis and treatment is more common in the united states than elsewhere world - wide, with approximately 1/3 of children and youth given a diagnosis of adhd receiving consistent treatment within the past year. more prescriptions, primarily pscyhostimulants, are given to patients in the south and midwest regions of the united states compared with the northeast. within the united states, caucasian children and those from higher income groups are more likely to receive prescriptions and to use medication consistently than children and youth from minority background [20, 21 ]. the most common age to begin medication is between 5 and 9 years, more among boys than girls. one quarter to one third of patients receive a single prescription [23, 24 ]. treatment often is short - term or intermittent, with one half to two thirds discontinuing within one year [22, 23 ]. once - daily extended release preparations improve the duration of stimulant use compared with immediate - release agents [20, 25, 26 ]. children diagnosed by psychiatrists are less likely to receive a prescription within the initial six months after diagnosis than those identified by primary care physicians, even after adjustment for comorbid conditions. psychiatrists are more likely to provide titration of stimulant doses with a lower initial dose, a higher maximal dose, and three or more visits in the first 90 days, suggesting increased monitoring. presence of comorbid disorders, especially bipolar disorder, schizophrenia, or autism decreased the use of adhd drug use, but increased the use of other categories of psychotropics, prescribed primarily by psychiatrists and neurologists. the techniques for medication initiation used by specialists may be one reason that specialist care leads to increased duration of use over time. while efficacy and safety of pharmacological agent are important for continued use, the high - rate of non - refill following initial prescription, as well as the variability by geography, provider characteristics and the child s age, ethnicity, and socio - economic status suggests a nuanced approach to medication adherence is warranted. patterns of continued medication use for adhd vary considerably, depending on sample characteristics and method of measurement. in community samples using prescription databases, such as those described in the preceding paragraph, the mean time to discontinuation ranges from 3 to 5 months, however, when single prescriptions are removed, duration of use is considerably longer [17, 29, 30 ]. cohort studies following participants enrolled in industry sponsored trials document low rates of discontinuation due to adverse effects, 12 % over 12 months on oros methylphenidate, 15- 21 % over 24 months on mixed amphetamine salts, 11 % discontinued atomoxetine over more than 2 years. consistent use was documented for 86 % of participants at 12 months in a cohort study examining effectiveness of oros methylphenidate. in general, estimates from surveys and clinical samples suggest that 36 - 68 % of children consistently use adhd medications once initiated ; consistent use declines over time from approximately 50 % at 2 years to 36 % at 5 years. fluctuations in use are common ; some children use medications regularly, others discontinue, and still others stop and restart medications, sometimes more than once [13, 17 ]. alongside the multiple patterns of ongoing medication use, there often are multiple changes in dose and formulation. while treatment guidelines may encourage daily use, parent interviews and self report surveys and cohort studies suggest that up to 40 % of families prefer to target behavior and learning at school and do not use medications on weekends and during school vacations. empirically based models of health behavior offer the opportunity to examine social, cognitive, and experiencial factors that influence medication adherence, cognitive behavioral models such as the health beliefs model take into account characteristics of the specific disorder, patient beliefs and attitudes, and the efficacy and safety profile of the medication, while a model such as the trans - theoretical model of change, describes the importance of experience and associated changes in attitude over time. while the concept of treatment adherence has evolved in the past 20 years, it remains poorly operationalized and measured, leading to a variety of interpretations by patients and providers [32 ]. for young children with adhd, healthcare decisions are usually made by the parent and therefore the parent s perceptions and experiences are of central importance. in general, medication adherence connotes the child s and parent s participation and engagement in using a medication regimen believed to be beneficial by both the family and the clinician who prescribes. the therapeutic partnership maintained by ongoing patient - provider transactions over time is essential for long - term adherence [32 ]. for a chronic condition such as adhd, where successful outcomes are more likely to occur with continued medication use, challenges can arise with development as the young person becomes an adolescent. ideally the patient - provider relationship also undergoes adjustments as the family transfers healthcare decision - making from the parent to the youth. however, adolescents are less willing to use medications for adhd than are their parents [33 ]. generally speaking we can organize factors that are associated with use of medication for adhd into parent or family characteristics, child characteristics, practitioner or health system factors and medication - related factors (table 1). parent beliefs about adhd and attitudes toward treatment are significant determinants of initiation. for example those parents who view the child s difficulties as a medical disorder that requires a biological intervention will agree with using medication and encourage long term use. many however prefer implementing behavioral strategies and other non - medication strategies, such as dietary changes, exercise or counseling [36, 37 ]. families are more accepting of a trial of stimulants when the diagnostic process has been thorough, and included comprehensive psychological testing. some parents feel that medication is unacceptable as treatment of adhd behaviors, and do not choose to use it for their child. many parents have mixed feelings about starting medication, and often weigh concerns about adverse effects and social disapproval against potential behavioral and academic benefits [12, 38 ]. increased knowledge about adhd, and associated attitudes that using medication is safe, effective and socially acceptable increases willingness to use medication [33 ]. however while knowledge may increase willingness to try medication treatment, it is not clearly associated with long term use. child characteristics that increase long term use of medication are younger age when starting, more severe adhd symptoms, additional learning and behavior conditions, and past experiences that medication is effective with few adverse effects [12, 38 ]. in general children are less likely to use medication when there are severe behavior problems at home and the child is unwilling [12, 40 ]. many children and youth on medication do recognize benefits for improved schoolwork and peer relationships [4143 ]. among adolescents, the belief that medication is effective, in combination with few experiences of adverse effects, increases willingness to use medication [33 ]. some youth feel ostracized socially due to adhd behaviors and therefore find taking medications helpful while others may avoid treatment because of fears medication use will create stigma [33, 42, 44 ]. university - aged young people with adhd described greater medication use when they made the treatment decision themselves, often with the goal to increase academic success. not surprisingly, as adolescents mature, their adhd symptoms become less obvious, and their priorities change, patterns of medication use for their adhd symptoms are likely to change. parents describe that a good relationship with their healthcare provider helped in the decision of starting treatment in their children. those who trust their health care provider and in general parents and pediatricians have a similar, but not identical, understanding of what adhd is, and the treatment priorities. families often consult with friends and relatives about the advice doctors provide ; where these informal consultations concur with the provider s advice to use medication, it is more likely to happen. barriers to ongoing medication use include the cost of prescriptions, while limited insurance coverage and taking time off work can interfere with ongoing monitoring and care. pediatricians note that providing care for children with special needs can be time - consuming, with additional phone calls to schools and mental health professionals outside of usual office hours. the physician s ability to clarify treatment preferences, discuss available options and maintain a treatment alliance with the family is crucial to success. ease of use, simplified dose regimen, and cost are also important [33 ]. the range of formulations for psychostimulants has burgeoned over the last decade, especially in the united states compared with other jurisdictions. a recent review of adhd treatment studies examined adhd medication use greater than 12 months, and found studies of four psychostimulant preparations (methylphenidate immediate release, amphetamine, oros methylphenidate, and mixed amphetamine salts) and a discontinuation trial evaluating atomoxetine. while best evidence suggests that psychostimulants preparations are all efficacious compared to placebo, very few head - to - head trials among stimulant preparations have been done. however, the once daily formulation of oros methylphenidate is more likely to be taken consistently than immediate release methylphenidate which requires multiple doses daily for efficacy [25, 26 ]. while the psychostimulants and atomoxetine both reduce adhd symptoms, and improve social and academic functioning in placebo controlled trials, not all children benefit equally well. another comparison of two agents examined oros methylphenidate and atomoxetine over several weeks, with the conclusion that oros methylphenidate is more efficacious for core adhd symptoms. overall a substantial proportion of treatment discontinuation in the first few months is due to adverse effects of medication. while families often may tolerate some adverse effects over months to years, such as decreased appetite or mild insomnia, other symptoms such as stomach aches, or headaches may be less tolerable. a long - term comparison of dextroamphetamine with methylphenidate formulations, suggests dextroamphetamine agents are less well tolerated by girls than boys. in particular, psychological side effect such as mood changes, irritability, depression and subdued personality are frequent reasons for discontinuation. increased moodiness and irritability appears to be more common among preschool children than older ones. other reasons for changing or discontinuing medications are slowing of the child s growth, development of tics, rashes and other concerns the young person or family attribute to the medication. psychotic symptoms, are estimated to occur adhd medications at a rate of 1.5 events / 100 person years and suicidal ideation is estimated to occur 5/1357 (0.37 %) in pediatric patients taking atomoxetine. the most frequently cited reasons for discontinuing medication are the family s experience that the medications are not effective or the child is experiencing intolerable adverse effects. the clinician should help the family have realistic expectations for how adhd medication can help, and provide frequent monitoring in the initial stages to address emerging adverse effects, as two important methods to improve medication adherence. the primary reason to examine adhd medication adherence is to provide the best care possible for children and youth with adhd so that long - term mental health, psychosocial, and economic outcomes are optimized. however despite extensive examination, solid evidence that medication interventions provide lasting long - term benefits remains elusive. this conundrum is now being addressed using large - scale population - based administrative databases, linking data about prescription renewal rates with health care and educational services information. using complex statistical methods, these studies show promise for describing community patterns of health care use and associated outcomes. on the other hand, the specifics of individual child characteristics, including clinical diagnosis, interpersonal relationships of families and providers, and co - interventions received are rarely captured, offering little practical guidance to front - line practitioners. the details required to inform day - to - day practice require alternative research methods. for example, studies using mixed qualitative and quantitative methods have begun to answer complex questions about treatment adherence relevant for front line practitioners. converging evidence from multiple samples offers surprisingly similar descriptions of parent attitudes and beliefs about childhood adhd and its treatment with medication. indeed, one very important challenge for physicians arising from this body of evidence is to acknowledge the limitations of medication as a sole treatment for adhd. parents, even those who rely on medication to assist their children, identify areas of burden, have ongoing concerns associated with its use, and articulate the need to investigate additional ways to help their child reach their full potential. another significant challenge to current practice is to include children as active participants in treatment decisions. this is not only good ethical practice but also the best way to engage them in treatment. the opinions and preferences of children and youth about their adhd treatment are not yet widely described, but progress is being made. young children certainly have less involvement in their treatment decisions than adolescents, but when they are unwilling to take medication, their parents experience the burden of conflict and in turn, medication is likely to be discontinued. developing a thorough understanding of young people s concerns about their mental health treatment, identifying and responding to their priorities and addressing their concerns is important for optimizing outcomes. over time inattention and overactivity may become less of a focus for intervention and other issues may emerge as more significant. maintaining the treatment alliance remains key as medication use may once again become a useful option at a later point in time. when thinking about the long - term course of mental health interventions for a young person with adhd, it can be important to recognize that early adhd symptoms are a risk marker for concurrent and subsequent difficulties. the initial psychoeducation about what is adhd and options for treatment is a crucial first step in providing care, but one that is not yet standardized. the process of seeking help for the behavior and learning problems associated with adhd is complex and families often depend on informal as well as formal sources of information within their community. therefore examining the best content to provide and the best ways to communicate it remains important. widely disseminated, accurate general knowledge about adhd may also diminish stigma and misconceptions about potential long - term consequences of medication. the general need for such mental health literacy extends to educators as well as health care workers and to other trusted persons to whom parents and youth go for answers. primary care practitioners play an important role as educators and gatekeepers in the process of identification and treatment of adhd. adhd medication treatment needs to be initiated in a way that encourages adherence and ongoing monitoring should be provided to maximize benefit and minimize adverse effects. as technology is integrated into the provision of healthcare services in general, it will also become useful for those with adhd. prior to technical development however, development and evaluation of standardized methods will be required. these may include decision aids to assist in informing parents and youth about options and enhance shared decision making, and monitoring algorithms with reminders to collect key information, address psychological as well as physiological adverse effects and evaluate for potential emergent concurrent disorders. pharmacies already use automated prescription renewal systems and these could be adapted for daily use by the parent or by the young person. experiences of adverse effects remain a significant and frequent reason for discontinuation of medications. as in other areas where personalized medicine is under discussion, there may be genetic biomarkers on the horizon to assist in identifying which agents are likely to be most effective and easier to tolerate for individual persons. increased understanding about how the brain changes with development and with prolonged treatment will bring new knowledge about how to ensure brain health over many years. medication is a key component of evidence - based care for children with adhd, and patterns of treatment adherence are complex. despite the fact that many young people show impaired functioning well into adolescence, many who begin medication treatment either stop and start medications over several years or discontinue use altogether. explanations for poor adherence to adhd medication treatment include patient perceptions expressing many negative responses to the recommendation of medication use. in addition, the healthcare provider often is working with more than one person in the family who contributes to decisions. not infrequently, patients and families perceive less than optimum effectiveness and tolerance of adverse effects, both of which lead to discontinuation. in this context, the clinician s ability to maintain a positive working alliance is crucial. also important is monitoring for concurrent or emergent difficulties ; these problems may become the priority rather than the adhd symptoms, perhaps requiring alternative or additional interventions. as we learn more about brain health and development, additional ways to help young people care for themselves will emerge ; we may see an increased focus on sleep hygiene, stress management, diet and exercise, factors known to support positive mental health. overall increased awareness of the need for shared decision making, with the young person as well as the parents and enhanced monitoring will remain key to enhancing adhd medication adherence and optimizing long - term outcomes. | safe and effective medication for attention deficit hyperactivity disorder (adhd) is available and recommended as first - line treatment for the core symptoms of inattention, overactivity and impulsiveness. despite impaired functioning during adolescence, many discontinue medication treatment. for children, healthcare decisions are usually made by the parent ; older youth make their own decisions. beliefs and attitudes may differ widely. some families understand that adhd is a neurobiological condition and accept that medication is indicated, for others, such treatment is unacceptable. converging evidence describes negative perceptions of the burden associated with medication use as well as concerns about potential short and long term adverse effects. indeed experiences of adverse effects are a frequent explanation for discontinuation among youth. ways to improve shared decision making among practitioners, parents and youth, and to monitor effectiveness, safety and new onset of concurrent difficulties are likely to optimize outcomes. |
zambia is a landlocked country located in southern africa with a population of about 10.8 million. fifty one percent of the population is made up of women and children. over 70% of the population lives below the poverty datum line. the gross per capita income is $ 630, and 13% of central government expenditure is allocated to health care (unicef zambia statistics). according to the zambia demographics and health survey in 2007, it is estimated that 14.3% of the sexually active age group (1549) is living with human immunodeficiency virus (hiv), 12.3% males and 16.1% females. children have been much affected by the hiv / aids epidemic in zambia, where over 30,000 children are hiv positive. while perinatal transmission accounts for the majority of new pediatric hiv infections, in countries such as zambia, where hiv prevalance is high, sexual exposure remains an important risk factor in children in the postweaning period. while hiv transmission rates attributable to sexual abuse are unknown, pediatric victims of sexual abuse are at a higher risk of hiv transmission due to physical trauma and due to the fact that multiple exposures often occur prior to discovery of the abuse in a pilot study conducted at the university teaching hospital (uth) in lusaka, zambia in 2003, 99% of sexually abused children reporting to the gynecology ward were females, which also places them at a higher risk for hiv acquisition. although epidemiologic data for the prevalence of child sexual abuse (csa) in zambia is not available, murray. found that csa is a significant concern in the community in lusaka. defilement was mentioned by 40% of women and 30% of children asked to list problems affecting children in the community. among the children. in 2007, slonim - nevo and mukuka surveyed 3,360 adolescents (defined as age 10 to 19 years) and found that 9% of adolescents reported a family member touching their breasts or genitals, 3% reported sexual intercourse, 2% reported oral sex, and 1% reported anal sex by a family member. females were more likely to have been touched sexually than their male counterparts, but males were more likely than females to have had sexual intercourse or oral sex with a family member. literature from countries surrounding zambia documents the existence of a csa epidemic in the region. prevalence studies rely on cross - sectional study design, most often surveying school children about their experiences of sexual abuse. in a review article of child sexual abuse in subsaharan africa, lalor. report that between 3.2 and 7.1% of all respondents report unwanted or forced sexual intercourse before the age of 18 years. jewkes. surveyed 11, 735 south african women between the ages of 15 and 49 years about their history of rape during childhood. overall, 1.6% reported unwanted sexual intercourse before the age of 15 years of age. 85% of child rape occurred between the age of 10 and 14 years and 15% between the ages of 5 and 9 years. in a study in zimbabwe, birdthistle reports that among unmarried, sexually active adolescents, 52.2% had experienced forced intercourse at least one time. 37.4% of first sexual intercourse acts were forced. in a study of 487 university students in tanzania, collings and madu surveyed a sample of 640 female university students in south africa and found that 34.8% had experienced contact sexual abuse before the age of 18 years. another study among high - school students in south africa, found that almost 20% were victims of parental or guardian sexual abuse. additional research suggests that the prevalence of child sexual abuse in subsaharan africa is similar to other countries across the world. in the second quarter of 2003, zambian police handled 300 cases of child rape, and some experts believe that for every case reported another 10 go unreported. the number of reported cases and the realization that these cases were likely to be the tip of iceberg, in combination with high hiv prevalence led to the identification of the need to establish a comprehensive multidisciplinary centre to increase public awareness of child sexual abuse and to improve management of sexually abused children with an emphasis on preventing hiv acquisition. in zambia, most reported child sexual abuse (csa) cases come to the attention of medical personnel because of symptomatic sexually transmitted diseases (stds). limited services were offered for sexually abused youth and no postexposure prophylaxis (pep) was available in the public sector. in 2003, a pilot study was conducted at the university teaching hospital (uth) to investigate the feasibility of giving pep to sexually abused children in zambia. the study was done within the department of obstetrics and gynaecology. in this study, 23% of eligible children were able to complete a 28-day course of pep. prior to this study, there was a lack of awareness of child sexual abuse and a lack of recognition of child sexual abuse cases. there were no protocols for how to address the needs of victims, and there was poor or no coordination between the various professionals involved in the management of sexually abused children. uth is situated in lusaka, the capital of zambia with a population close to 2 million. uth houses the only medical school in the country and the schools of registered nurses and midwifery. most of the professionals in zambia ; medical personnel, social workers, psychiatrists, psychologists, lawyers, and magistrate, are found in lusaka. it was therefore important that a one stop centre with a multidisciplinary approach be established in lusaka. the one stop centre would then act as a centre for developing appropriate protocols for the management of child sexual abuse in zambia as well as become a training institution for the rest of the country. the diagram below (figure 1) depicts the previous system for management of a child who had been sexually abused, along with some of the associated flaws and potential delays due to the lack of a centralized, coordinated service. when a child had been sexually or physically abused, the majority are reported either to the victim support unit within the police or, if the child had been physically injured or had a medical symptom, for example a genital discharge, or to a local health facility. a few children presented to a nongovernmental organization such as the young womens ' christian association (ywca). the processing goals of a child sexual abuse case involved care and protection of the child, investigation of the background to the abuse, and apprehension and prosecution of the offender. as a result, the child was likely to have been interviewed (and even examined or inspected) on more than one occasion, often by people without the requisite skills. all too often the result was that the child was further traumatized, and the guardian and child were put much inconvenience when both were already highly distressed. the need to visit multiple sites for evaluation also led to critical delays in the administration of pep as well as an increased risk of loss - to - follow - up. clearly, efforts towards the development of systems and training of professionals to more adequately work with sexually abused youth were in need. the literature suggests that one stop centres decrease the trauma experienced by the child and the caregiver [14, 15 ]. developing a centre that encompasses all aspects of care required for sexually abused children is likely to reduce the strain of reporting on families and assure proper follow - up care. the university teaching hospital in lusaka undertook the mission to develop a one - stop centre to address the multidisciplinary needs of sexually abused youth. this paper will present the process of implementing such a center in a low - resource environment, and discuss the challenges and lessons learned. the uth proposed an intervention with a multidisciplinary approach to increase and improve case reporting, management and services for child sexual abuse patients with special emphasis on hiv prevention. a management team was put in place composed of clinical heads from the departments of pediatrics, obstetrics and gynecology, and surgery. the team evaluated the management of sexually abused children prevailing in zambia and identified gaps and weaknesses in the medical management, legal framework, and media reporting. a technical team composed of members of the zambia society for the prevention of child abuse and neglect (zaspcan) comprising a doctor, a psychiatrist, a psychologist, a lawyer, a police officer, and a journalist was tasked to review zambian laws pertaining to child sexual abuse, review the existing protocols on the medical management of child sexual abuse, review the literature on management of traumatized children, and lastly, to review the reporting on child sexual abuse in both electronic and print media. after a comprehensive consultative process with local and international professionals, strengths and weakness of the existing system were identified. in order to address many of the problems identified with the system, the one - stop centre, a multidisciplinary clinic where families could access all necessary services in one child - friendly location, it was established that in order to implement the one - stop centre, there needed to be identification and training of the professionals who manage sexually abused children. a manual for the management of sexually abused children was produced which will be used to train all professionals staffing a one - stop centre. the draft manual was circulated to key personnel in the medical, psychosocial, police, legal, and media communities to review and validate the various components to see that that they were in compliance with both the social norms and standards of care as well as provided protection to the children. the medical interview and the physical examination of a sexually abused child were covered, as well as how to complete medical legal forms and the collection of forensic specimens. hiv testing and counseling, treatment and management of stis, medical complications seen with csa, and post - exposure prophylaxis (pep) administration were also included. emphasis was placed on rapid hiv identification and testing and counseling of those presenting within 72 hours of the abuse in order to provide prompt post - exposure prophylaxis (pep) (figure 2). the psychosocial component of the manual included safety / confidentiality procedures, psychosocial manifestations of sexual abuse, short - and long - term effects of the abuse, posttraumatic stress disorder (ptsd), disclosure and reasons for refusal to disclose the sexual abuse, and challenges in child counseling. included in this section were ; definitions of csa, definition of a child, children 's rights, how to treat child witnesses, ratification and domestication of international law instruments, dealing with child offenders, and how to preserve evidence. prior to the development of the manual, child sexual abuse was reported in the media without following any guidelines. though in other countries reporters do not form part of the team in child advocacy centres (cac), they were included in the technical team as hostile reporting was damaging children both physically and mentally. media representatives were also considered important in increasing public awareness of child sexual abuse to increase the number of cases that were being reported. once the manual was completed, trainings utilizing the new manual were conducted for the professionals who would be staffing the one - stop centre. as csa is widely believed to be underreported and most cases presented only after symptoms or complications developed, a series of public sensitization activities, including school debates, were conducted to increase public awareness of child sexual abuse and to increase awareness of the importance of early reporting and where to report. in most western countries, child advocacy centers (cacs) are not located within medical institutions and offer a more comprehensive package to include physical abuse as well as child neglect [17, 18 ]. we chose to establish the multidisciplinary centre within the pediatric department because most of the sexually abused children came to the attention of the health workers because of medical complications and in order to offer pep to abused children, which was only available at the uth. the centre would not provide services for isolated physical abuse cases nor neglected children. the one - stop centre was established in the pediatric department on 26th april 2006. a location was selected where there is minimal foot traffic and there are no conspicuous notices indicating its function to help preserve the confidentiality of the children and their guardians attending the center. the centre included a physical examination room and several interview rooms including one with a two - way mirror, microphone, and speakers which allows one person to interview (usually a medical person) the child whilst the police officers and counselors take notes from another room. special care was taken to provide comfortable and child - friendly waiting facilities (tv set, toys, and educational materials). since there is an extreme shortage of doctors, the clinical officer trained in forensic and medical examination abroad was appointed to coordinate the medical management at the centre. in western countries, the clinical officer is supported by a director who is a senior pediatrician and a middle - grade doctor. the clinical officer examines the child, prescribes medications as indicated for the sexually abused children, and refers to the consultant if assistance is needed. the centre is also staffed by one police officer from the victim support unit section of the local police, one social worker, and three nurses. to round out the multidisciplinary vision of the one - stop centre, the director and psychiatrist began working with boston university to add a range of psychosocial assessment tools to strive for comprehensive, multidisciplinary assessments as documented in the literature as the gold - standard in childs sexual abuse care. the assessments were chosen based on results from a local qualitative study conducted in lusaka as well as local input from psychiatrists, mental health professionals, nurses, and clinical officers. intake interviews are conducted with the caregiver and child separately (if the child is able). a medical / laboratory panel includes the following tests : rapid hiv antibody tests, rapid plasma reagin, pregnancy, hepatitis b, and forensic specimens (high vaginal swab for wet prep, gram stain and culture to identify gonorrhea, trichomonas, and spermatozoa). mental health assessments for the youth include the post - traumatic stress disorder reaction index, the strengths and difficulties questionnaire, and my feelings about the abuse. this last measure specifically examines the construct of shame, which is considered to be critical in the zambian culture. the mental health assessment administered to the caregivers about the abused child is the child behavior checklist. a systematic flow has been designed to promote excellence in the care of sexually abused youth. family registers at uth main desk and receives a treatment form the family is then directed to the one stop centre where they are greeted by the social worker and/or nurses counselors. youth and their caregivers are immediately asked if the abuse happened within the last 72 hours. if the abuse occurred within 72 hours of presentation and the child is hivnegative on rapid test, the child is eligible for pep. if abuse occurred within 72 hours, the child is immediately brought to a nurse to take the necessary blood tests, and administer pep if appropriate. if the child is pubertal, in addition to pep, they are given emergency contraception. after blood tests and pep administration, the intake forms and the questionnaire for assessment of level of trauma are completed by the nurse or social worker. a physical exam is completed by the clinical officer and/or the consultant, and the uth treatment form and police medical forms are completed.if abuse did not occur within 72 hours, the child / care - giver is interviewed by one of the staff, blood tests are performed, a physical exam of the child is conducted and the uth treatment and police form (issued at the centre) are completed by the clinical officer. the police officer stationed at the centre also completes the relevant potion of the police form.if a child is hiv positive, they are referred to the paediatric antiretroviral therapy (art) clinic for further assessment, management, and follow up.if a child is found to be pregnant, she is referred to the antenatal and/or prevention of mother to child hiv transmission (pmtct) clinic for further assessment, management, and follow up. if abuse occurred within 72 hours, the child is immediately brought to a nurse to take the necessary blood tests, and administer pep if appropriate. if the child is pubertal, in addition to pep, they are given emergency contraception. after blood tests and pep administration, the intake forms and the questionnaire for assessment of level of trauma are completed by the nurse or social worker. a physical exam is completed by the clinical officer and/or the consultant, and the uth treatment form and police medical forms are completed. if abuse did not occur within 72 hours, the child / care - giver is interviewed by one of the staff, blood tests are performed, a physical exam of the child is conducted and the uth treatment and police form (issued at the centre) are completed by the clinical officer. the police officer stationed at the centre also completes the relevant potion of the police form. if a child is hiv positive, they are referred to the paediatric antiretroviral therapy (art) clinic for further assessment, management, and follow up. if a child is found to be pregnant, she is referred to the antenatal and/or prevention of mother to child hiv transmission (pmtct) clinic for further assessment, management, and follow up. drugs used for pep were zidovudine 240 mgs / m in combination with lamivudine 4 mg / kg (combivir) twice daily for 28 days. no syrups were available initially leaving the very young children without any pep options until later when syrup formulations were made available. initially, a two - drug regimen was recommended as effective though currently a 3-drug regimen is in place in accordance with current guidelines. to gain support from local policy makers (parliamentarians, ministry of health, local and international organizations) several meetings were held to explain the concept of a one - stop centre to emphasize the need for multidisciplinary care for sexually abused youth and to request financial support for such a centre. a data collection and management system was developed with help from boston university, and a monitoring and evaluation specialist was put in place. monthly reports are provided to the uth as well as biannually to funders. in the period between january 2006 and december 2008 2863 the one - stop centre has improved the followup of children, with 52% of eligible children completing a 28-day course of pep, compared to 23% in the pilot study conducted in zambia in 2004 - 2005 (table 1). considering that one - stop centres do not exist in most poorly - resourced countries, the first step was to look at centres established in developed countries and see how they could be adapted to suit the local needs the environment and the limited resources available. unlike most, one - stop centres in developed countries, which are located away from hospitals [17, 18 ], the center was established within the hospital where most senior medical professionals are found. however, most often, they have to deal with the acutely ill and have little time to audit the performance of the centre. the most significant challenge continues to be a lack of both monetary and human resources in the setting of numerous competing demands. because of the gaps in the zambian medical training curriculum which does not include child sexual abuse topics, there was a lack of experienced local medical professionals available to conduct the trainings. for this purpose, consultants with clinical experience in managing sexually abused children were recruited from abroad to come and train the medical team, and selected members of the local team were sent for training abroad. the most difficult task was to find a team which was prepared to allocate time not only to training but also to spearheading the implementation process. these professionals were already overburdened with treating the severely ill due to the hiv / aids pandemic and had little time to take on other equally important duties. it is hoped that as the number of medical professionals increase and once a critical number of professionals have been trained, abused children will be able to receive services in the primary health centres, and the uth centre will assume a coordinating and training role and act as a referral centre for complicated cases. the establishment and training of the team would not have been possible without collaboration, funding, and technical assistance from international organizations and individuals. with their assistance, protocols to guide the operations of the one - stop centre were developed. for this, the local team reviewed available data, and with technical assistance from outside sources, adapted it to meet the local needs. the main goal of the one - stop centre was to protect sexually abused children from acquiring hiv infection. the drugs used for pep are those used in the treatment of hiv and aids. this is a huge challenge as currently there is a shortage of drugs for those who require treatment. it is therefore important that the national drug budget takes into account drugs for pep as this is an important strategy to prevent hiv infection. future research will need to explore other, more cost - effective regimens of drugs to be used for pep in poor resource settings, as was done in the pmtct program. single - dose nevirapine and short - course zidovudine regimens were identified which were more cost - effective, but also efficacious at preventing maternal to child hiv transmission. follow up of children to ensure their completion of a 28-day course of pep is a great challenge. currently, when a child qualifies for pep, a 7-day course of drugs is given, and the child is advised to come for review a week later or earlier if there are any side effects. upon review, if the child has taken the medication and has had no adverse effects, he / she is given the remaining 21-day course of drugs and scheduled for review again at the completion of treatment. even though followup improved from 23% to 52% with the establishment of the one - stop centre, few children report back on day 28, and negligible numbers return at 3, 6, and 12 months to repeat hiv testing as per protocol. various methods have been used to encourage the initial 7- and 28-day reviews, such as reminder phone calls and diary cards, with limited improvement (table 1). one potential barrier to followup is lack of money for transport to the uth, which is often far from the child 's home. it is hoped that once services have been decentralized to the primary health centres which are based in the community, follow up will improve as it will reduce transport costs to and from uth. police and legal services are grossly limited by shortage of transport and resources, including human resources, required for effective forensic investigations. the legal system is hostile to an abused child in that there are no child - friendly courts, most prosecutors are not familiar with csa, and doctors are not keen to give expert opinion in court. the one stop centre has been trying to address these issues by conducting trainings and seminars for all those involved in the prosecution of child sexual abuse. one stop centres have proved to be effective in improving the management of sexually abused children [2022 ]. this paper demonstrated a process used to develop such a centre in a low - resource environment. in order to establish a one stop centre in a developing country, it is important to get the support of the relevant stakeholders (policy makers, lawyers, magistrates, police, health workers, and influential networks in the communities). mobilization of financial resources is essential in the initial stages as most medical systems in developing countries are overburdened with acute illnesses with no resources to invest in preventative strategies such as hiv / aids. the one - stop centres should be established within a health institution where the majority of patients initially present, and the concentration of senior health care providers is based, who would then be responsible for developing and modifying protocols, training health care workers based in rural areas, and maintaining a database which would help guide future policies and identify areas where future csa related research may be needed. it is not feasible to establish one - stop centers in all places in zambia as in the present format they would be extremely expensive. in order to create a sustainable program throughout zambia, the multidisciplinary concept should be adapted to work within the current health care system. in the future, once there is a cadre of health care workers trained in the identification and treatment of child sexual abuse available, the services should be established as close to the community as possible. this is especially important in poorly resourced countries where caretakers may fail to report abuse or be adherent to the followup regimen because of lack of transport funds. curricula at the health institutions need to be adapted to include child sexual abuse to ensure professionals are equipped with the knowledge and skills to care for children who have been sexually abused at graduation. it remains critical for the uth centre as well as other large tertiary institutions where the centres are established to gain the support from the government to sustain these necessary services and reduce reliance on external funding. | objective. to improve care of sexually abused children by establishment of a one stop centre at the university teaching hospital. methodology. prior to opening of the one stop centre, a management team comprising of clinical departmental heads and a technical group of professionals (health workers, police, psychosocial counselors lawyers and media) were put in place. the team evaluated and identified gaps and weaknesses on the management of sexually abused children prevailing in zambia. a manual was produced which would be used to train all professionals manning a one stop centre. a team of consultants from abroad were identified to offer need based training activities and a database was developed. results. a multidisciplinary team comprising of health workers, police and psychosocial counselors now man the centre. the centre is assisted by lawyers as and when required. uth is offering training to other areas of the country to establish similar services by using a trainer of trainers model. a comprehensive database has been established for lusaka province. conclusion. for establishment of a one stop centre, there needs to be a core group comprising of managers as well as a technical team committed to the management and protection of sexually abused children. |
extrarenal calyces are rare anomalies of the upper urinary tract. only 20 cases were reported so far in the literature. the rarity of this anomaly and the complexity of possible associated anomalies often makes the preoperative diagnosis difficult. they are known to be associated with other anomalies, including bifid kidney and renal dysplasia. to the best of our knowledge, only 3 cases have been reported in the literature wherein, it was associated with pelviureteric junction obstruction (pujo).[24 ] when associated, management options depend on the renal function and presenting symptoms. herein, we are reporting a case where routine preoperative imaging did not throw any light on the diagnosis and was an intraoperative surprise. a 9-year - old male child presented to us with complaints of pain in the left flank for 1 month. an ultrasound of the abdomen showed severe hydronephrosis of the left kidney with a parenchymal thickness of 2 mm with upper ureteric dilation. a radionuclide scan showed a poorly functioning left kidney with a split renal function of 7% with obstructed curves. an ante grade dye study was done, which showed significant stasis in the hydronephrotic kidney and the ureter was not visualized. intraoperatively, a large dilated kidney with papery thin cortex was found with 5 pseudopodia - like projections (extrarenal calyces) arising from it and connected to the pelvis ; pujo was noted. the rest of the ureter appeared normal. in view of the complex nature of the lesion and poorly functioning kidney, a right nephroureterectomy was performed [figure 1 ]. the presence of extrarenal calyces is a very rare anomaly of the upper urinary tract that was first described in 1925. a few cases were reported in the literature and the exact etiology of this condition is unclear. it has been hypothesized that the anomaly could be due to a disparity resulting from slow development of the metanephric tissue or to a relatively rapid development of the ureteric bud. a kidney with extrarenal calyces is usually associated with other anomalies, such as bifid kidney, renal ectopia, horseshoe kidney, and renal dysplasia. it might be an incidental finding diagnosed at autopsy or may present with complications, such as hydronephrosis secondary to associated pujo, as in our case. because the incidence of this anomaly is quite low and the clinical presentation is very similar to classic pujo, a high degree of suspicion should be kept, especially when preoperative radiologic findings are not classical and show distorted calyceal appearance on preoperative imaging studies. this would safeguard against inadvertent injury of the calyces when operating on a well - functioning kidney. but, when they are associated with complications, such as pujo, as in our case, the management is not standardized. (2006) mentioned the successful management of pujo associated with extrarenal calyces in a pelvic kidney using transperitoneal laparoscopic reconstruction. the management options may include the following : (1) to do a classic pyeloplasty and follow the patient, risking failure of pyeloplasty and recurrent urinary tract infection (uti) (as was done in the previous case) ; (2) to resect the pelvis and do a ureterocalicostomy with fusion of the calyces (again associated with extensive dissection and suturing) ; or (3) to go ahead with a single - stage nephroureterectomy, thereby preventing the child from further recurrent uti. the management strategies are controversial and should be decided on individual merits and surgeon competence. however, in kidneys with well - preserved function, a renal sparing procedure is worth attempting. | extrarenal calyces are one of the rare renal anomalies associated with the collecting system. their association with renal ectopia or horse shoe kidney is known. but, here in, we are reporting an intraoperative surprise, where extrarenal calyces were associated with pelviureteric junction obstruction and routine preoperative imaging did not show any light on the diagnosis. |
the main terminology used to describe heart failure (hf) is based on measurement of the left ventricular ejection fraction (lvef). historically, hf was divided in patients with normal lvef [hf with preserved ef (hfpef), typically with a lvef 50% ] and those with reduced lvef [hf with reduced ef (hfref), typically with a lvef 50% of at least one principal coronary artery at coronary angiography. dyspnea was quantified according to the new york heart association (nyha) functional class. systemic hypertension was defined as a systolic blood pressure 140 mmhg or a diastolic blood pressure 90 mmhg without treatment ; hypercholesterolemia in patients without atherosclerotic cardiovascular disease as a total plasma cholesterol or ldl cholesterol above 5 mmol / l and 3 mmol / l, respectively, or in patients with atherosclerotic cardiovascular disease or diabetes above 4 mmol / l and 2.5 mmol / l, respectively ; obesity as a body mass index 30 kg / m and chronic renal insufficiency as an estimated glomerular filtration rate by modification of diet in renal disease formula 50% of at least one principal coronary artery at coronary angiography. dyspnea was quantified according to the new york heart association (nyha) functional class. systemic hypertension was defined as a systolic blood pressure 140 mmhg or a diastolic blood pressure 90 mmhg without treatment ; hypercholesterolemia in patients without atherosclerotic cardiovascular disease as a total plasma cholesterol or ldl cholesterol above 5 mmol / l and 3 mmol / l, respectively, or in patients with atherosclerotic cardiovascular disease or diabetes above 4 mmol / l and 2.5 mmol / l, respectively ; obesity as a body mass index 30 kg / m and chronic renal insufficiency as an estimated glomerular filtration rate by modification of diet in renal disease formula < 60 ml / min. clinical followup was assessed in june 2016 by phone interview of patient 's general practitioner / cardiologist, patient, or family. biomarkers and posology of furosemide were not normally distributed, and results are, therefore, presented as medians with interquartile ranges. the study population was categorized into three groups : patients with hfref, patients with hfmref, and patients with hfpef. wallis tests or anova for continuous variables and test for categorical variables, using bonferroni corrections for multiple comparisons. regression model was used to adjust for the effect of age on the differences in baseline characteristics among groups. allcause mortality was summarized using kaplan meier survival curve, and log rank test was used for initial comparisons. all analyses were performed using standard statistical software spss version 20 (spss inc., a total of 482 patients were referred for invasive coronary angiography for assessment of hf from june 2012 through june 2013, who constituted the study population. there were 258 (53%), 115 (24%), and 109 (23%) patients with hfref, hfmref, and hfpef, respectively. distribution of left ventricular ejection fraction across the population of patients with heart failure referred for invasive coronary angiography. patient age increased, whereas prevalence of lbbb, brain natriuretic peptide level, and the use of betablocker and furosemide decreased from hfref to hfpef (table 1). characteristics of patients with heart failure and reduced, mild range, or preserved ef acei, angiotensin converting enzyme inhibitor ; arb, angiotensin ii receptor blocker ; bnp, brain natriuretic peptide ; copd, chronic obstructive pulmonary disease ; crt, cardiac resynchronization therapy ; egfr, estimated glomerular filtration rate ; icd, implantable cardiac defibrillator ; lbbb, left block bundle branch. patients with hfpef or hfmref were older, were more likely to have nyha stage 2 dyspnea, had a higher systolic blood pressure, were less likely to have acei / arb and spironolactone, had lower furosemide dose, and had lower haemoglobin than those with hfref. except for acei / arb treatments, these differences remained significant after adjustment for the age difference between groups. patients with hfref or hfmref were more likely to be male and to have less history of hypertension than those with hfpef. after adjustment for the age difference between groups, only sex difference between groups remains significant. overall, and after adjustment for age differences, cardiovascular risk factors and medical history were similar in the three groups of patients. survival data were available for 452 of the 482 patients (94%), with a mean (sd) followup of 32.2 14.3 months. there were 17 (7%), 9 (8%), and 4 (4%) patients lost for followup among patients with hfref, hfmref, and hfpef, respectively (p = 0.390). the survival rate was no different among patients with hfref, hfmref, and hfpef (figure 2). (p = 0.061) ; 18, 15, and 13% at 1 year (p = 0.337) ; and 33, 32, and 32% at the time of followup (p = 0.903). meier survival curves for patients with heart failure and reduced, mild, range or preserved ejection fraction. the unadjusted hazard ratios for death in the group of patients with hfmref and hfpef as compared with the patients with hfref were 0.88 (95% confidence interval, 0.58 to 1.35 ; p = 0.560) and 0.95 (95% confidence interval, 0.63 to 1.43 ; p = 0.820), respectively. after adjustment for age, the likelihood of survival was still the same among groups (hazard ratio for death among patients with hfmref as compared with patients with hfref, 0.78 ; 95% confidence interval, 0.51 to 1.20, p = 0.259 and hazard ratio for death among patients with hfpef as compared with patients with hfref, 0.78 ; 95% confidence interval, 0.51 to 1.18, p = 0.242). patient age increased, whereas prevalence of lbbb, brain natriuretic peptide level, and the use of betablocker and furosemide decreased from hfref to hfpef (table 1). characteristics of patients with heart failure and reduced, mild range, or preserved ef acei, angiotensin converting enzyme inhibitor ; arb, angiotensin ii receptor blocker ; bnp, brain natriuretic peptide ; copd, chronic obstructive pulmonary disease ; crt, cardiac resynchronization therapy ; egfr, estimated glomerular filtration rate ; icd, implantable cardiac defibrillator ; lbbb, left block bundle branch. patients with hfpef or hfmref were older, were more likely to have nyha stage 2 dyspnea, had a higher systolic blood pressure, were less likely to have acei / arb and spironolactone, had lower furosemide dose, and had lower haemoglobin than those with hfref. except for acei / arb treatments, these differences remained significant after adjustment for the age difference between groups. patients with hfref or hfmref were more likely to be male and to have less history of hypertension than those with hfpef. after adjustment for the age difference between groups, only sex difference between groups remains significant. overall, and after adjustment for age differences, cardiovascular risk factors and medical history were similar in the three groups of patients. survival data were available for 452 of the 482 patients (94%), with a mean (sd) followup of 32.2 14.3 months. there were 17 (7%), 9 (8%), and 4 (4%) patients lost for followup among patients with hfref, hfmref, and hfpef, respectively (p = 0.390). the survival rate was no different among patients with hfref, hfmref, and hfpef (figure 2). the respective mortality rates were 15, 9, and 9% at 6 months (p = 0.061) ; 18, 15, and 13% at 1 year (p = 0.337) ; and 33, 32, and 32% at the time of followup (p = 0.903). meier survival curves for patients with heart failure and reduced, mild, range or preserved ejection fraction. the unadjusted hazard ratios for death in the group of patients with hfmref and hfpef as compared with the patients with hfref were 0.88 (95% confidence interval, 0.58 to 1.35 ; p = 0.560) and 0.95 (95% confidence interval, 0.63 to 1.43 ; after adjustment for age, the likelihood of survival was still the same among groups (hazard ratio for death among patients with hfmref as compared with patients with hfref, 0.78 ; 95% confidence interval, 0.51 to 1.20, p = 0.259 and hazard ratio for death among patients with hfpef as compared with patients with hfref, 0.78 ; 95% confidence interval, 0.51 to 1.18, p = 0.242). as previously described,3 the distribution of the lvef across our population of patients with hf referred for invasive coronary angiography observed a unimodal bell shaped including 24% of hfmref, which is substantially more than previous big series.4, 5, 6 anyway, the bellshaped distribution is relatively constant and we can assume that the prevalence of hfmref depends on the patient recruitment method.3 the new esc terminology was done because patients with lvef that ranges from 40 to 49% were in a grey area between hfref and hfpef and, consequently, were excluded from hf clinical trials of hf.4 identifying hfmref as a separate group has the aim of stimulating research into the underlying characteristics, pathophysiology, and treatment of this group of patients. the main question is to know if differentiation of patients with hf based on lvef can make the difference between underlying aetiologies, demographics, and comorbidities with the ulterior motive of a common response to therapies. in our cohort of hf patients referred for coronary angiography, except for sex where patients with hfpef are more likely to be female than those with hfref and hfmref, we found that patients with hfmref and hfpef share the same clinical profile : they are older, are more likely to have nyha stage 2 dyspnea, have a higher systolic blood pressure, are less likely to have spironolactone, have lower furosemide dose, and have lower haemoglobin than those with hfref., our study shows that patients with hfmref have the same clinical profile as patients with hfpef, which is different from hfref mainly for age, symptoms, and systolic blood pressure. finally, patients with hfref, hfmref, and hfpef share the same cardiovascular risk factors and medical history. previous large cohorts of patients with hf have reported that hfpef and hfref have different epidemiological and etiological profiles. compared with hfref, patients with hfpef are older, more often women, are more likely to be obese, have lower haemoglobin, and more commonly have a history of hypertension and atrial fibrillation, while a history of myocardial infarction is less common.7, 8 several previous studies that have focused on hfmref reported intermediate clinical characteristics of patients with hfmref, between those of hfref and hfpef : patients were younger and more predominantly male compared with those with hfpef.4, 6 several cardiovascular risk factors were shared among hfmef, hfref, and hfpef, but patients with hfmef were more likely to have hypertension compared with those with hfref. the recent study of kapoor. found that patients with hfmref are closer to patients with hfpef in terms of mean age and comorbidities as anaemia, atrial fibrillation, diabetes, hypertension, or renal disease.6 these results confirm the difference in demographics and comorbidities among patients with hf, according to their lvef, and suggest that our study has probably not enough power to highlight slight differences. interestingly, patients with hfmref were more likely to have ischaemic heart disease compared with those with hfpef, and similar to those with hfref4 suggesting that ischemic burden could play a role in the time course of a potential transition between hfpef and hfref. the role of coronary artery disease in the course of hfpef and the transition from hfpef to hfref is supposed to be determinant : patients with coronary artery disease have greater deterioration of lvef and may have been more likely to transition to hfref following a myocardial infarction.9 these findings led the authors to suggest that hfmref could constitute a subset of hfpef enriched with coronary artery disease and representing an early stage of hfref.4 our results contradict this hypothesis by showing the same prevalence rates of myocardial infarction and artery coronary disease among the three groups of patients with hf. beyond the difference of phenotype between groups of patients with hf, our study shows that patients have the same mortality over a followup period of 2.7 years, whatever their group of ef. previous data suggest that the mortality rate of hfmref is intermediate between that of hfref and hfpef. these data show a graded relationship between lower ef and higher risk of events.3, 5 finally, in regard of these data and in terms of outcomes, chronic stable hfmref resembles hfpef more than hfref. our results are slightly different showing the same prognosis and the high mortality rate among the three groups of patients with hf. there is only a trend of increased mortality among patients with hfref as compared with those with hfmref and hfpef, but this trend disappears in the first year to join the general prognosis of hf.4 these results confirm that beyond the simple ef, symptoms and congestive signs of hf carry a poor prognosis. hf is a heterogeneous disorder resulting of the interaction of multiple aetiologies, comorbidities, and cardiovascular risk factors, where imbalance can lead to maintain or decrease of lvef. the discrepancy among studies in terms of prevalence, comorbidities, clinical profile, and prognosis of patients with hfmref among large cohorts suggests that the link between pathophysiology of hf and lvef is not so linear and simple. hf includes multiple diverging patientoriented time trajectories, resulting in a wide spectrum of different phenotypes.10 following any form of cardiac dysfunction, each patient will follow his individual trajectory within a large spectrum of lvef, depending on the number of disease modifiers.10 this questions if the patients with hfmref are truly a distinct pathophysiologic entity or a transitional phenotype between hfref and hfpef as suggested by previous big cohort.6 the new esc classification of hf does not imply that patients will inevitably progress from hfpef to hfmef then hfref. the recent study of nadruz. regarding patients with previously reduced and then recovered ef suggests that mild range lvef is not necessarily a transition step of the progression from normal lvef to hfref or vice versa.11 hfref, hfmref, and hfpef are probably different phenotypes of the same disease whose final common pathway is the decrease of cardiac output and the onset of congestive signs leading to neurohormonal activation and poor prognosis. the recognition of a new group for the patients with midrange lvef occurred due to historical development of hf trials rather than due to a strong pathophysiological basis for a third entity in hf.4 an ideal hf classification system would group together pathophysiologically similar individuals who may respond in the same way to clinical management and targeted therapy. any classification that can guide treatment will be useful in clinical practice. among measurements and indices of the left ventricular pump, lvef has emerged as one of the most sensitive ones leading to the most therapeutic evidence, including effective pharmacological and device therapies. preserved in patients with hfmref and hfpef ? other measures of myocardial function such as myocardial deformation imaging may be better parameters.12, 13 as previously discussed, the sample of our study, as compared with large clinical trials in hf, is probably too small for allowing enough power to discriminate slight differences among groups of patients with hf. trends of difference in the prevalence rates of obesity, hypertension, and sleep disorder breathing among groups suggest that a bigger sample would be able to show a significant difference. however, we think that these differences among studies are the results of the heterogeneity of the different phenotypes of the same disease. our study is an observational study, and therefore unmeasured confounding factors may influence the observed associations and prognosis. thus, our study does not integrate echocardiographic and haemodynamic features, as well as right ventricular function, which are key determinants in clinical practice for phenotyping and assessing prognosis of patients with hf. we did not distinguish the subset of patients with previously reduced and then recovered ef, which have been recently described as a distinct group from the residual population with similar clinical characteristics but better prognosis.11 the difference in prognosis between these subsets of patients with hfmref may explain some reported discrepancies on prognosis in patients with hfmref. finally, we did not distinguish compensated and decompensated hf, or changes in medical therapy over time, which have a strong impact on prognosis. we did not distinguish cardiovascular mortality from allcause mortality, which can be different among groups of hf and according to the lvef, and could explain the difference of prognosis observed in previous studies. as previously discussed, the sample of our study, as compared with large clinical trials in hf, is probably too small for allowing enough power to discriminate slight differences among groups of patients with hf. trends of difference in the prevalence rates of obesity, hypertension, and sleep disorder breathing among groups suggest that a bigger sample would be able to show a significant difference. however, we think that these differences among studies are the results of the heterogeneity of the different phenotypes of the same disease. our study is an observational study, and therefore unmeasured confounding factors may influence the observed associations and prognosis. thus, our study does not integrate echocardiographic and haemodynamic features, as well as right ventricular function, which are key determinants in clinical practice for phenotyping and assessing prognosis of patients with hf. we did not distinguish the subset of patients with previously reduced and then recovered ef, which have been recently described as a distinct group from the residual population with similar clinical characteristics but better prognosis.11 the difference in prognosis between these subsets of patients with hfmref may explain some reported discrepancies on prognosis in patients with hfmref. finally, we did not distinguish compensated and decompensated hf, or changes in medical therapy over time, which have a strong impact on prognosis. we did not distinguish cardiovascular mortality from allcause mortality, which can be different among groups of hf and according to the lvef, and could explain the difference of prognosis observed in previous studies. despite overlapping features with hfref, as cardiovascular risk factors and medical history, patients with hfmref have a different clinical profile, which is nearly the same than patients with hfpef, except for sex. given the failure of clinical trials in the field of hfpef, these results question the relevance of this new classification of hf to stimulate research into the new group of patients with hfmref. | abstractaimsthe main terminology used to describe heart failure (hf) is based on measurement of the left ventricular ejection fraction (lvef). lvef in the range of 4049% was recently defined as hf with midrange ef (hfmref) by the 2016 european society of cardiology guidelines. the purpose of our study was to assess the clinical profile and prognosis of patients with hf according to this new classification.methods and resultsa total of 482 patients referred for hf were retrospectively included over a period of 1 year. there were 258 (53%), 115 (24%), and 109 (23%) patients with hf with reduced ef (hfref), hfmref, and hf with preserved ef (hfpef), respectively. patient age increased, whereas left block bundle branch, brain natriuretic peptide level, and the use of betablocker and furosemide decreased from hfref to hfpef. after adjustment for the age, patients with hfpef and hfmref were more likely to have nyha stage 2 dyspnea, had a higher systolic blood pressure, were less likely to have spironolactone, had lower furosemide dose, and had lower haemoglobin than those with hfref. cardiovascular risk factors and medical history were similar in the three groups of patients. there was a 33% death rate after a mean followup of 32.2 14.3 months. the survival was the same among patients whatever the group of hf (p = 0.884).conclusionspatients with hfref, hfmref, and hfpef share the same cardiovascular risk factors, medical history, and prognosis. patients with hfmref have a different clinical profile, which is nearly the same as patients with hfpef, except for sex. these results question the relevance of this new classification of hf to stimulate research into this new group of patients. |
there is a tide in the affairs of men, which taken at the flood, leads on to fortune. omitted, all the voyage of their life is bound in shallows and in miseries. proactive measures include avoiding known risks by not settling in or venturing into hazardous environments, and mitigating them by constructing dams, levees, and other protective infrastructure. proactive mitigation also includes creating disaster warning systems, infrastructure to facilitate rapid evacuation, and emergency shelters in advance of need. proactive measures are necessarily costly because they both consume and sequester resources for an indefinite period of time. this cost is particularly psychologically burdensome because it is impossible to predict with any certainty precisely when a threat will become a crisis or a disaster. in contrast, reactive measures are much less expensive in the short term. delivering emergency food, water, shelter, medical care, and post - disaster evacuation have the theoretical advantage of being deployable anywhere, ideally from just a few locations. the appeal of post - disaster response is that the overhead is low and the resources can be used reliably on a regular basis. unfortunately, what is not factored into this approach is the vastly more expensive loss of property and lives that proactive mitigation would have prevented. the reasons for disasters such as hurricane katrina can only be understood by inquiring into the nature of technology and the human minds that wield it. one way to look at this progression is in terms of timescales. to begin, the ' trivial timescale ' is bounded by day - to - day activities such as bathing, unstructured socializing, and going to work. the second timescale is the ' personal timescale ', which allows for planning and actions that occur over the time course of roughly two human generations (defined here as 44 years). such planning includes selecting and mastering a livelihood, procreating and rearing children to adulthood, and setting aside resources to be used in old age and for the benefit of offspring. the vast majority of people are incapable of significant understanding or planning beyond the personal timescale ; indeed, there is little evolutionary reason for human minds to function in timescales longer than this. the third timescale is the ' historical timescale ', which extends from the present to the beginning of human history. very few of us spend any time in this timescale, and the emotional connection to events going back more than two human generations is slight. the fourth and most powerful timescale is the ' cosmic timescale ', namely the period of time from the beginning to the end of the universe. galileo, newton, and einstein exemplify the power of thinking in the cosmic timescale. these disciplines have facilitated the enormous and powerful technologies we now wield, from splitting the atom to our emerging mastery over the very fabric of life. disaster is largely an artifact in the transition from hunter - gatherer to town and urban dweller. homo sapiens began their relentless advance across the face of the earth about 100,000 years ago. agriculture began to emerge about 12,000 years ago, the first human settlements 11,000 years ago, and the first cities only 7000 years ago. from an evolutionary standpoint this is a trivial amount of time. the widespread application of the scientific method and development of the mathematics of statistics and probability occurred less than 400 years ago an evolutionarily insignificant amount of time. our entry as a species into the historical and cosmic timescales spawned both our reliance on technology and our vulnerability to disaster. accordingly, it is likely that disasters will always be part of human life for at least two reasons : human settlement has been undertaken in environments where the hazards are not completely understood ; and known hazards are underestimated or ignored. however, our concern here is primarily with situations in which people abuse reason and technology and take extreme risk at great cost in terms of lives and property. here, history is perhaps the best teacher, for it is rich in the story of why catastrophes like katrina occur nearly 2000 years ago, in 79 ad pompeii was a thriving roman port city on the bay of naples. pompeii and the surrounding communities of herculaneum, stabiae, oplontis, sora, tora, taurania, cossa, and leucopetra were of roughly the same character and economic importance to rome as are new orleans, biloxi, gulfport, mobile, bay st. pompeii had been continuously inhabited since it was founded in the 6th century bc. in 62 ad the damage from the earthquake was so extensive that 14 years later reconstruction was still underway, although most of the residential and business structures had been rebuilt apparently on a grander scale than before. finally, on the afternoon of 24 august at about 13:00 hours mount vesuvius erupted. pompeii, much like new orleans, was largely evacuated, and like in new orleans only about 10% of the population (2000 souls) remained in the city for the final cataclysm. this is remarkable because this eruption of vesuvius was the first time in recorded history that a vertical eruption of this magnitude and its accompanying pyroclastic flow were documented (by pliny the younger). by the evening of the 24th two additional eruptive surges this had exactly the same effect on the remaining population as did the flooding of new orleans ; it was as impossible to move through the ash to escape via either land or sea. between 07:00 and 08:00 hours on the morning of the 25th, the fourth and fifth eruptive surges occurred and these were lethal to both the city and its inhabitants. these surges moved at a rate of 200300 km / hour, tearing off roofs, fragmenting lighter structures, and transiently raising the ambient temperature to over 200c. so, what is the relevance of pompeii to our contemporary response to risks and hazards of similar magnitude ? vesuvius last erupted in 1944, at which time there was significant loss of life and property. since that time the area has been densely repopulated, and the town of san sebastiano was rebuilt on its former site directly atop the lava flow that destroyed it in 1944 ! currently there are 3 million people living in an area of high risk and approximately 600,000 living in the ' zona rosa ', virtually in the same geographic area occupied by pompeii, herculaneum, and their sister communities. the logistics of the timely evacuation of 3 million people with little or no warning, living at a density greater than that of the inhabitants of hong kong, are incomprehensible. at this time, engineering to protect the population at risk should vesuvius experience another catastrophic eruption does not exist. because of the impossibility of timely evacuation in the event of a sudden eruption, those who choose to live and work in the shadow of the volcano today are more likely to lose their lives than were the inhabitants of pompeii nearly 2000 years ago. this is a classic example of people choosing to continue occupying hazardous zones where effective proactive risk mitigation is not technologically possible. proactive, protective technology exists that can reduce the threat of disaster along the gulf coast and presumably other areas to a negligible or acceptable level and is in use around the world. the multibillion dollar deltawerk seawalls constructed in the netherlands and the thames barrier in london are examples of the kind of engineering that could protect new orleans and other at - risk us cities from future hurricane storm surges. radically improved building codes that mandate structural resistance to high winds and flying debris, such as those implemented in florida after hurricane andrew, would greatly reduce the loss of homes and businesses, even in the face of category 4 hurricanes. a retrovirus infecting a few individuals in a remote corner of africa can bloom into a relentless worldwide epidemic. a disaster half a world away can cripple or potentially even destroy our increasingly unified technologic civilization. as a species we must make the jump from the trivial and personal timescales to the historical and cosmic ones, or we shall perish. eleven thousand years ago we made the incredible jump from hunter - gatherers to agricultural town dwellers. the wisdom of this decision is challenged with each failure in foresight that leads on to disaster. from the moment we began to seek our fortune in the realm of the historical and cosmic timescales, we embarked on the ultimate voyage. to quote shakespeare once again, ' on such a full sea are we now afloat. and what will be our choice on to fortunes or bound in shallows and miseries ? | unique among animals, humans survive not by superb physical adaptation to our environment, but rather by intelligent, large - scale adaptation of the environment to our needs. we build houses with climate control systems that mimic the environment of sub - saharan africa. we safely live in environments where the temperature never rises above freezing and where the level of the sea is higher than the land we farm and build vast cities upon. we live in tropical rainforests teeming with hostile organisms, and atop arid, life - poor mountains and plains, at elevations in excess of 5000 m. as this editorial is written, a few of us live in space, circling the earth in an environment of hard vacuum, searing heat, and cryogenic cold. the sole endowment for our survival that evolution has bestowed upon us is reason and technologic civilization, which is its product. all human habitations, and all life on earth for that matter, are under continuous threat of some kind. violent weather, earthquakes, volcanic eruptions, and even meteorite impacts represent threats of varying degrees of risk. |
congenital central hypoventilation syndrome (cchs) is a life - threatening disorder characterized by an idiopathic failure of the automatic control of breathing, particularly during sleep. in cchs, patients breathe with insufficient depth due to impaired sensitivity to hypercapnea and hypoxia. hirschsprung 's disease (hd), also known as congenital aganglionic megacolon, is characterized by the absence of ganglion cells in any part of the bowel wall. the absence of ganglion cells in hirschsprung 's disease inhibits normal peristalsis, resulting in a functional intestinal obstruction. the combination of cchs and hd was reported first by haddad in 1978 and it has been extremely rare with approximately 60 cases reported in the worldwide literature (1, 2). previous studies described that de novo mutation of the phox2b gene was involved in cchs pathogenesis (3, 4). many patients with cchs are heterozygous for a polyalanine expansion mutation in the second polyalanine repeat residue of the phox2b gene, with one allele having 20 repeats and the affected allele having 25 - 33 repeats of the polyalanine sequence (3, 4). some authors reported that a subset of patients had other mutations in the phox2b gene (5, 6). in korean population, only one case has been reported, however, genetic analysis has not been performed (7). here, we report a haddad syndrome with the phox2b gene mutation for the first time in a korean. this 41-week - gestation male was born to a 30-yr - old mother with good health by normal spontaneous vaginal delivery in november 2009. there was neither family history of congenital anomalies nor maternal exposure to drugs during pregnancy. the baby had apgar scores of 7 and 5 at 1 and 5 min, respectively. the baby was immediately transferred to our hospital for further evaluation and management after stabilization. for the next few days, we tried extubation several times, but failed to succeed because the baby suffered from recurrent apnea and hypercarbia leading to the clinical suspicious of cchs. on day 8 of life, a cranial ultrasound study was performed and it did not show evidence of hemorrhage or mass lesions. during the second day of life, it was noted that the patient had distended abdomen and was unable to pass meconium without rectal stimulation. a gastrografin contrast enema study showed multiple meconium plugs in the colon suggesting meconium plug syndrome (fig. meconium was passed and abdominal distension was relieved after contrast study. on day 6 of life, he showed sustained abdominal distension with poor bowel movement. a barium contrast enema study was needed to rule out other causes of lower gastrointestinal obstruction. he was noted to have long segment hd with a transition zone at mid descending colon. seromuscular biopsies of the bowel taken 3 cm proximal and distal to the transition zone revealed the presence of ganglion cells in the proximal biopsy specimen only. the cytogenetic study was performed clinically in the infant and his parents and their karyotypes were normal. genomic dna of the patient and his parents was isolated from peripheral blood using the wizard genomic dna purification kit according to the manufacturer 's instructions (promega, madison, wi, usa). amplification of the coding sequence of the phox2b gene by polymerase chain reaction (pcr) was carried out as previously described with minor modifications (4). polymerase chain reaction - single strand conformational polymorphism (pcr - sscp) was performed as previously described (8). pcr products in the patient, his parents, and normal controls were electrophoresed through 8 and 10% polyacryamide gels (fig. a mutant band was found, however, his parents had no mutant band. to determine the sequences of the dna sample showing the mutant band, direct sequencing was performed by macrogen inc, in korea however, the quality of the result was not clear. to get apparent result of sequencing, paisley, united kingdom) and 10 single colonies were collected for extracting plasmid dna. direct sequencing revealed expanded alleles containing polyalanine 26 repeats in exon 3 of the phox2b gene, further supporting the diagnosis cchs (fig. importantly, we were able to prove a phox2b gene mutation containing polyalanine 26 repeats in this patient. hirschsprung 's disease often produces intestinal obstruction in the neonate, characterized by abdominal distention, bilious vomiting, and failure to pass meconium within the first 48 hr. cchs is characterized by relatively normal ventilation when the infant is awake and hypoventilation, even apnea, during sleep. haddad syndrome is interpreted not so much as a combination of two diseases but rather as a complicated form of cchs. when hd is associated with cchs, it is different from classical hd, characterized by extensive intestinal aganglionosis (9). the incidence of cchs is extremely low in hd cases, however, the incidence of hd in cchs cases varies from 16% to 50% according to previous studies (3, 5, 6, 9). additional symptoms are ophthalmic abnormalities, esophageal dysmotility, sensorineural hearing loss, neural crest tumors (ganglioneuromas, neuroblastomas), and dysmorphic facial features (3, 5, 9). however, additional symptoms were not found in our case. according to previous report in korean, a haddad syndrome patient also had no additional symptoms except congenital ptosis (7). (3), and its rate was over 90% among cchs patients (4, 6). polyalanine expansions are the most common mutations in patients with cchs, though rare mutations have been reported such as frameshift mutation, nonsense mutation or missense mutation (3 - 5). in our case, his parents showed no phox2b gene mutation, suggesting de novo mutation of the phox2b gene in haddad syndrome. interestingly, genotype - phenotype correlation has been described in cchs patients with the phox2b gene mutation (4). increasing polyalanine repeat mutation size is associated with a more severe clinical phenotype (4). (10) described that haddad syndrome (cchs + hd type) showed usually expansions of + 6 and + 7 alanines, and frameshift or missense phox2b mutations may predispose to neuroblastoma. clinical phenotype of our patient was severe, presenting long segment hd and no capacity to maintain sufficient oxygenation and ventilation for at least 30 - 60 min of sleep after removing mechanical ventilation support (4). however, our case with expansion of + 6 alanines had no additional symptoms like neural crest tumors. though the symptoms of cchs and hd were severe, no additional symptom in the patient with this mutation type was in agreement with the hypothesis by trochet. this result supports that the assay for phox2b polyalanine repeat mutation represents a highly sensitive and specific technique for confirming the diagnosis of cchs and estimating the prognosis. in summary, this report presents a rare case of haddad syndrome with de novo mutation of the phox2b gene in korean for the first time. considering the difficulty of diagnosis of this syndrome because of overlapped of symptoms and signs in an infant | congenital central hypoventilation syndrome with hirschsprung 's disease, also known as haddad syndrome, is an extremely rare disorder with variable symptoms. recent studies described that congenital central hypoventilation syndrome had deep relation to the mutation of the phox2b gene in its diagnosis and phenotype. we report a newborn male infant with clinical manifestations of recurrent hypoventilation with hypercapnea and bowel obstruction. these clinical manifestations were compatible with congenital central hypoventilation syndrome and hirschsprung 's disease, and polyalanine 26 repeats in the phox2b gene supported the diagnosis of congenital central hypoventilation. we described a first case of haddad syndrome in korean and its clinical and genetic characteristics were discussed. |
this modification adds a methyl group (ch3) to the 5 position of cytosine in a dna sequence and is inheritable through cell division.1,2 for mammalian cells, it occurs only at cpg sites (cytosines paired with guanines). dna methylation plays an essential role for both normal and cancerous cell development36 and is closely related to significant processes, including x - chromosome inactivation, genomic imprinting, and tumor growth.710 in a genome, there are different types of methylation patterns, including hypermethylation, hypomethylation, and hemimethylation. hypermethylation occurs when samples in one group (eg, cancer patients) have more methylation than the samples in another group (eg, normal individuals). hypomethylation occurs when samples in one group have less methylation than the samples in another group. hemimethylation means that at a cpg site, only one strand of the dna is methylated (denoted m in fig. recent research studies1113 show that, on a number of genes, hemimethylation may occur as a same - strand cluster (fig. 1b) with only two cpg sites, or a different - strand cluster with more than two cpg sites (fig. the cluster pattern means that two or more consecutive cpg sites are methylated only on one dna strand, and not on the other strand, as shown in figure 1a. the polarity (or reverse) hemimethylation clusters imply that, at the two consecutive cpg sites, the methylation patterns on the positive and negative strands are mu and um, respectively, as shown in figure 1b. mu and um means that, at two adjacent cpg sites, the first site is hemimethylated as mu on the positive and negative strands, while the next adjacent site is hemimethylated as um on two strands, which has a reversed pattern. the hemimethylation patterns shown in figure 1 are like the footprints of dna demethylation (ie, methyl groups are removed) in cancer.12 this footprint role exists because hemimethylation is a transitional state between being methylated and having no methylation at specific cpg sites. therefore, the identification of hemimethylation is important for understanding both methylation events and the establishment of different methylation patterns. a major experimental limitation in hemimethylation studies has been the difficulty in obtaining methylation signals from the two complementary strands of dna molecules for all cpg sites in an entire genome. previous hemimethylation studies can only obtain the hemimethylation data for a few genes using the traditional sanger sequencing and hairpin sequencing methods.1113 even though microarray technologies can obtain methylation levels genome - wide, they can not produce methylation signals on two dna strands separately. however, the next - generation sequencing (ngs) technology,14,15 combined with the bisulfite conversion technique (ie, c is converted to u and then becomes t), makes it possible to obtain methylation signals at the cpg site level on both dna strands in an entire genome.1619 during the last several years, a number of pioneering research groups have successfully used the bisulfite - converted methylation sequencing method on either arabidopsis thaliana or human samples.16,1825 using bisulfite - converted methylation sequencing data, we can detect both the gain and loss of methylation by investigating hemimethylation patterns on two complementary strands. nevertheless, the ngs technology produces a large amount of data.26 the quality of bisulfite sequencing data may be poor because of incomplete bisulfite conversion, genome variation, and sequencing errors.27 all of these features make processing and analyzing data challenging when identifying hemimethylation patterns. to address this challenge, this pipeline can identify hemimethylated cpg sites and characterize different patterns in an entire genome. hmpl locates hemimethylated sites in each of the two different samples and then compares them. in the next section part i (preprocessing) utilizes available software packages in three steps : sequencing data quality assessment, trimming, and alignment (ie, steps 13 of the workflow). this part includes data parsing and summary reports (ie, steps 4 and 5 of the work - flow). hmpl code and resource files can be downloaded from the following web link : http://hal.case.edu/~sun/hmpl/hmpl.zip. step 1 : assess sequencing qualities using fastqc.28 fastqc is a software package for assessing sequencing qualities by generating basic and informative diagnostic plots for sequencing data. this package provides a modular set of analyses for users to obtain a quick impression as to whether or not there are any obvious and serious problems before they start any downstream data analysis. fastqc produces basic statistics plots and summary reports for (1) per base sequence quality, (2) sequence quality scores, (3) summary of per base sequence content, (4) per sequence gc content, (5) per base n content, (6) sequence length distribution, (7) duplicate sequences, (8) overrepresented sequences, (9) adapter content, (10) kmer (or k - base) content, and (11) per tile sequence quality. quite often, sequencing quality is very low at the 3 end in sequencing reads, and raw reads may include adapter sequences. therefore, hmpl has included the quality trimming and adapter - trimming step. in particular, dynamic trimming (the trim function provided in the software package brat29) and fixed - number - base trimming options are provided for quality trimming. step 3 : align reads using brat - bw31 and obtain methylation ratios at all cytosine sites. after trimming, alignment is done using brat - bw.31 after alignment, the methylation level (or ratio) at each cytosine (or c) site is obtained using the acgt - count function of the brat - bw package. the acgt - count function provides two options for generating output files : (1) the counts of a, c, g, and t at each cytosine site and (2) the methylation level for each cytosine site. in the hmpl package, we have chosen the second option of the acgt - count function. at each cytosine site, the methylation level is calculated as the ratio of the count of c (or the number of sequencing reads with methylated cytosine) to the count of c and t (or the total number of reads covering that site). the acgt - count function produces methylation levels for positive and negative strands in two separate output files. each output file includes the following columns for each cytosine site (ie, each row) : chromosome, start position, end position, total number of sequencing reads, methylation level, and dna strand (+ or). step 4 : identify hemimethylation patterns and compare these patterns in two samples. in this step, a hemimethylated cpg site is defined as a singleton if its adjacent cpg sites within d bases (eg, d = 100, a user - specified distance) are not hemimethylated. a hemimethylation cluster consists of at least two cpg sites that are all hemimethylated, and any two adjacent cpg sites in this cluster are within d bases. starting from the first cpg site on a chromosome, for each hemimethylated cpg site, hmpl checks if the next cpg site is hemimethylated. if it is hemimethylated and is within a d - base region of the previous cpg site, these two are grouped together as a cluster and we continue to check the next (or third) cpg site ; otherwise, this hemimethylated site is defined as a singleton. hemimethylation clusters may have the following patterns : (1) consecutive cpg sites hemimethylated in the same dna strand (eg, the three cpg sites in fig. 1a), (2) the polarity or reverse hemimethylation cluster with only two cpg sites (as shown in fig. 1b), and (3) consecutive cpg sites methylated on different strands and with more than two cpg sites, eg, the methylation of three cpg sites are muu and umm on the positive and negative strands, respectively (as shown in fig. hmpl can also compare hemimethylated singleton sites and clusters of two samples. because high - throughput sequencing data may include sequencing and alignment errors first, the user may determine a coverage cutoff value b (b > 0, eg, b = 5) depending on the sequencing quality and coverage level. on each strand, there must be at least b sequencing reads to cover a specific cpg site in order for hmpl to check whether or not this site is hemimethylated. second, if the methylation level of a specific cpg site at one strand (eg, the positive strand) is larger than the cutoff value h0 (eg, h0 = 0.9), it is identified as m (methylated) ; if the methylation level is lower than the cutoff value l0 (eg, l0 = 0.1), it is identified as u (unmethylated). using the above criteria, hmpl defines a cpg site as mu (ie, methylated on the positive strand and unmethylated on the negative strand), um, mm, or uu. in the case of identifying hemimethylation clusters. if a dataset has poor sequencing quality and low coverage, the results of using different cutoff values may be very different. therefore, we recommend that users select very stringent cutoff values for h0 and l0 to reduce the false positive discovery rate because of poor sequencing quality and low coverage. for example, the user may use h0 = 0.9 or 0.95 instead of 0.8 and l0 = 0.1, or 0.05 instead of 0.2. if the user s dataset has good sequencing quality and high coverage, changing the cutoff values may not affect the results significantly. step 5 : provide genetic annotation and report findings for each hemimethylated cpg site, hmpl provides the following genetic information. gene : if a hemimethylated cpg site is located on a gene, hmpl will report the name of that gene.pomoter : if a hemimethylated cpg site is within a d - base long region (eg, d = 1000) of the promoter of a gene (ie, d - base before the transcription starting site of a gene), hmpl will report the name of that gene. gene : if a hemimethylated cpg site is located on a gene, hmpl will report the name of that gene. pomoter : if a hemimethylated cpg site is within a d - base long region (eg, d = 1000) of the promoter of a gene (ie, d - base before the transcription starting site of a gene), hmpl will report the name of that gene. the hmpl uses raw sequencing reads (in fastq format) as input in step 1 and step 2. in steps 3, 4, and 5, more detailed information about the input and output files of hmpl can be found in the user manual, which can be downloaded from http://hal.case.edu/~sun/hmpl/hmpl.user.manual.pdf. the preprocessing step of hmpl (part i) can be implemented with the following command (the command options of pre.hmpl.pl are explained in table 1). [options ] the preprocessing pipeline ties the software and source code together with the appropriate dataflow to ensure that the correct output is achieved. users need to have perl, python 2.6, r, fastqc, and brat - bw software installed on their system. if users have finished the hemimethylation pre - processing pipeline and have obtained the files of combined cpg sites, they may only run the parsing analysis using the part ii of hmpl (ie, parse.hmpl.pl). the usage of part ii is given below (the command options of parse.hmpl.pl are explained in table 2). we have prepared a small dataset with 5 million sequencing reads (a fastq.fastq file), the human chromosome 22 reference sequence (a fasta.fa file), and the example scripts. users may align these 5-million reads to chromosome 22 and then identify hemimethylated sites using both part i and part ii of hmpl. users simply need to download the data, install the hmpl package, and then change the data path / directory in the example script accordingly to run the hmpl. it takes about 11 minutes to run this small dataset using a linux computer with 4 gb ram. in addition, in order for users to explore the different options and arguments of hmpl, we have prepared a file named readme. this file includes example scripts of running the hmpl using different command options and also explains to the user what to expect in the screen output. the small dataset, example scripts, and the readme file can be downloaded from the following web link : http://hal.case.edu/~sun/hmpl/hmpl.zip. in order to show the running time of hmpl in practice, we use two human example datasets. each dataset has ~50 million raw sequencing reads, which will be introduced in detail in the results section. using the linux server with dual quad - core 2.66 ghz xeon e5430 processor that has 4 gb ram for each core, it takes ~4 hours to run part i (the pre - processing part) of the hmpl, pre.hmpl.pl, if the reference index is provided. if the index file were not provided, it would first take about three to four additional hours to build a reference index for the whole human genome, which has about 3 billion bases (~3 gb data). therefore, it is more efficient to first build a reference index for the alignment tool brat - bw before running the hmpl. it requires ~19 minutes to run part ii of hmpl (the parsing pipeline, parse.hmpl.pl) when using uncombined input with the default coverage setting. the uncombined input means that positive and negative strand methylation levels of all cpg sites are provided as two separate files. if the positive and negative strand methylation data are combined, it will only take 15 minutes for hmpl to generate the results. if users have a faster linux server or high - performance computing clusters that have more memory and computing power, it will take much less time (eg, a couple of hours) to run the hmpl preprocessing pipeline (pre.hmpl.pl), and it will take just a few minutes to get the results of parsing and comparing two samples using the hmpl part ii (parse. part i (preprocessing) utilizes available software packages in three steps : sequencing data quality assessment, trimming, and alignment (ie, steps 13 of the workflow). this part includes data parsing and summary reports (ie, steps 4 and 5 of the work - flow). hmpl code and resource files can be downloaded from the following web link : http://hal.case.edu/~sun/hmpl/hmpl.zip. step 1 : assess sequencing qualities using fastqc.28 fastqc is a software package for assessing sequencing qualities by generating basic and informative diagnostic plots for sequencing data. this package provides a modular set of analyses for users to obtain a quick impression as to whether or not there are any obvious and serious problems before they start any downstream data analysis. fastqc produces basic statistics plots and summary reports for (1) per base sequence quality, (2) sequence quality scores, (3) summary of per base sequence content, (4) per sequence gc content, (5) per base n content, (6) sequence length distribution, (7) duplicate sequences, (8) overrepresented sequences, (9) adapter content, (10) kmer (or k - base) content, and (11) per tile sequence quality. quite often, sequencing quality is very low at the 3 end in sequencing reads, and raw reads may include adapter sequences. therefore, hmpl has included the quality trimming and adapter - trimming step. in particular, dynamic trimming (the trim function provided in the software package brat29) and fixed - number - base trimming options are provided for quality trimming. step 3 : align reads using brat - bw31 and obtain methylation ratios at all cytosine sites. after trimming, alignment is done using brat - bw.31 after alignment, the methylation level (or ratio) at each cytosine (or c) site is obtained using the acgt - count function of the brat - bw package. the acgt - count function provides two options for generating output files : (1) the counts of a, c, g, and t at each cytosine site and (2) the methylation level for each cytosine site. in the hmpl package, we have chosen the second option of the acgt - count function. at each cytosine site, the methylation level is calculated as the ratio of the count of c (or the number of sequencing reads with methylated cytosine) to the count of c and t (or the total number of reads covering that site). the acgt - count function produces methylation levels for positive and negative strands in two separate output files. each output file includes the following columns for each cytosine site (ie, each row) : chromosome, start position, end position, total number of sequencing reads, methylation level, and dna strand (+ or). step 4 : identify hemimethylation patterns and compare these patterns in two samples. in this step, a hemimethylated cpg site is defined as a singleton if its adjacent cpg sites within d bases (eg, d = 100, a user - specified distance) are not hemimethylated. a hemimethylation cluster consists of at least two cpg sites that are all hemimethylated, and any two adjacent cpg sites in this cluster are within d bases. starting from the first cpg site on a chromosome, for each hemimethylated cpg site, hmpl checks if the next cpg site is hemimethylated. if it is hemimethylated and is within a d - base region of the previous cpg site, these two are grouped together as a cluster and we continue to check the next (or third) cpg site ; otherwise, this hemimethylated site is defined as a singleton. hemimethylation clusters may have the following patterns : (1) consecutive cpg sites hemimethylated in the same dna strand (eg, the three cpg sites in fig. 1a), (2) the polarity or reverse hemimethylation cluster with only two cpg sites (as shown in fig. 1b), and (3) consecutive cpg sites methylated on different strands and with more than two cpg sites, eg, the methylation of three cpg sites are muu and umm on the positive and negative strands, respectively (as shown in fig. hmpl can also compare hemimethylated singleton sites and clusters of two samples. because high - throughput sequencing data may include sequencing and alignment errors first, the user may determine a coverage cutoff value b (b > 0, eg, b = 5) depending on the sequencing quality and coverage level. on each strand, there must be at least b sequencing reads to cover a specific cpg site in order for hmpl to check whether or not this site is hemimethylated. second, if the methylation level of a specific cpg site at one strand (eg, the positive strand) is larger than the cutoff value h0 (eg, h0 = 0.9), it is identified as m (methylated) ; if the methylation level is lower than the cutoff value l0 (eg, l0 = 0.1), it is identified as u (unmethylated). using the above criteria, hmpl defines a cpg site as mu (ie, methylated on the positive strand and unmethylated on the negative strand), um, mm, or uu. in the case of identifying hemimethylation clusters. if a dataset has poor sequencing quality and low coverage, the results of using different cutoff values may be very different. therefore, we recommend that users select very stringent cutoff values for h0 and l0 to reduce the false positive discovery rate because of poor sequencing quality and low coverage. for example, the user may use h0 = 0.9 or 0.95 instead of 0.8 and l0 = 0.1, or 0.05 instead of 0.2. if the user s dataset has good sequencing quality and high coverage, changing the cutoff values may not affect the results significantly. step 5 : provide genetic annotation and report findings for each hemimethylated cpg site, hmpl provides the following genetic information. gene : if a hemimethylated cpg site is located on a gene, hmpl will report the name of that gene.pomoter : if a hemimethylated cpg site is within a d - base long region (eg, d = 1000) of the promoter of a gene (ie, d - base before the transcription starting site of a gene), hmpl will report the name of that gene. gene : if a hemimethylated cpg site is located on a gene, hmpl will report the name of that gene. pomoter : if a hemimethylated cpg site is within a d - base long region (eg, d = 1000) of the promoter of a gene (ie, d - base before the transcription starting site of a gene), hmpl will report the name of that gene. the hmpl uses raw sequencing reads (in fastq format) as input in step 1 and step 2. in steps 3, 4, and 5, more detailed information about the input and output files of hmpl can be found in the user manual, which can be downloaded from http://hal.case.edu/~sun/hmpl/hmpl.user.manual.pdf. the preprocessing step of hmpl (part i) can be implemented with the following command (the command options of pre.hmpl.pl are explained in table 1). 1 -p -r [options ] the preprocessing pipeline ties the software and source code together with the appropriate dataflow to ensure that the correct output is achieved. users need to have perl, python 2.6, r, fastqc, and brat - bw software installed on their system. if users have finished the hemimethylation pre - processing pipeline and have obtained the files of combined cpg sites, they may only run the parsing analysis using the part ii of hmpl (ie, parse.hmpl.pl). the usage of part ii is given below (the command options of parse.hmpl.pl are explained in table 2). 1 [options ]. in order for users to test the hmpl, we have prepared a small dataset with 5 million sequencing reads (a fastq.fastq file), the human chromosome 22 reference sequence (a fasta.fa file), and the example scripts. users may align these 5-million reads to chromosome 22 and then identify hemimethylated sites using both part i and part ii of hmpl. users simply need to download the data, install the hmpl package, and then change the data path / directory in the example script accordingly to run the hmpl. it takes about 11 minutes to run this small dataset using a linux computer with 4 gb ram. in addition, in order for users to explore the different options and arguments of hmpl, we have prepared a file named readme. this file includes example scripts of running the hmpl using different command options and also explains to the user what to expect in the screen output. the small dataset, example scripts, and the readme file can be downloaded from the following web link : http://hal.case.edu/~sun/hmpl/hmpl.zip. in order to show the running time of hmpl in practice, we use two human example datasets. each dataset has ~50 million raw sequencing reads, which will be introduced in detail in the results section. using the linux server with dual quad - core 2.66 ghz xeon e5430 processor that has 4 gb ram for each core, it takes ~4 hours to run part i (the pre - processing part) of the hmpl, pre.hmpl.pl, if the reference index is provided. if the index file were not provided, it would first take about three to four additional hours to build a reference index for the whole human genome, which has about 3 billion bases (~3 gb data). therefore, it is more efficient to first build a reference index for the alignment tool brat - bw before running the hmpl. it requires ~19 minutes to run part ii of hmpl (the parsing pipeline, parse.hmpl.pl) when using uncombined input with the default coverage setting. the uncombined input means that positive and negative strand methylation levels of all cpg sites are provided as two separate files. if the positive and negative strand methylation data are combined, it will only take 15 minutes for hmpl to generate the results. if users have a faster linux server or high - performance computing clusters that have more memory and computing power, it will take much less time (eg, a couple of hours) to run the hmpl preprocessing pipeline (pre.hmpl.pl), and it will take just a few minutes to get the results of parsing and comparing two samples using the hmpl part ii (parse. the hmpl pipeline can be used to compare any two samples of bisulfite sequencing data. in this paper, we demonstrate the use of hmpl using publicly available bisulfite - treated methylation sequencing datasets for cell lines mcf10a and mcf7.25 these two samples are breast cancer cell lines. because a number of hypermethylated genes have been reported for breast cancer cells35 and there are hemimethylation patterns reported in individual genes,1113 it is very likely that many cpg sites have been hemimethylated in these two cell lines. the bisulfite methylation sequencing data of mcf10a and mcf7 and more information about these two cell lines can be found from the corresponding references.25,36 the sequencing reads of mcf10a and mcf7 are generated using the reduced representative bisulfite sequencing (rrbs) protocol.16 there are 54,295,326 and 50,054,248 sequencing reads for mcf10a and mcf7, respectively, and the read length is 50-base for each dataset. in order to identify hemimethylation singletons and clusters in both mcf10a and mcf7 and compare these two samples, we have run both part i (pre.hmpl.pl) and part ii (parse.hmpl.pl) of the hmpl. in this section, we mainly focus on showing the hemimethylation result, which is the summary of the hmpl part ii (parse.hmpl.pl) output. for a detailed description of the output files, see table 3. the hemimethylated singleton and cluster patterns are compared and summarized in figure 3. in this figure, i. singleton means comparing the singleton hemimethylated cpg sites in mcf10a and mcf7. consecutive polarity cluster shows the results of comparing the polarity (or reverse) clusters that include two consecutive cpg sites ; no other cpg sites are located between these two sites. non - consecutive polarity cluster means comparing the polarity (or reverse) clusters that include two cpg sites that are not consecutive. there is at least one cpg site located between these two sites, but there are either no sequencing reads (or data) or no hemimethylation sites between them. iv. non - polarity cluster refers to the clusters that do not have the polarity (or reverse) pattern (eg, the patterns shown in fig. figure 3 and table 4 show the results of comparing mcf10a with mcf7, which are summarized based on the output files named compare as shown in the last row of table 3. the comparison results indicate that the hemimethylation patterns between the non - tumorigenic sample mcf10a and tumorigenic sample mcf7 are different at some cpg sites and/or genomic regions. our pipeline hmpl has provided gene annotation files for all cpg sites by giving names of genes in which hemimethylated cpg sites are located, as well as the names of genes in whose promoter regions the hemimethylated cpg sites are located. the results of comparing mcf10a and mcf7 show that there are more polarity (or reverse) clusters (eg, fig. 1b) than the single - strand hemimethylation clusters (eg, fig. 1a). this may be as a result of the fact that the methylation sequencing data we have used are generated by the rrbs protocol,16 which only sequences a small percentage of cpg sites in a human genome. in fact, there are ~5% of the cpg sites with at least 3 coverage in the rrbs data we have analyzed. if we use the whole genome bisulfite sequencing (wgbs) data, it is very likely that more single - strand hemimethylation clusters would be identified. currently, we are not aware of any wgbs data for either mcf10a or mcf7. therefore, we have used the available rrbs data to demonstrate the usage of hmpl. even though rrbs data are not ideal for identifying all hemimethylation sites in an entire genome, we have found many hemimethylation singletons and clusters, which show the capability of our hmpl package. in fact, hmpl can be used to compare any two samples with data generated using either the rrbs or wgbs protocol. in order to see if the genes with hemimethylated cpg sites are biologically important or meaningful, we have further investigated the 532 genes by comparing them with oncogenes, breast cancer methylated genes, and transcription factors. the comparison results show that seven of these genes are methylated, 17 are oncogenes, and 62 are transcription factors. we have also conducted the gene set enrichment analysis (gsea) for these 532 genes using the gsea software package and the molecular signature database provided by the broad institute.37 the analysis results show that 87 genes are significantly represented in (or overlapped with) 10 cancer modules (with p - value < 0.05), which are gene sets that are significantly changed in a variety of cancer conditions. these 87 genes and the 10 cancer modules they belong to are provided in table 5. a detailed description of these 10 modules can be found online.38 the 532-gene list is included in the hmpl.zip file that can be downloaded from the following web link : http://hal.case.edu/~sun/hmpl/hmpl.zip. there are a number of alignment tools for bisulfite methylation sequencing data, such as brat,29 brat - bw,31 bsmap,39 bs seeker,40 bismark,41 methylcoder,42 rmapbs,43 pash,44 and batmeth.45 a comprehensive list of these tools can be found at omictools.com.46 among all these available tools, we have tested brat - bw, brat, and bsmap. we have found that they provide similar results, but brat - bw is faster. brat - bw is user - friendly and has the following useful features : (1) it can align both single - end and paired - end reads, (2) it can produce the acgt count for all cytosines in a genome, (3) it can account for overlapping paired - end reads, and (4) it can check strands. if users prefer another alignment tool, they can obtain the alignment results and methylation levels using their preferred alignment tool, and then run part ii of the hmpl to obtain hemimethylated singletons and clusters. if necessary, this can be done with some minor format changes of their alignment output files. reformatting is easy because the parsing pipeline of hmpl (ie, part ii) only requires data with the following columns that most alignment tools provide for two dna strands : chromosome, start position, end position, total number of sequencing reads, methylation level, and dna strand. the cutoff values used in hmpl, especially the ones provided in table 2 for the parsing pipeline (ie, part ii), should be determined depending on the sequencing quality and coverage. if the user has a sequencing dataset with very good quality and high coverage, changing the cutoff value may not significantly affect the results. however, if the sequencing dataset has low coverage and poor quality, changing the cutoff values may lead to very different results. for this case, we recommend that users set up very stringent cutoff values to reduce false discovery rates. we set up the default values based on findings in previous publications, some basic and common knowledge of methylation sequencing data, and our experience with bisulfite sequencing data ; however, every dataset is different. we suggest that the users first determine the quality and coverage of their data, and then try different values to see if their results are dramatically different. if results are dramatically different, users may choose results that are obtained based on stringent cutoff values, especially when they plan to do experimental validation. using results based on stringent cutoff values can ensure a high validation rate and then the user may expand their validation list by adding more hemimethylated singletons or clusters from the list obtained with less stringent cutoff values. ideally, it is best to have a list of known hemimethylated and nonhemimethylated sites to study the true and false discovery rates (or sensitivity and specificity) of hmpl. for the nonhemimethylated sites, we can choose the cpg sites that are located in the housekeeping genes, which are relatively stable and not likely to be methylated on either strand. in fact, housekeeping genes have been used for the purpose of negative control in previous methylation studies.4749 for the example dataset mcf7, using the coverage cutoff of 5 (that is, at least five reads to cover each strand) and other default settings, there are 532 genes with at least three hemimethylated sites. we compare these 532 genes with the 205 known housekeeping genes used in a previous study,49 and there is no overlap ; therefore, none of these 532 genes are housekeeping genes. as for the known hemimethylated sites, to the best of our knowledge, no available list of genes or sites can be used as a positive control. this is because genome - wide hemimethylation study is rarely done, and a few hemimethylated sites have been experimentally validated. however, we have decided not to use this approach because there is little knowledge about genome - wide hemimethylation patterns. with little known information, a simulation would be very arbitrary and the simulated data would not reflect true unknown patterns. the purpose of our hmpl development is to provide an exploratory tool for this research topic. for genes identified with a number of hemimethylated cpg sites, users may do further investigation by studying their biological functions and relationships with other genes using pathway analyses. first, the hmpl is designed for identifying hemimethylation only at cpg sites, but not at any non - cpg sites (eg, chg and chh sites, where h represents a, c, or t in a dna sequence). if users are interested, our algorithms may be modified to study the hemimethylation of non - cpg sites. second, our pipeline is developed for identifying hemimethylated sites (or clusters) by comparing two samples, but it is not designed for comparing multiple samples in two or more groups. because the hemimethylation study is an area with little research, hmpl is good for preliminary studies. as for the topic of identifying hemimethylation patterns in multiple samples and in multiple groups, more sophisticated statistical methods may be used, and our group is working on projects related to this approach. for the first step of the hmpl workflow, we have used the software package fastqc. even though fastqc is not designed for bisulfite - treated methylation sequencing data if users find some serious sequencing quality issues in their data, we recommend that they check data more thoroughly using other available software packages, such as saap - rrbs,50 bseqc,51 and methyqa,27 before they interpret their hmpl results. therefore, it is important to develop a software package to identify such patterns. to address this need, we have developed a new software package, hmpl, which includes both preprocessing and data parsing. for two samples, each with 50 million reads, it takes a few hours for hmpl to align the sequencing reads, and it only takes a few minutes to process the methylation level data. if users have obtained their coverage and methylation ratio data, part ii of hmpl can identify hemimethylation patterns in minutes. | dna methylation (the addition of a methyl group to a cytosine) is an important epigenetic event in mammalian cells because it plays a key role in regulating gene expression. most previous methylation studies assume that dna methylation occurs on both positive and negative strands. however, a few studies have reported that in some genes, methylation occurs only on one strand (ie, hemimethylation) and has clustering patterns. these studies report that hemimethylation occurs on individual genes. it is unclear whether hemimethylation occurs genome - wide and whether there are hemimethylation differences between cancerous and noncancerous cells. to address these questions, we have developed the first - ever pipeline, named hemimethylation pipeline (hmpl), to identify hemimethylation patterns. utilizing the available software and the newly developed perl and r scripts, hmpl can identify hemimethylation patterns for a single sample and can also compare two different samples. |
acute pancreatitis is characterized by the onset of parenchymal and peripancreatic fat necrosis with associated inflammation in a previously healthy individual. acute pancreatitis should be suspected in patients with acute severe abdominal pain and can be classified based on severity. the atlanta classification divides acute pancreatitis into two groups : interstitial edematous acute pancreatitis and necrotizing acute pancreatitis. the first category is characterized by pancreatic parenchymal and peripancreatic inflammation without necrosis, while the latter category involves inflammation and some degree of necrosis. the diagnosis of drug - induced acute pancreatitis first requires a diagnosis of acute pancreatitis. elevations in several biomarkers are indicative of pancreatitis, including serum lipase and amylase that are secreted in bulk by pancreatic acinar cells and thus are most commonly measured. other laboratory tests with diagnostic implications include serum trypsinogen, pancreatic proteases, c - reactive protein, interleukin-6 and interleukin-8. the next step in diagnosing drug - induced pancreatitis requires ruling out more common etiologies such as gallstone pancreatitis and ethanol - induced acute pancreatitis. the history should focus on previous symptoms and any record of gallstones, ethanol abuse, hypercalcemia, hypertriglyceridemia and trauma. serum amylase, lipase, triglyceride level, calcium level and liver function tests should be ordered. abdominal and endoscopic ultrasounds should be performed to evaluate for gallstones and other obstructive possibilities such as tumors of the pancreas head. orlistat is a pancreatic lipase inhibitor which is used to treat obesity. due to the increasing prevalence of obesity the indications of orlistat are body mass index (bmi) > 30 without any risk factors or bmi > 27 with additional risk factors such as hypertension, hyperlipidemia or diabetes. orlistat has been reported to be associated with adverse events such as hepatic cholestasis, subacute hepatic failure and hepatic necrosis. however, placebo - controlled studies have shown no relationship with acute pancreatitis [4, 5, 6, 7, 8, 9 ]. only four cases of acute pancreatitis associated with orlistat have been reported in the literature, and one of those cases did not demonstrate an increase in serum amylase [10, 11, 12 ]. we report an interesting case caused by orlistat use caught in the early stages of acute pancreatitis through imaging ; in addition, the case had significantly elevated serum amylase levels. a 54-year - old male with a history of hypertension was admitted to the emergency department with complaints of abdominal pain, nausea and vomiting lasting for 24 h. it was learned from his medical history that he had been using irbesartan for 7 years, and in addition to this drug, orlistat treatment had been started 7 days before for obesity. the obese - looking patient had a height of 1.72 m, a weight of 94 kg and a bmi of 31. he was afebrile with a blood pressure of 140/90 mm hg and a pulse of 102 bpm. the results were : white blood cells 12,600/l, serum amylase 2,409 u / l, c - reactive protein 136 mg / l and lactate dehydrogenase 835 u / l. abdominal computed tomography revealed peripancreatic fat tissue edema and a heterogeneous appearance of the pancreas (fig. on the third day after admission, abdominal pain, nausea and vomiting were improved. serum amylase levels were normal and the patient was told to take specific measures addressed to his obesity. alcohol use and gallstones are responsible for the development of more than 80% of acute pancreatitis cases. drug - induced acute pancreatitis has a good prognosis and can be diagnosed by exclusion. absence of gallstones, lack of a history of alcohol use with normal serum lipid and calcium levels and history of a recently started new medication support the diagnosis [13, 14 ]. orlistat blocks intestinal fat absorption by inhibiting gastric and pancreatic lipase and thus can cause symptoms such as rectal pain, oily feces, frequent defecation, nausea and vomiting. all these lead to a modification of the patient 's behavior and eating habits to avoid eating fat, with the patient consequently losing weight. orlistat is metabolized in the gastrointestinal tract and its direct destructive effect is shown in intestinal villi of animal models. however, the mechanism in the development of acute pancreatitis is not known ; a possible mechanism may be direct toxicity or hypersensitivity such as in other drug - induced acute pancreatitis. we report a case of drug - induced acute pancreatitis about 1 week after initiation of orlistat. the patient in our case had a very high serum amylase level, although imaging studies showed that he was in a very early stage of edematous pancreatitis. serum amylase levels were found to be normal in a previously reported case of acute pancreatitis associated with orlistat. therefore, acute pancreatitis must be kept in mind when faced with newly developed abdominal pain in patients under orlistat treatment. in addition, imaging techniques (such as hepatobiliary ultrasound and abdominal computed tomography) should be requested. more attention must be paid when planning to prescribe orlistat to patients if there are risk factors for acute pancreatitis (alcohol use, height, serum calcium and lipid levels). | orlistat is a pancreatic lipase inhibitor which is used to treat obesity. due to the increasing prevalence of obesity, orlistat use is thought to rise progressively. we report an interesting case caused by orlistat use caught in the early stages of acute pancreatitis through imaging ; in addition, the case had significantly elevated serum amylase levels. a 54-year - old male who had a history of orlistat treatment started 7 days before was admitted to the emergency department with complaints of abdominal pain, nausea and vomiting lasting for 24 h. abdominal computed tomography revealed peripancreatic fat tissue edema and a heterogeneous appearance of the pancreas. based on these findings, it was concluded that edematous pancreatitis was in its initial stage. orlistat is a drug that is increasingly widespread use due to obesity. more attention must be paid when planning to prescribe orlistat to patients if there are risk factors for acute pancreatitis (alcohol use, height, serum calcium and lipid levels). |
multidrug resistance (mdr) remains the major obstacle to success in a wide variety of advanced malignancies, particularly in solid cancers. the overexpression of p - gp is one of the important mechanisms involved in mdr [13 ]. p - gp - mediated cellular uptake and the efflux of anticancer drugs from cancer cells are responsible for the cell elimination of the drugs which in turn decreases their therapeutic effect. therefore, one of the best methods of reversing mdr and increasing the efficacy of anticancer drugs is to use the potent p - gp inhibitors modulating p - gp function and/or expression. it is an important step to use assays to evaluate the effect of candidate drugs as inhibitors of p - gp function in vivo after the drugs were found to have an mdr - reversing effect in vitro. for nonsurgical solid cancer patients, there are currently no direct assays measuring intratumoral drug concentrations or the alteration of p - gp function and/or expression. thus, alternative approaches to evaluate the degree of functional inhibition of p - gp in vivo will be a useful aid in the development of mdr modulators. p - gp is also expressed in normal tissues in which they seem to have an important role in the protection against xenobiotics. several authors reported that p - gp was expressed in lymphocytes including cd3, cd4, and cd8 t cells, as well as the cd56 natural killer (nk) cells, with the highest expression levels and activities observed in cd56 cells followed by cd8 cells [5, 6 ]. however, the physiological roles that p - gp plays in these cells are unclear. the p - gp in the lymphocytes appears functionally identical to that observed in multidrug resistant cells ; they have the same substrate and antagonist specificities [5, 7, 8 ]. since the expression and function of p - gp in cd56 cells are the highest, is rho123 accumulation widely used in cd56 populations as a surrogate indicator to evaluate the degree of functional inhibition of p - gp in clinical trials of p - gp inhibitors, for example, the work of tariquidar and zosuquidar. as mouse nk cells do not express cd56, we used rho123 accumulation in cd8 cells as a surrogate indicator to evaluate the reversal activity by p - gp reversors in the mouse mdr tumor - bearing model. the search for mdr modulators has extended to the natural products and their derivatives ; natural source compounds have become the most widely used of fourth - generation p - gp inhibitors because they are less toxic and more potent than the disappointing first- and second - generation mdr modulators [1214 ]. ch, manufactured by salification from cepharanthine, which is a biscoclaurine alkaloid, extracted from stephania cepharantha hayata has a variety of biological activities (figure 1) [1517 ]. recently, it has been reported that ch has an mdr - reversal effect, and p - gp inhibition is one of the reversal mechanisms of mdr in vitro [18, 19 ] ; however, the effect was not evaluated in vivo. we present here a method to detect the effect of the inhibitor of the p - gp function on rho123 accumulations of the cd8 cells in whole blood from the mdr tumor - bearing mouse. this method was first used to develop and validate the efficacy of ch against p - gp functions in the mouse model for potential use in subsequent clinical evaluations. the experiments were carried out on 24-month - old balb / c male mice, obtained from the henan experimental animal center (zhengzhou, china). the animals had been raised from birth in a specific pathogen - free environment and were handled according to the institutional guidelines complying with chinese legislation. hca / fap variants were developed from the parental hca hepatocarcinoma cells, which were obtained from keygen biotechnology co. ltd. (nanjing, china). by step - by - step, continuous exposure to increasing doses of fap, the overexpression of p - gp in the variant was confirmed by the western blot analysis and the cells that have been previously described. ch was supplied by the henan academy of medical and pharmaceutical sciences (zhengzhou, china). adriamycin (adr) was purchased from hisun pharmaceutical co., ltd (zhejiang, china). cisplatin (cddp) was purchased from qilu pharmaceutical co., ltd (shandong, china). 5-flurouracil (5-fu) was purchased from xudong haipu pharmaceutical co., ltd (shanghai, china). rho123 was purchased from sigma - aldrich (st. louis, mo, usa) and dissolved in pbs at a concentration of 1 mg / ml. the ficoll - histopaque mouse lymphocyte separation medium was obtained from solarbio science & technology co., ltd (beijing, china). the pe antimouse cd8 antibody (clone 53 - 6.7) and pe rat igg2, isotype ctrl were obtained from biolegend (biolegend corp. hca / fap cells were collected from the ascitic fluid of balb / c mice harboring 57 day - old ascitic tumor. the 1 10 hca / fap cells were injected intramuscularly in the right axilla of balb / c male mice selected for the experiment on day 0. the next day, the animals were randomized and divided into different groups ; each group comprised 10 mice. to study the effects of rho123 administration on peripheral blood cd8 cells, the retention of fluorescence in these cells was investigated as described previously with some modifications and a dose - response curve established. on the 8th day after the hca / fap injection, the mice received a single intravenous (i.v.) injection of rho123. the doses used were 0.5, 1.0, 2.5, 5.0, and 7.5 mg / kg, and the volume of administration was 10 l / g. one hour after administration, peripheral blood was collected and cell suspension was prepared as described below. for the investigation of the effects of p - gp inhibitors ex vivo, blood samples were collected as described below on the 8th day after hca / fap injection. ch was then added at final concentrations of 10.0, 5.0, and 2.5 m ; ver was added at a final concentration of 5.0 m, an equal volume of the vehicle was added as control. the samples were incubated at 37c for one hour before rho123 was added at a final concentration of 50 ng / ml, and the tubes were incubated further at 37c for one hour. cell suspension was prepared as described below. to study p - gp function in vivo, rho123 was injected with or without ch or ver as described before with small modifications on the 8th day after hca / fap injection. briefly, mice, respectively, received a single intravenous (i.v.) injection of the vehicle as control ; 2.5, 5.0, and 10.0 mg / kg of ch or 2.5 mg / kg of ver followed one hour later by a single i.v. after one hour, blood samples were collected and cell suspension was prepared as described below. peripheral blood was collected from the orbital sinus of each mouse in a tube containing k2edta as an anticoagulant. mononuclear cells were obtained by the separation of anticoagulated blood by a ficoll - histopaque density - gradient centrifugation and transferred to 1.5 ml eppendorf tubes and kept on ice. cells were washed twice with ice - cold phosphate buffered saline (pbs, ph 7.4). after that, the pellet was gently resuspended in ice - cold pbs and adjusted to a concentration of 1 10 cells / ml. single - cell suspensions were incubated with either the pe - conjugated antimouse cd8 antibody or pe - conjugated rat igg2, isotype ctrl as a negative control. after staining for 30 minutes in the darkness at 4c, the cells were washed twice with ice - cold pbs and then resuspended in pbs and kept on ice in the dark until analyzed as previously described. a life - gate based on forward scatter (fsc) and side scatter (ssc) parameters were made to analyze only viable cells ; other gates were made to determine the subpopulations. amplifier settings for fsc and side ssc were used in linear mode and those for fluorescence channels were used in a logarithmic mode. fluorescence compensation was manually set for fl1 channel (rho123) and fl2 channel (pe) with single rho123-stained cells and pe - stained cells separately. multicolor flow cytometry analyses were used to evaluate the proportions of the cd8 population and the mean fluorescence intensity (mfi) of rho123 in the population. cells were analyzed on epics - xl mcl, and data were analyzed with expo32 adc software (beckman coulter, fullerton, calif, usa). to evaluate the antitumor effect of fap chemotherapy protocol plus various concentrations of ch or ver in vivo as previously described. twenty four - hours after the hca / fap injection, the mice were randomly divided into different groups and treated as follows : group i : control (normal saline i.v. injected consecutively from day 1 to 9),group ii : 10 mg / kg of ch alone i.v. injected from day 1 to 8, group iii : fap chemotherapy : 4.2 mg / kg of adr and 85.0 mg / kg of 5-fu i.v. injected on day 1, 2.8 mg / kg of cddp i.v. injected from day 1 to 8, group iv : 2.5 mg / kg of ch i.v. injected from day 1 to 8 with fap chemotherapy, group v : 5.0 mg / kg of ch i.v. injected from day 1 to 8 with fap chemotherapy, group vi : 10.0 mg / kg of ch i.v. injected from day 1 to 8 with fap chemotherapy, group vii : 2.5 mg / kg of ver i.v. injected from day 1 to 8 with fap chemotherapy. control (normal saline i.v. injected consecutively from day 1 to 9), 10 mg / kg of ch alone i.v. injected from day 1 to 8, fap chemotherapy : 4.2 mg / kg of adr and 85.0 mg / kg of 5-fu i.v. injected on day 1, 2.8 mg / kg of cddp i.v. injected from day 1 to 8, 2.5 mg / kg of ch i.v. injected from day 1 to 8 with fap chemotherapy, group v : 5.0 mg / kg of ch i.v. injected from day 1 to 8 with fap chemotherapy, 10.0 mg / kg of ch i.v. injected from day 1 to 8 with fap chemotherapy, 2.5 mg / kg of ver i.v. injected from day 1 to 8 with fap chemotherapy. the tumor length (l) and width (w) were measured, and the tumor weight (wr) was calculated as follows : (1)wr = 12 l w2. the percent of tumor growth inhibition was calculated on day 8 by comparing the average values of the treated groups with those of the tumor - bearing control group. for all the experiments, data are presented as the mean sd. statistical comparison between the groups with different doses or concentrations tests for significant differences among the groups were done using one - way anova with multiple comparisons (fisher 's pairwise comparisons) using spss 13.0 (ibm software, somers, ny, usa). administration of rho123 (0.57.5 mg / kg) in mice produced a dose - dependent fluorescence fashion in peripheral blood cd8 cells (figure 2). all doses were well tolerated without significant toxicity. the lower dose of rho123, 2.5 mg / kg, was selected to investigate the effects of ch on the accumulation of rho123 in peripheral blood cd8 cells. using this dose, rho123 accumulations were increased after administration of either ver (positive control) or ch and presented a clear dose - dependent relationship with ch (2.510 mg / kg) compared with vehicle control, as shown in figure 3. the efficacy of the p - gp reversors was not equal ; ver was less efficient and potent than ch. there has similar result in ex vivo blood samples ; the mfis of cd8 + cells in the samples treated with the vehicle, 5.0 m of ver and 2.5, 5.0 and 10.0 m of ch were 14.4 0.3, 20.0 0.3, 22.9 0.5, 25.4 1.0 and 29.8 0.9, respectively. the mfi was related to the presence of ch in a concentration pattern in ex vivo blood samples, as shown in figure 4. this demonstrated that p - gp is active in cd8 cells and can be modulated by ch in vivo and ex vivo. 2.5 mg / kg of ver or 2.5, 5.0, and 10.0 mg / kg of ch were evaluated in the hca / fap tumor - bearing mouse. the fap combination chemotherapy plus 10.0 mg / kg of ch provided the maximum antitumor activity of the treatments studied. compared with the fap chemotherapy group, the antitumor effect was increased in a dose - dependent manner when the fap combined chemotherapy plus various concentrations of ch was used, as shown in table 1. hepatocellular carcinoma (hcc) represents the third leading cause of cancer - related death. chemotherapy consisting of fap was commonly used to treat hcc in clinical situations ; however, hcc is often resistant to these drugs. therefore, one of the key approaches is to overcome drug resistance in the success of chemotherapy against hcc. previous studies of p - gp inhibitors such as ver, cyclosporine a, and its analog psc833 [2628 ] have been limited because of its serious toxicity at the time of dosage for reversing drug resistance. an important step to develop the mdr reversors targeting p - gp is to determine the inhibition level of p - gp in the preclinical animal model. an ideal approach is to detect the real concentrations of the investigated drug directly in vivo, but it is almost impossible, even in the animal model. if there is no related data on the inhibition level of p - gp, it is difficult to interpret the efficacy of p - gp inhibitors in pre- or clinical trials. recently, the functional study of p - gp was of great concern. among lymphocytes in the mouse, cd8 cells expressed the highest levels of p - gp followed by cd4 > cd3 [6, 21, 2932 ]. since blood samples are easy to be collected, we selected peripheral blood cd8 cells as a surrogate indicator to investigate the alteration of p - gp activities before and after the administration of p - gp inhibitors and then evaluate the reversal effect of the drug in a solid - tumor - bearing mouse. rho123 is a fluorescent substrate and has been shown to be a sensitive reagent for the detection of p - gp function by flow cytometry [14, 33 ]. some authors reported that rho123 was a substrate for mrp and p - gp, but leite. reported that classical mrp1 reversors were unable to enhance rho123 accumulation in lymphocytes suggesting that mrp1 might not be active or rho123 is not a good substrate for mrp1 expressed in these cells therefore, the alteration of rho123 fluorescence intensities in these cells responded to the alteration of the p - gp function. in this study, we demonstrated that the mean fluorescence intensities of cd8 cells were related to the dose of rho123 in a dose - dependent manner suggesting that it is a good choice to use the accumulation of rho123 in cd8 cells as a surrogate indicator for the study of p - gp inhibitors. we further studied the modulation effect of ch in vivo and ex vivo ; the results showed that the mean fluorescence intensities of cd8 cells were related to ch in a dose- or concentration - dependent manner either in vivo or ex vivo. this suggested that the activities of p - gp were inhibited by ch. in our previous study, it was shown that the enhancement of cytotoxicity of fap against cultured hca / fap cells by ch occurs in vitro ; the efflux rate of rho123 in hca / fap cells was 2 times less than that of hca cells after 1 hour explosure to ch plus fap. mice is 0.47 times of clinical dosing of fap protocol, that is, 85.0 mg / kg of 5-fu, 4.2 mg / kg of adr, and 2.8 mg / kg of cddp. in the present study, we established an hca / fap solid tumor mouse model and assessed the antitumor effect of fap combination protocol plus ch. the i.v. dosing for a mouse is 42.50 mg / kg of 5-fu, 2.10 mg / kg of adr, 1.40 mg / kg of cddp. dose of 10.0 mg / kg ch partly inhibited the tumor growth, but the effect was weak ; this is consistent with conclusions drawn from other authors ' reports : a single i.v. dose of 5.0 mg / kg ch has no antitumor effect (data not shown). when fap combined chemotherapy plus various concentrations of ch was used, the antitumor effect was increased in a dose - dependent manner. therefore, with this correlation with the results of the inhibition of p - gp function by ch in cd8 cells, we think that the antitumor activity recovered by ch in a dose - dependent manner was related to the inhibition of the p - gp function. all of the efficacious combination protocols with ch appeared to be well tolerated, as indicated by the minimal changes in body weights of the various treatment groups (as shown in table 2). since an ld50 of ver for the i.v. administration in mouse was reported to be 7.6 mg / kg, but a single i.v. dose of 5.0 mg / kg ver caused the death of 60% of animal in the treated balb / c mice (data not shown), we selected the dose of 2.5 mg / kg as a positive control. there is no mortality in the fap treated group ; we think the 20% mortality in the fap plus ver treated group was caused by the increasing toxicity when ver combined with adr. although one can argue that what occurs at the level of peripheral blood cd8 cells may not accurately reflect the real inhibition level of p - gp in the solid tumor in mouse, tc - sestamibi scans performed in some studies have shown effective p - gp inhibition in both normal tissues and solid tumors [9, 38 ], suggesting the inhibition level of p - gp was modulated simultaneously. we have to note that no model is perfect and there are obvious differences between humans and mice ; the balb / c mouse is unique for its features, making even more difficult extrapolations to human conditions. with regard to the results obtained, we conclude that ch is a potent p - gp inhibitor for clinical application. although limitations may exist in the surrogate assay in cd8 cells in the mouse model, we are convinced that this assay can provide us with much useful information in screening potential p - gp inhibitors in vivo. | the purpose of this study was the use of rhodamine 123 (rho123) accumulation in peripheral blood cd8+cells as a surrogate indicator to evaluate the modulating effect of p - glycoprotein (p - gp) inhibitors in the multidrug resistance (mdr) tumor - bearing mouse model. rho123 was administered to mice, and the fluorescence level in cd8 + cells was measured. cepharanthine hydrochloride (ch) and verapamil (ver), two p - gp inhibitors, were administered to mice 1 hour prior to rho123 administration in vivo or added to peripheral blood 1 hour prior to rho123 addition ex vivo. the tumor inhibition effect of 5-fluorouracil / adriamycin / cisplatin (fap) protocol plus ch was also investigated. a concentration- or dose - response relationship was shown between the concentration and dose of ch and rho123 accumulation or the antitumor activity. in conclusion, the measurement of rho123 accumulation in cd8 + cells provides a surrogate assay for the screening of candidate p - gp inhibitors in preclinical trials, and ch is effective in modulating p - gp - mediated mdr in vivo. |
the fundamental principle of bonding to dental hard tissues is based on micromechanical interlocking of the adhesive resin with dentin / enamel surfaces.1,2 while bonding to enamel depends on the micromechanical retention to the etched substrate, bonding to dentin relies on hybridization with the exposed collagen mesh.3 the current dental adhesive systems rely on different strategies for bonding to the dental substrates. in etch - and - rinse systems, a conditioner (usually 3040% phosphoric acid) selectively dissolves the hydroxyapatite crystals and creates spaces for infiltration;3,4 the acid gel needs to be removed and the dentin kept moist for adhesion.5,6 however, managing the proper moisture condition is a critical procedure. in order to reduce the technique - sensitivity and simplify the bonding procedures, self - etching adhesive were introduced.4,7,8 the concept of self - etching is based on the use of a non - rinse acidic primer that simultaneously etch and penetrate the dental substrates,9 presenting either one or two application steps. in two - step systems, the priming step is followed by application of free - solvent adhesive resin.8 although two - step systems generally present adequate bonding performance, new single - step systems are constantly introduced into the market. these systems are mixtures of acidic resin monomers, additives, solvents and water.9,10 water is required to enable dissociation of the acidic monomers responsible for etching.9 however, excess water might interfere with the polymerization of the adhesives.11 thus, solvents, such as ethanol and acetone are added to accelerate water elimination.9 most manufacturers recommend that self - etching adhesive should be applied to dry substrate. therefore, the water contained in the adhesive needs to be sufficient to ionize the acid monomers without, however, compromising the polymerization process.9 the first generation single - step self - etching adhesives were presented in two bottles that needed to be mixed before use. this mode of presentation permits water and acidic monomers to be in different bottles, hence the ionization process does not occur before the components of the two bottles were mixed, allowing the addition of a high water content. however, some recently introduced one - step self - etching systems present only one bottle in order to simplify the procedure by eliminating the mixing of solutions. these systems probably present lower water content and therefore might need water from the substrate for proper ionization. despite the adequate performance on dentin, one - step self - etching adhesives present limitation in properly bonding to enamel, probably due to the relatively high ph compared with phosphoric acid.1,4 conventional bonding tests usually do not allow the evaluation of dentin and enamel substrates in the same specimen. shear bond strength tests with reduced bonding area have been widely used because this reduction allows regional mapping and preparing multiple specimens from the same tooth.12 the experimental setup of this methodology allows both substrates to be evaluated in the same specimen. in addition, the orientation of enamel prisms and dentin tubules obtained with this type of preparation are close to the orientation presented in the lateral walls of tooth cavities in clinical situation.13 the aim of this study was to evaluate the bond strength of three one - step adhesive systems to dry or moist dentin and enamel. the null hypotheses tested were : (1) the moisture condition of substrate would not affect the bond strengths ; and (2) no significant differences in bond strength between enamel and dentin would be detected. thirty human third molars stored in 0.05% thymol saline solution for no more than 3 months were used. in order to obtain two halves, the teeth were sectioned to the mesio - distal axis, parallel to the long axis of tooth, using a slow speed diamond saw under water cooling. each half was embedded in polystyrene resin to facilitate handling, keeping the buccal / lingual surfaces exposed. the surfaces were wet - ground with 320-grit sic papers (carburundum, vinhedo, sp, brazil) until a flat surface on both enamel and dentin was obtained, with sufficient area to build up resin composite cylinders (1 mm in diameter), as shown in figure 1. the dental surfaces were wet - polished with 600-grit sic papers to standardized the smear layer. the adhesive systems used in this study, with their batch numbers, manufacturers and compositions are listed in table 1. the substrate surface was extensively air - dried for 30 s with oil - free compressed air. the surface was either kept dry (no water rinsing) or left wet after rinsing it for 10 s with 2.5 l of distilled water using a pipette (micropipet, pipetman, gilson, ny, usa). for application of adper prompt, one drop of each bottles a and b were mixed, applied to dental surface and agitated for 15 s. an additional layer was applied, gentle air - dries and light polymerized for 10 s. for ibond samples, three consecutive coats were applied, left undisturbed for 30 s and then gently air - dried. this adhesive was light polymerized for 20 s. for application of xeno iii, equal amounts of bottles a and b were mixed, applied to tooth substrates and left undisturbed for at least 20 s. after brief air - drying, the adhesive was light polymerized for 10 s. after the adhesive procedures, polyvinyl siloxane (aquasil, dentsply detrey, konstanz, germany) molds with a cylinder - shaped orifice (1 mm in inner diameter 2 mm in height) were individually placed onto the dentin and enamel surfaces. the composite resin filtek z-350 (3 m espe, st. the resin composite was light polymerized for 20 s. light polymerization procedures were performed using the quartz - tungsten - halogen curing unit optilux 501 (demetron kerr, orange, ca, usa) with approximately 650 mw / cm irradiance. two cylinders were made on both dentin and enamel substrates for each tooth (n = 4 per specimen), as shown in figure 2. the embedded specimens were attached to the testing device and each resin composite cylinder was tested on a mechanical testing machine (emic dl 2000, so jos dos pinhais, pr, brazil). a thin steel wire (0.2 mm in diameter) was looped around each cylinder and a shear load was applied to the base of the cylinder at a crosshead speed of 0.5 mm / min until failure (figure 2). the average value of the two bonded cylinders for each substrate in the same specimens was recorded as the shear bond strength for that specimen. data were submitted to three - way anova (dental substrate moisture condition adhesive system) and the tukey test at a 95% confidence level. the fractured specimens were mounted on aluminum stubs, coated with gold (scd 050, baltec, vaduz, liechtenstein) and evaluated by scanning electron microscopy (jsm-5600lv, jeol, tokyo, japan). the failure modes were classified as follows : type 1 adhesive failure between bonding agent and substrate ; type 2 cohesive failure within the substrate ; type 3 - cohesive failure within the composite resin ; type 4 - cohesive failure within the adhesive ; type 5 mixed failure. the statistical analysis showed the factors substrate (p<.01) and adhesive system (p<.05) were significant, as was the interaction between adhesive system and moisture condition (p<.01). the results of the tukey s test for the interaction and substrate are shown in tables 1 and 2, respectively. in the moist substrate, ibond presented higher values in the moist condition, whereas xeno iii showed a better performance on the dry substrate. the evaluated one - step self - etching adhesives showed higher shear bond strength to dentin than to enamel. the failure modes of the tested samples to enamel and dentin are shown in figures 3 and 4, respectively. in enamel adper prompt presented some cohesive failures within the adhesive, mainly under the dry substrate condition. for dentin, the cohesive failures within the adhesive were predominant, except for xeno iii applied to moist dentin. irrespective of the moisture condition or adhesive system used, the shear bond strength to enamel was lower than to dentin. enamel has a higher mineral content than dentin, and it requires a solution with lower ph to etch it.4,14,15,16 despite the relatively low ph of the one - step self - etching adhesives used in this study, they may have etched the enamel in a less effective manner compared with dentin. this may have resulted in incomplete penetration of the adhesive resin and creation of a more heterogeneous inter - diffusion zone, reducing in bond strengths. another hypothesis is that the re - precipitation of calcium phosphates may occur into partially desmineralized interfibrillar spaces, impairing the adhesive penetration.17 similar results of better performance of one - step self - etching adhesives bonded to dentin than enamel are reported by burrow using micro - shear testing. regarding the moisture conditions of substrates, only ibond presented higher bond strength values when applied on the moist substrates. this result may indicate that this adhesive needs more water to effectively dissociate the acidic monomers and etch the substrate. ibond is presented in a single bottle, which means that the acidic monomers and the solvents (including water) are together in the same solution. this presentation does not allow a high water fraction to be mixed in the solution. furthermore, acetone is used as a co - solvent to aid in eliminating the excess water.9,19 the presence of acetone may allow the use of ibond on moist substrates, without compromising its polymerization and performance. however, about 60% of the failures occurred cohesively within the adhesive on dentin. the higher intrinsic water content of dentin may increase the water fraction and compromise the effective polymerization of the adhesive.20 on the other hand, xeno iii presented the highest values on the dry substrate. this mode of presentation permits water and the acidic monomers to be in different bottles. thus, the ionization process does occur before the components of the two bottles are mixed, allowing the addition of a high water content. the possibility of having more water allows this adhesive to efficiently etch both enamel and dentin. despite presenting ethanol as co - solvent to accelerate water elimination, a higher amount of water, can interfere with the adhesive polymerization and reduce the bond strength.2022 in dentin, this effect was more pronounced and there were cohesive failures within the adhesive, mainly under the dry dentin condition. again, this probably occurred because of the higher intrinsic water content of this substrate. the excess of water may also compromise the diffusion of adhesive into the etched substrate.23,24 thus, more mixed and adhesive failures were detected. this system, like xeno iii, is presented in two bottles ; which allows a high amount of water in it composition. the water content of adper prompt seems to be more balanced, and the adhesive presented similar values under both moisture conditions. however, the bond strength values of adper prompt were similar to the lowest values obtained with xeno iii (moist) and ibond (dry). furthermore, it was observed that this adhesive presented more cohesive failures within the adhesive than the others. this can be explained by the absence of a co - solvent, which makes it difficult for the excess water to be eliminated and compromises the performance of this adhesive.9,25 the self - etching adhesives were developed in an endeavor to reduce the technical sensitivity of etch - and - rinse adhesive systems. the main limitation of the latter is determining the optimal moisture condition for adhesive application after rinsing dentin to eliminate the phosphoric acid gel.25 the present study demonstrated that that the moisture condition is also essential for the proper performance of some one - step self - etching adhesives. however, the optimal moisture condition seems to be material dependent and should be investigated in further studies. based on the results of the present study, it can be concluded that the : xeno iii and ibond presented the highest bond strength on dry and moist substrates, respectively. | objectivesthe aim of this study was to evaluate the bond strength of one - step adhesive systems to dry or moist dental substrate.methodsthirty human third molars were sectioned into two halves, in the mesio - distal direction, parallel to the long axis of the tooth. each half was embedded in a polystyrene resin cylinder so that the buccal / lingual surface remained exposed. this exposed surface was abraded to obtain both flat exposed enamel and dentin. the samples were randomly allocated according to the adhesive system (xeno iii, adper prompt and ibond) and moisture condition (dry and moist). the substrates were air - dried for 30 s for dry condition, while the moist substrates were re - wet with 2.5 l of distilled water after drying. after the adhesive procedures, two resin composite cylinders were build - up on dentin and enamel substrates, totaling four per sample. a shear load was applied to the samples at a crosshead speed of 0.5 mm / min until failure. data were statistically analyzed by three - way anova and the tukey test (=0.05).resultsthe evaluated one - step adhesives showed higher bond strength to dentin than enamel. the ibond presented better bond performance to moist substrate and xeno iii to dry substrate. the moisture condition did not interfere in the performance of adper prompt. the xeno iii and ibond presented higher bond strength than the other adhesives to both dry and moist substrates.conclusionsthe moisture condition of substrate interfered in the performance of one - step self - etching adhesives and the best moisture condition was material dependent. |
. it is characterised not only by the movement abnormalities, but also by cognitive impairment and abnormal behaviour, as well as weight problems and sleep disturbances. hd patients experience a complex variety of movement problems, which include not only chorea, but also akinesia and bradykinesia. a number of studies have assessed impairment of simple motion sequences in hd patients as a way of quantifying the progression of symptoms [2, 7, 12, 15 ]. sequential hand movements in hd patients have been examined at various stages of the condition, with markedly slower execution of movements by the hd patients when compared with controls. as well as performing movements more slowly, hd patients are also slower in switching from one movement to the next. garcia ruiz and colleagues studied the degree of bradykinesia and timing in genetically confirmed hd individuals compared with controls, using the four capit timed tasks previously used for pd. these results have been reproduced in more recent studies that reported significantly reduced tapping rates in manifest hd patients as compared to controls, but not premanifest individuals ; unified huntington s disease rating scale (uhdrs) motor scores and duration of the disease were highly correlated with the tapping results, something which did not correlate with the cag repeat lengths [3, 1315 ]. longitudinal studies have shown a significant decline in tapping rate over a period of 3 years in manifest hd patients, and a strong correlation between uhdrs scores and the motor tests. furthermore, the variability of finger tapping (using target intervals of 600 and 1,200 ms) correlated with an index of the probability of motor onset, estimated from cag length and age. quick and easy - to - use hand tapping devices have enabled the number of taps in 30 s, and the variability in tapping rhythm and fatigue over the testing period, to be measured. initial cross - sectional testing of hd patients using an early model of such a device showed that the tapping frequency correlated significantly with the motor uhdrs and independence scores. longitudinal data from a small cohort followed over 10 years revealed that this correlation was maintained over time, suggesting the technique may provide an objective measure of disease progression. recently, a large study, trackhd, used a force transducer to measure paced finger tapping in premanifest and manifest hd patients ; significant differences between premanifest and manifest patients were found, again suggesting that finger - tapping measures are an important way of monitoring the progression of the disorder. here we present a pilot study, using a new portable device in which the sensors are activated purely by contact and are independent of force ; this facilitates the rapid collection of finger - tapping data, and allows the possibility of introducing modifications of the basic task that may have diagnostic utility. because it provides information not merely about average tapping rates, but about the statistical distribution of the individual intervals between responses, it enables the behaviour to be characterised more specifically and quantitatively, sometimes revealing aberrant patterns of response that would not be detected by conventional average measures ; this is likely to aid monitoring and diagnosis. the present study was approved by the local regional ethics committee and was conducted in the john radcliffe hospital, oxford, uk. all had a positive genetic test for hd, and were evaluated by an experienced clinician (ahn) using a standard neurological examination and the uhdrs to ascertain whether individuals had motor manifestation of huntington s disease (table 1). there were two experimental groups : eight in a manifest group (mg) with overt motor signs, and a small aged - matched group of three controls (c). all participants gave their informed consent after the procedures had been explained to them.table 1characterisation of the manifest (m) patient group and controls (c)m (n = 8)c (n = 3)age : mean sd54.50 10.0854.33 7.81cag repeats42.17 1.80n / auhdrs (motor score) (mean, range)31.75 (1181)n / atotal functional capacity12.00 0.71n / avf (mean, range)26.86 (1745)n / ahand tapping (reciprocal median latency)mean se (s) single4.08 0.604.95 0.85 alternating3.18 0.414.06 0.53 pronating2.14 0.303.49 0.74 (sd of main distribution)mean se (s) single0.75 0.140.35 0.12 alternating0.49 0.060.44 0.21 pronating0.41 0.030.41 0.07except where stated, all figures are mean 1se characterisation of the manifest (m) patient group and controls (c) except where stated, all figures are mean 1se the tapping pad (fig. 1 left : ober consulting poland) has two touch sensors each of 60-mm diameter, their centres 115-mm apart ; each has an associated blue led providing feedback whenever one of the sensor active fields is touched. the sensors are activated purely by contact, independent of force, as they work by monitoring electrical impedance, with a threshold that is automatically adjusted to the background noise level. left and right taps are recorded independently, at a sampling frequency of 1 khz (resolution 1 ms), for a fixed period of 30s. three protocols were used, and in each case the subject used their dominant hand : (1) alternation : the instructions were to tap the left and right sensors alternately as rapidly as possible using the tip of the index finger ; (2) pronation / supination : alternately as before and as rapidly as possible, but with the hand in the supine position when tapping medially side, but prone on when tapping laterally, using the finger nail rather than finger tip ; (3) single : tapping the right sensor only, with the index finger. for each task, the successive intervals between taps were recorded, generating histograms of the distribution of intervals between consecutive taps. the statistics of these inter - tap intervals for hand tapping was then analysed using the computer application spic (saccadic programming and instrumentation computer), calculating best - fit parameters (and) of the later (linear approach to threshold with ergodic rate) model.fig. 1left the portable device for recording hand tapping. right a typical reciprobit plot of hand - tapping interval data from one subject in this study tapping alternately on the two sides : responses for right and left movements are combined. the points represent a cumulative histogram of the latencies (intervals between taps), plotted on a probability scale as a function of reciprocal latency left the portable device for recording hand tapping. right a typical reciprobit plot of hand - tapping interval data from one subject in this study tapping alternately on the two sides : responses for right and left movements are combined. the points represent a cumulative histogram of the latencies (intervals between taps), plotted on a probability scale as a function of reciprocal latency the present study was approved by the local regional ethics committee and was conducted in the john radcliffe hospital, oxford, uk. all had a positive genetic test for hd, and were evaluated by an experienced clinician (ahn) using a standard neurological examination and the uhdrs to ascertain whether individuals had motor manifestation of huntington s disease (table 1). there were two experimental groups : eight in a manifest group (mg) with overt motor signs, and a small aged - matched group of three controls (c). all participants gave their informed consent after the procedures had been explained to them.table 1characterisation of the manifest (m) patient group and controls (c)m (n = 8)c (n = 3)age : mean sd54.50 10.0854.33 7.81cag repeats42.17 1.80n / auhdrs (motor score) (mean, range)31.75 (1181)n / atotal functional capacity12.00 0.71n / avf (mean, range)26.86 (1745)n / ahand tapping (reciprocal median latency)mean se (s) single4.08 0.604.95 0.85 alternating3.18 0.414.06 0.53 pronating2.14 0.303.49 0.74 (sd of main distribution)mean se (s) single0.75 0.140.35 0.12 alternating0.49 0.060.44 0.21 pronating0.41 0.030.41 0.07except where stated, all figures are mean 1se characterisation of the manifest (m) patient group and controls (c) except where stated, all figures are mean 1se 1 left : ober consulting poland) has two touch sensors each of 60-mm diameter, their centres 115-mm apart ; each has an associated blue led providing feedback whenever one of the sensor active fields is touched. the sensors are activated purely by contact, independent of force, as they work by monitoring electrical impedance, with a threshold that is automatically adjusted to the background noise level. left and right taps are recorded independently, at a sampling frequency of 1 khz (resolution 1 ms), for a fixed period of 30s. three protocols were used, and in each case the subject used their dominant hand : (1) alternation : the instructions were to tap the left and right sensors alternately as rapidly as possible using the tip of the index finger ; (2) pronation / supination : alternately as before and as rapidly as possible, but with the hand in the supine position when tapping medially side, but prone on when tapping laterally, using the finger nail rather than finger tip ; (3) single : tapping the right sensor only, with the index finger. for each task, the successive intervals between taps were recorded, generating histograms of the distribution of intervals between consecutive taps. the statistics of these inter - tap intervals for hand tapping was then analysed using the computer application spic (saccadic programming and instrumentation computer), calculating best - fit parameters (and) of the later (linear approach to threshold with ergodic rate) model.fig. right a typical reciprobit plot of hand - tapping interval data from one subject in this study tapping alternately on the two sides : responses for right and left movements are combined. the points represent a cumulative histogram of the latencies (intervals between taps), plotted on a probability scale as a function of reciprocal latency left the portable device for recording hand tapping. right a typical reciprobit plot of hand - tapping interval data from one subject in this study tapping alternately on the two sides : responses for right and left movements are combined. the points represent a cumulative histogram of the latencies (intervals between taps), plotted on a probability scale as a function of reciprocal latency figure 1 (right) shows a typical distribution plot from one of the participants in the study. as usually found for saccadic and evoked manual reaction times, distributions of the intervals between taps in this task are skewed, with a long tail of longer latencies, and in general the reciprocal of reaction time, or promptness, follows a gaussian distribution (and is therefore more amenable to statistical analysis). consequently if inter - tap interval distributions are plotted cumulatively, on a probit scale, using a reciprocal abscissa (a reciprobit plot), they will be expected to generate a straight line, as seen on fig. 1 (right). such a distribution can be fully described by just two parameters : these are, its mean (which is also the reciprocal of the median latency), and, its standard deviation. a large value of corresponds to increased promptness or speed of response, and thus a shorter interval ; because of the reciprocal relationship, the units for these two parameters are s or hz. the best - fit values of the parameters can be determined automatically by minimisation of the kolmogorov the observed distribution of reciprocal inter - tap intervals has the expected normal distribution, but in some cases significant, idiosyncratic deviations are observed. figure 2a demonstrates a bimodal pattern of response, possibly analogous to the population of 2b) is the existence of a distinct sub - set of aberrantly long inter - tap intervals (around 600700 ms), probably due to inattention : in this case they represent only some 5% of all trials. 2d shows responses from one manifest hd patient with remarkably long inter - tap intervals, and very marked differences between the three types of task. an important point to note is that many of these characteristic features would have been entirely missed by the conventional technique of simply counting the total number of taps in a fixed time - period : they are only revealed by quantitative analysis of the distribution of individual intervals.fig. 2examples of unusual latency distributions. a a subject showing marked bimodality in the alternation task, with one group of responses around 300 ms and another around 450 ms : responses in each direction are very similar. b a subject showing sporadic inattention in the pronation task : in both directions a small group of responses (top right) is markedly delayed relative to the main distribution. c marked left / right difference ; responses to the right are about 40 ms faster (alternation task). d a manifest hd patient with exceptionally slow responses, affecting all three types of movement, but most marked for the pronation / supination task examples of unusual latency distributions. a a subject showing marked bimodality in the alternation task, with one group of responses around 300 ms and another around 450 ms : responses in each direction are very similar. b a subject showing sporadic inattention in the pronation task : in both directions a small group of responses (top right) is markedly delayed relative to the main distribution. c marked left / right difference ; responses to the right are about 40 ms faster (alternation task). d a manifest hd patient with exceptionally slow responses, affecting all three types of movement, but most marked for the pronation / supination task best - fit values of and were estimated for all participants and conditions. figure 3 shows the average values of the parameters broken down by type of response and category of participant (controls and manifest). when comparing the three different tasks for the hd participants, is significantly different between the alternation and pronating tasks (p = 0.0011) and between pronating and single (p = 0.0024), and is significantly different for pronating versus alternating (p = 0.045), but no other comparisons are significant at p = 0.05. when comparing the patients with the controls, for all three tasks the patients show the expected reduction in (equivalent to increased time between taps), but because of the very small group sizes in this pilot study, the only difference seen in fig. 3, that is significant at p = 0.05 (unpaired t test), is for with single tapping (p = 0.036).fig. 3average values of (left) and (right) analysed by type of response and category of participant (c control, m manifest hd). error bars show 1se average values of (left) and (right) analysed by type of response and category of participant (c control, m manifest hd). error bars show 1se it is instructive to plot and as a scatter plot for the two groups (fig. 4). what is then seen is an apparent segregation between the manifest patients (m) and the controls ; once again, it would obviously be desirable to have more participants than was available for this preliminary evaluation.fig. 4scatter plot of versus for the two groups of participants performing single tapping ; despite the very small numbers in this pilot study, the manifest hd patients (red) seem to form a distinct cluster from the controls (black) scatter plot of versus for the two groups of participants performing single tapping ; despite the very small numbers in this pilot study, the manifest hd patients (red) seem to form a distinct cluster from the controls (black) finally, some idea of the test s predictive validity can be seen by looking at the correlation between the observed values of and and a more general measure of the patients neurological status, the total motor score (tms). regression analysis reveals r values that are significant or near - significant for (pearson : p = 0.063, 0.008 and 0.065 (fig. 5) for alternation, pronation and single tapping, respectively), but not for.fig. 5relation between and in each of three types of test, and the total motor score, across all manifest hd subjects and controls (the latter are assigned a tms score of zero) relation between and in each of three types of test, and the total motor score, across all manifest hd subjects and controls (the latter are assigned a tms score of zero) figure 1 (right) shows a typical distribution plot from one of the participants in the study. as usually found for saccadic and evoked manual reaction times, distributions of the intervals between taps in this task are skewed, with a long tail of longer latencies, and in general the reciprocal of reaction time, or promptness, follows a gaussian distribution (and is therefore more amenable to statistical analysis). consequently if inter - tap interval distributions are plotted cumulatively, on a probit scale, using a reciprocal abscissa (a reciprobit plot), they will be expected to generate a straight line, as seen on fig. 1 (right). such a distribution can be fully described by just two parameters : these are, its mean (which is also the reciprocal of the median latency), and, its standard deviation. a large value of corresponds to increased promptness or speed of response, and thus a shorter interval ; because of the reciprocal relationship, the units for these two parameters are s or hz. the best - fit values of the parameters can be determined automatically by minimisation of the kolmogorov the observed distribution of reciprocal inter - tap intervals has the expected normal distribution, but in some cases significant, idiosyncratic deviations are observed. figure 2a demonstrates a bimodal pattern of response, possibly analogous to the population of 2b) is the existence of a distinct sub - set of aberrantly long inter - tap intervals (around 600700 ms), probably due to inattention : in this case they represent only some 5% of all trials. 2d shows responses from one manifest hd patient with remarkably long inter - tap intervals, and very marked differences between the three types of task. an important point to note is that many of these characteristic features would have been entirely missed by the conventional technique of simply counting the total number of taps in a fixed time - period : they are only revealed by quantitative analysis of the distribution of individual intervals.fig. 2examples of unusual latency distributions. a a subject showing marked bimodality in the alternation task, with one group of responses around 300 ms and another around 450 ms : responses in each direction are very similar. b a subject showing sporadic inattention in the pronation task : in both directions a small group of responses (top right) is markedly delayed relative to the main distribution. c marked left / right difference ; responses to the right are about 40 ms faster (alternation task). d a manifest hd patient with exceptionally slow responses, affecting all three types of movement, but most marked for the pronation / supination task examples of unusual latency distributions. a a subject showing marked bimodality in the alternation task, with one group of responses around 300 ms and another around 450 ms : responses in each direction are very similar. b a subject showing sporadic inattention in the pronation task : in both directions a small group of responses (top right) is markedly delayed relative to the main distribution. c marked left / right difference ; responses to the right are about 40 ms faster (alternation task). d a manifest hd patient with exceptionally slow responses, affecting all three types of movement, but most marked for the pronation / supination task best - fit values of and were estimated for all participants and conditions. figure 3 shows the average values of the parameters broken down by type of response and category of participant (controls and manifest). when comparing the three different tasks for the hd participants, is significantly different between the alternation and pronating tasks (p = 0.0011) and between pronating and single (p = 0.0024), and is significantly different for pronating versus alternating (p = 0.045), but no other comparisons are significant at p = 0.05. when comparing the patients with the controls, for all three tasks the patients show the expected reduction in (equivalent to increased time between taps), but because of the very small group sizes in this pilot study, the only difference seen in fig. 3, that is significant at p = 0.05 (unpaired t test), is for with single tapping (p = 0.036).fig. 3average values of (left) and (right) analysed by type of response and category of participant (c control, m manifest hd). error bars show 1se average values of (left) and (right) analysed by type of response and category of participant (c control, m manifest hd). error bars show 1se it is instructive to plot and as a scatter plot for the two groups (fig. 4). what is then seen is an apparent segregation between the manifest patients (m) and the controls ; once again, it would obviously be desirable to have more participants than was available for this preliminary evaluation.fig. 4scatter plot of versus for the two groups of participants performing single tapping ; despite the very small numbers in this pilot study, the manifest hd patients (red) seem to form a distinct cluster from the controls (black) scatter plot of versus for the two groups of participants performing single tapping ; despite the very small numbers in this pilot study, the manifest hd patients (red) seem to form a distinct cluster from the controls (black) finally, some idea of the test s predictive validity can be seen by looking at the correlation between the observed values of and and a more general measure of the patients neurological status, the total motor score (tms). regression analysis reveals r values that are significant or near - significant for (pearson : p = 0.063, 0.008 and 0.065 (fig. 5) for alternation, pronation and single tapping, respectively), but not for.fig. 5relation between and in each of three types of test, and the total motor score, across all manifest hd subjects and controls (the latter are assigned a tms score of zero) relation between and in each of three types of test, and the total motor score, across all manifest hd subjects and controls (the latter are assigned a tms score of zero) the aim of this study was to evaluate the potential use of a new device for quantitative assessments in disorders, such as hd in which hand - tapping tapping is abnormal. it has two key features : first, the force required to be exerted by the subject to register a response is very low (essentially zero) ; this makes it feasible to use variations on the basic alternation task that can pose a greater challenge to a patient with relatively mild impairment. figure 2d provides a particularly clear example of this, when comparing ordinary alternation with the pronation task : the difference in for the two types of task is equivalent to over 450 ms of latency difference. the second potential benefit is that by providing sequential information about individual responses in each direction, a great deal of data are generated in a short period of time, from which much more can be calculated than the average response time that has previously been conventionally used. for simple tapping, the parameter seems to provide particularly clear discrimination between subject groups (fig. 3, right), and it is possible that a combination of and together may be helpful in this respect (fig. 4). because responses in the two directions are not conflated, lateral asymmetries (fig 2c), suggesting a relatively one - sided functional impairment, become obvious and can be quantified. other kinds of idiosyncrasies, not previously noted (such as the bimodality of fig. another advantage of the device is that it is small, lightweight and portable and the software is easy - to - use allowing its potential ease of introduction to a clinical environment. this is, in other words, a simple tool that has the potential to address a general problem in studying not only in hd, but also other neurodegenerative disorders : a lack of objective and genuinely quantitative neurological tests, by which disease progress can be monitored and treatments evaluated with respect to the particular needs of individual patients. obviously much more data are needed to provide true validation, and to discover what aspects of the interval distributions will be most useful for this process, and this work is currently in hand. | measuring the rate of finger tapping is a technique commonly used as an indicator of impairment in degenerative neurological conditions, such as huntington s disease. the information it provides can be greatly enhanced by analysing not simply the overall tapping rate, but also the statistical characteristics of the individual times between each successive response. recent technological improvements in the recording equipment allow the responses to be analysed extremely quickly, and permit modification of the task in the interest of greater clinical specificity. here we illustrate its use with some pilot data from a group of manifest hd patients and age - matched controls. even in this small cohort, differences in the responses are apparent that appear to relate to the severity of the disease as measured by conventional behavioural tests. |
gt(rosa)26sor, b6;129-gt(rosa)26sor / j and b6;129s2-scarb1/j and fvb / nj wild - type mice were obtained from the jackson laboratory (bar harbor, me). mice were bred and maintained at the comparative bioscience center of the rockefeller university according to guidelines established by the institutional animal committee. adenoviral constructs encoding human and murine homologues of the four hcv entry factors (cd81, scarb1, cldn1 and ocln) and mutants thereof were created using the adeasy adenoviral vector system (agilent technologies, santa clara, ca) according to the manufacturer s instructions. plasmids encoding chimeric hcv genomes, including jc1flag2(p7fluc2a) and bicistronic hcv genomes expressing cre, were linearized with xbai and transcribed using megascript t7 (ambion, austin, texas). rna was electroporated into huh-7.5 cells using a ecm 830 electroporator (btx genetronics) and infectious virus was collected from supernatants 4872 h after transfection. gt(rosa)26sor, b6;129-gt(rosa)26sor / j and b6;129s2-scarb1/j and fvb / nj wild - type mice were obtained from the jackson laboratory (bar harbor, me). mice were bred and maintained at the comparative bioscience center of the rockefeller university according to guidelines established by the institutional animal committee. adenoviral constructs encoding human and murine homologues of the four hcv entry factors (cd81, scarb1, cldn1 and ocln) and mutants thereof were created using the adeasy adenoviral vector system (agilent technologies, santa clara, ca) according to the manufacturer s instructions. plasmids encoding chimeric hcv genomes, including jc1flag2(p7fluc2a) and bicistronic hcv genomes expressing cre, were linearized with xbai and transcribed using megascript t7 (ambion, austin, texas). rna was electroporated into huh-7.5 cells using a ecm 830 electroporator (btx genetronics) and infectious virus was collected from supernatants 4872 h after transfection. | hepatitis c virus (hcv) remains a major medical problem. antiviral treatment is only partially effective and a vaccine does not exist. development of more effective therapies has been hampered by the lack of a suitable small animal model. while xenotransplantation of immunodeficient mice with human hepatocytes has shown promise, these models are subject to important challenges. building on the previous observation that cd81 and occludin (ocln) comprise the minimal human factors required to render mouse cells permissive to hcv entry in vitro, we attempted murine humanization via a genetic approach. here we show that expression of two human genes is sufficient to allow hcv infection of fully immunocompetent inbred mice. we establish a precedent for applying mouse genetics to dissect viral entry and validate the role of scarb1 for hcv uptake. we demonstrate that hcv can be blocked by passive immunization, as well as show that a recombinant vaccinia virus (rvv) vector induces humoral immunity and confers partial protection against heterologous challenge. this system recapitulates a portion of the hcv life cycle in an immunocompetent rodent for the first time, opening opportunities for studying viral pathogenesis and immunity and comprising an effective platform for testing hcv entry inhibitors in vivo. |
this multi - center trial was conducted at 13 sites with subjects aged 845 years with autoimmune type 1 diabetes for less than 3 months and with evidence of -cell function evidenced by stimulated c - peptide > 0.2 pmol on a 2-h mixed meal tolerance test (mmtt). autoimmune type 1 diabetes was defined by the presence of any of four islet autoantibodies within 14 days of diagnosis (gad, insulinoma - associated protein 2, or islet cell autoantibodies [icas ]). subjects were otherwise healthy without major systemic illness nor allergic or autoimmune conditions requiring treatment with immunosuppressive agents or steroids. the protocol was approved by the type 1 diabetes trialnet steering committee, the data and safety monitoring board (dsmb), and regulatory authorities ; human subject approval was obtained at participating sites prior to study initiation. the study was a three - arm, randomized, double - masked, placebo - controlled clinical trial conducted by type 1 diabetes trialnet. roche pharmaceuticals provided mmf, dzb, and placebo, but had no involvement in study management, data collection and analysis, or manuscript preparation. there were 126 subjects randomized to receive mmf alone (with dzb placebo), mmf and dzb in combination, or control (mmf placebo and dzb placebo), stratified within clinical center. by error, among the last six sites to join the study, 12 subjects assigned to receive mmf alone inadvertently received dzb - alone, thus resulting in an imbalance in the group sample sizes. the results from these 12 subjects mmf or matched placebo was administered daily at a dose of 600 mg / m (maximum 2,000 mg / day) in 23 divided doses for 2 years. dzb or matched placebo was given by intravenous infusion at study day 0 and two weeks later at a dose of 1 mg / kg. all subjects were to be followed for at least 2 years under the intention - to - treat principle, including those who did not receive the full course of assigned therapy. because both drugs reduce the ability to fight viral infections, screening for cytomegalovirus (cmv), and epstein - barr virus (ebv) thereafter, ebv - negative subjects were followed monthly, and ebv - positive subjects were followed at 3-month intervals. visits included assessment of diabetes care, adverse events, and laboratory measurements to assess medication side effects. in the case of an acute infection, an infectious disease committee developed treatment algorithms for common infections and provided consultation as needed. a 4-h mmtt was conducted at baseline and 2 years, and a 2-h mmtt at 3, 6, 12, and 18 months with timed sample collection at 1530 min intervals. c - peptide levels were measured using a two - site immunoenzymometeric assay (tosoh 600 ii analyzer). a1c was measured quarterly using ion - exchange high performance liquid chromatography (variant ii, bio - rad diagnostics). biochemical autoantibodies (gad-65, ica-512, miaa) were measured using radio - immunobinding assays ; icas were measured using indirect immunofluorescence. potential participants were screened for antibodies to hepatitis b surface antigen, hepatitis c, and human immunodeficiency virus using enzyme immunoassays that, if positive, resulted in exclusion from the study. antibodies to cmv ebv were measured using indirect immunofluorescence (anti - ebv vca igm) and enzyme immunoassay (anti - cmv igg and igm ; anti - ebv vca and ebna igg). cmv and ebv viral load was measured using real time quantitative pcr (lightcycler system ; roche applied science). the prespecified primary analyses were based on the intention - to - treat cohort that included all subjects randomized correctly to the three specified treatment groups. the primary outcome was the geometric mean difference between active- and placebo - treated subjects of the area under the stimulated c - peptide curve over the first 2 h of a 4-h mmtt conducted at the 2-year visit in an ancova model adjusting for the baseline c - peptide, age, and sex. the 2-h c - peptide area under the curve (auc) (pmol / ml/120 min) was computed using the trapezoidal rule from timed measurements of c - peptide during each mmtt (including the basal). the auc mean (pmol / ml) equals the auc divided by the interval of time. the log([mean c - peptide ] + 1) transformation of the baseline and follow - up auc mean was used to allow for mean c - peptide values close to zero and to normalize the distribution of the residuals (6). data from all 13 centers contributed to the primary and secondary effectiveness analyses of the mmf plus dzb combination and its respective control group. however, owing to the randomization error, subjects received mmf alone in only seven centers. thus, these analyses compare the mmf alone subjects only with the concurrently randomized control subjects from these centers. secondary analyses include assessment of differences between groups over time in a longitudinal normal errors repeated - measures model of the log([mean c - peptide ] + 1) values. the mean rate of change over 324 months was estimated using a mixed effects random coefficient model (18) using the log values. the cox proportional hazards model assessed the relative risk (hazard ratio) of the loss of the 2-h c - peptide < 0.2 pmol / ml (19). prespecified secondary outcomes also include : differences in a1c, insulin dose, hypoglycemic episodes, rates of infection, and adverse events over time. for assessment of safety, the percents of subjects with an event were compared among the three groups using fisher exact test. the rate of events per subject was compared between groups using the poisson model test (20). the target sample size of 120 subjects (40 per group) provided 85% power to detect a 65% difference in the geometric mean c - peptide for any one of the three possible pairwise comparisons among the three treatment groups using a test at the 0.05 level (one - sided, adjusted for three comparisons), with 10% loss to follow - up. owing to the randomization error, the protocol was modified to compare the mmf and dzb combination group versus all placebo subjects, and to compare the mmf only subjects versus the placebo subjects enrolled within the same clinical centers (7 of the original 13) in which the randomization was not affected, each using a test at the 0.025 level (one - sided, adjusted for two comparisons). the mmf plus dzb versus placebo comparison, with about 40 per group, provided 85% power to detect a 61% difference ; and the mmf alone versus placebo comparison, with about 30 per group, provided 80% power to detect a 67% increase, each allowing for 10% losses to follow - up. in april 2008, based on 41 and 47% of the planned total information, the dsmb recommended termination of the treatment phase of the study. at that time the conditional power of each comparison under the current trend in the data were less than 0.02% and under the original design assumptions, termination for futility led to less than a 1% increase in the probability of a type ii error (21). sites were notified on april 30, 2008, to immediately terminate treatment but continue to follow all subjects. this report is based on closed and locked data for all visits through april 30, 2008. nominal one - sided p values (without adjustment for multiple tests) are presented for analyses of primary and secondary effectiveness outcomes ; 2-sided p values for safety outcomes. the study was a three - arm, randomized, double - masked, placebo - controlled clinical trial conducted by type 1 diabetes trialnet. roche pharmaceuticals provided mmf, dzb, and placebo, but had no involvement in study management, data collection and analysis, or manuscript preparation. there were 126 subjects randomized to receive mmf alone (with dzb placebo), mmf and dzb in combination, or control (mmf placebo and dzb placebo), stratified within clinical center. by error, among the last six sites to join the study, 12 subjects assigned to receive mmf alone inadvertently received dzb - alone, thus resulting in an imbalance in the group sample sizes. mmf or matched placebo was administered daily at a dose of 600 mg / m (maximum 2,000 mg / day) in 23 divided doses for 2 years. dzb or matched placebo was given by intravenous infusion at study day 0 and two weeks later at a dose of 1 mg / kg. all subjects were to be followed for at least 2 years under the intention - to - treat principle, including those who did not receive the full course of assigned therapy. because both drugs reduce the ability to fight viral infections, screening for cytomegalovirus (cmv), and epstein - barr virus (ebv) was based on close surveillance rather than active prophylaxis. thereafter, ebv - negative subjects were followed monthly, and ebv - positive subjects were followed at 3-month intervals. visits included assessment of diabetes care, adverse events, and laboratory measurements to assess medication side effects. in the case of an acute infection an infectious disease committee developed treatment algorithms for common infections and provided consultation as needed. a 4-h mmtt was conducted at baseline and 2 years, and a 2-h mmtt at 3, 6, 12, and 18 months with timed sample collection at 1530 min intervals. c - peptide levels were measured using a two - site immunoenzymometeric assay (tosoh 600 ii analyzer). a1c was measured quarterly using ion - exchange high performance liquid chromatography (variant ii, bio - rad diagnostics). biochemical autoantibodies (gad-65, ica-512, miaa) were measured using radio - immunobinding assays ; icas were measured using indirect immunofluorescence. potential participants were screened for antibodies to hepatitis b surface antigen, hepatitis c, and human immunodeficiency virus using enzyme immunoassays that, if positive, resulted in exclusion from the study. antibodies to cmv ebv were measured using indirect immunofluorescence (anti - ebv vca igm) and enzyme immunoassay (anti - cmv igg and igm ; anti - ebv vca and ebna igg). cmv and ebv viral load was measured using real time quantitative pcr (lightcycler system ; roche applied science). the prespecified primary analyses were based on the intention - to - treat cohort that included all subjects randomized correctly to the three specified treatment groups. the primary outcome was the geometric mean difference between active- and placebo - treated subjects of the area under the stimulated c - peptide curve over the first 2 h of a 4-h mmtt conducted at the 2-year visit in an ancova model adjusting for the baseline c - peptide, age, and sex. the 2-h c - peptide area under the curve (auc) (pmol / ml/120 min) was computed using the trapezoidal rule from timed measurements of c - peptide during each mmtt (including the basal). the auc mean (pmol / ml) equals the auc divided by the interval of time. the log([mean c - peptide ] + 1) transformation of the baseline and follow - up auc mean was used to allow for mean c - peptide values close to zero and to normalize the distribution of the residuals (6). data from all 13 centers contributed to the primary and secondary effectiveness analyses of the mmf plus dzb combination and its respective control group. however, owing to the randomization error, subjects received mmf alone in only seven centers. thus, these analyses compare the mmf alone subjects only with the concurrently randomized control subjects from these centers. secondary analyses include assessment of differences between groups over time in a longitudinal normal errors repeated - measures model of the log([mean c - peptide ] + 1) values. the mean rate of change over 324 months was estimated using a mixed effects random coefficient model (18) using the log values. the cox proportional hazards model assessed the relative risk (hazard ratio) of the loss of the 2-h c - peptide < 0.2 pmol / ml (19). prespecified secondary outcomes also include : differences in a1c, insulin dose, hypoglycemic episodes, rates of infection, and adverse events over time. for assessment of safety, the two active groups are compared with the total control group enrolled. the percents of subjects with an event were compared among the three groups using fisher exact test. the rate of events per subject was compared between groups using the poisson model test (20). the target sample size of 120 subjects (40 per group) provided 85% power to detect a 65% difference in the geometric mean c - peptide for any one of the three possible pairwise comparisons among the three treatment groups using a test at the 0.05 level (one - sided, adjusted for three comparisons), with 10% loss to follow - up. owing to the randomization error, the protocol was modified to compare the mmf and dzb combination group versus all placebo subjects, and to compare the mmf only subjects versus the placebo subjects enrolled within the same clinical centers (7 of the original 13) in which the randomization was not affected, each using a test at the 0.025 level (one - sided, adjusted for two comparisons). the mmf plus dzb versus placebo comparison, with about 40 per group, provided 85% power to detect a 61% difference ; and the mmf alone versus placebo comparison, with about 30 per group, provided 80% power to detect a 67% increase, each allowing for 10% losses to follow - up. in april 2008, based on 41 and 47% of the planned total information, the dsmb recommended termination of the treatment phase of the study. at that time the conditional power of each comparison under the current trend in the data were less than 0.02% and under the original design assumptions, termination for futility led to less than a 1% increase in the probability of a type ii error (21). sites were notified on april 30, 2008, to immediately terminate treatment but continue to follow all subjects. this report is based on closed and locked data for all visits through april 30, 2008. nominal one - sided p values (without adjustment for multiple tests) are presented for analyses of primary and secondary effectiveness outcomes ; 2-sided p values for safety outcomes. supplemental figure a1 (available in the online appendix at http://care.diabetesjournals.org/cgi/content/full/dc09-1349) summarizes subject disposition - screening of 228 subjects, randomization of 126 subjects, and subsequent disposition. table 1 presents baseline characteristics for each active therapy group versus its respective control group. the groups were well - matched with 30% ages 812 years, 31% ages 1317 years, and 39% age 18 years. baseline characteristics of the treatment groups, including all 114 subjects correctly randomized means sd are presented for continuous variables. one subject did not receive the second infusion due to patient decision to continue study treatment. 80% or greater by capsule count up through last recorded visit starting with month 3. safety data are presented on all 114 properly randomized subjects, excluding the 12 who received the dzb - alone instead of mmf alone. of these, 60 completed an mmtt at 2 years and contributed to the primary outcome analysis. all randomized subjects received study treatment, and all but one received the two planned dzb / placebo infusions. median compliance with mmf daily capsules was estimated to be 75% in the mmf plus dzb group, 63% in the mmf alone group, and 71% in the control group over the treatment period based on capsule counts. mean mpa trough levels over 24 months were 4.5 3.4 (mcg / ml) and 5.8 4.0 (mcg / ml), respectively, for mmf plus dzb and mmf alone groups with expected trough range of 1.03.5 g / ml. treatment was terminated in 23 subjects due to adverse events (5), elevated liver enzymes (2), ebv pcr - positivity (8), treatment noncompliance (3), or loss to follow - up (5) (supplemental figure a1). control subjects lost c - peptide at a rate of 53.5% per year, and both the mmf alone and the mmf plus dzb treatment groups had comparable rates of loss, 46.4% and 48.1%, respectively. in the primary analysis (fig. 1a) at 2 years,;the geometric mean stimulated c - peptide auc was 0.28 pmol / ml (95% ci 0.190.37) in those treated with mmf plus dzb, compared with 0.27 (0.180.37) for their control subjects, p = 0.47 ; and 0.25 (0.140.37) in mmf alone treated subjects, compared with 0.23 (0.120.35) for their control subjects, p = 0.41. there was no statistical difference between treatment and control subjects over 2 years or during the early phase when dzb would have been more active. a) the geometric means and 95% cis for the 2-h auc stimulated c - peptide levels over time within each group. b) the cumulative incidence of decline in peak c - peptide to < 0.2 pmol / ml within each group. the relative hazard was 0.61 (95% ci : 0.281.33, p = 0.11) for mmf plus dzb vs. control, and 1.05 (0.502.19, p = 0.83) for mmf alone vs. control. c) ratio of geometric means for mmf plus dzb vs. control groups, with 95% cis, within subgroups of subjects defined at baseline. d) likewise for mmf alone vs. control (a1c 2nd tertile upper 95% confidence limit is 28.9). during follow - up, all but eight subjects had detectable levels of c - peptide. the auc mean c - peptide fell below 0.2 pmol / ml during follow - up in 12 mmf plus dzb, 16 mmf alone, and 17 control subjects. cumulative incidence of decline of peak c - peptide below 0.2 pmol / ml did not differ between groups (fig. the geometric mean ratio for mmf plus dzb versus control subjects was 1.02 (95% ci 0.651.59) and for mmf alone versus control subjects was 1.08 (0.572.02). figure 1c and d show that these mean ratios and confidence limits within subgroups, defined by baseline characteristics, are not nominally significantly different from 1, with the exception of the effect of mmf alone within the 10 subjects in the highest tertile of baseline a1c (p = 0.042). however, comparison of ratios among the a1c tertiles, and among all other subgroups, failed to reach significance demonstrating that variation among subgroups was within the realm of chance. similar results applied to subgroups defined from the mean levels of a1c and insulin dose over 24 months. dzb reduced cd4cd25 t - cell levels maximally at 4 weeks (depletion 83.9% and blocking 97.5%) and these recovered within 612 months. the month 24 c - peptide level was not associated with either percent reduction in cd4cd25 t - cells within the mmf plus dzb group, or mmf trough levels in either the mmf plus dzb or mmf alone groups. mirroring changes in c - peptide, daily insulin dose slowly rose from below 0.5 units / kg at baseline to 0.57 units / kg with mmf plus dzb versus 0.61 units / kg among control subjects (p = 0.17) ; and to 0.65 units / kg with mmf alone versus 0.62 units / kg among control subjects (p = 0.68) (fig. there were 19 serious adverse events (aes) reported for 14 subjects (34%) in the mmf and dzb group, nine in five subjects (16%) in the mmf alone group, and three in three subjects (7%) in the control group (online appendix table 1), p < 0.01. more grade 2 or higher aes occurred in mmf plus dzb subjects (167 or 4.1 events / subject) compared with mmf alone (117, 3.8 events / subject) or control subjects (133, 3.2 events / subject), p = 0.09 (online appendix table 1). contrary to expectations, there was no difference in the occurrence of infectious or gastrointestinal events among groups. eight individuals had asymptomatic reactivation of previous ebv infection using a sensitive pcr assay (five in mmf plus dzb, one in mmf, and two in control). neutropenia and leukopenia, both side effects of mmf and dzb, occurred approximately equally among the three groups. a slight excess of elevated liver enzymes occurred in the mmf plus dzb group. major hypoglycemic events were reported for 27 subjects, with an average of two each, with no difference among groups. supplemental figure a1 (available in the online appendix at http://care.diabetesjournals.org/cgi/content/full/dc09-1349) summarizes subject disposition - screening of 228 subjects, randomization of 126 subjects, and subsequent disposition. table 1 presents baseline characteristics for each active therapy group versus its respective control group. the groups were well - matched with 30% ages 812 years, 31% ages 1317 years, and 39% age 18 years. baseline characteristics of the treatment groups, including all 114 subjects correctly randomized means sd are presented for continuous variables. one subject did not receive the second infusion due to patient decision to continue study treatment. 80% or greater by capsule count up through last recorded visit starting with month 3. safety data are presented on all 114 properly randomized subjects, excluding the 12 who received the dzb - alone instead of mmf alone. of these, 60 completed an mmtt at 2 years and contributed to the primary outcome analysis. all randomized subjects received study treatment, and all but one received the two planned dzb / placebo infusions. median compliance with mmf daily capsules was estimated to be 75% in the mmf plus dzb group, 63% in the mmf alone group, and 71% in the control group over the treatment period based on capsule counts. mean mpa trough levels over 24 months were 4.5 3.4 (mcg / ml) and 5.8 4.0 (mcg / ml), respectively, for mmf plus dzb and mmf alone groups with expected trough range of 1.03.5 g / ml. treatment was terminated in 23 subjects due to adverse events (5), elevated liver enzymes (2), ebv pcr - positivity (8), treatment noncompliance (3), or loss to follow - up (5) (supplemental figure a1). mean auc c - peptide at entry was 0.70 0.33 pmol / ml. control subjects lost c - peptide at a rate of 53.5% per year, and both the mmf alone and the mmf plus dzb treatment groups had comparable rates of loss, 46.4% and 48.1%, respectively. in the primary analysis (fig. 1a) at 2 years,;the geometric mean stimulated c - peptide auc was 0.28 pmol / ml (95% ci 0.190.37) in those treated with mmf plus dzb, compared with 0.27 (0.180.37) for their control subjects, p = 0.47 ; and 0.25 (0.140.37) in mmf alone treated subjects, compared with 0.23 (0.120.35) for their control subjects, p = 0.41. there was no statistical difference between treatment and control subjects over 2 years or during the early phase when dzb would have been more active. a) the geometric means and 95% cis for the 2-h auc stimulated c - peptide levels over time within each group. b) the cumulative incidence of decline in peak c - peptide to < 0.2 pmol / ml within each group. the relative hazard was 0.61 (95% ci : 0.281.33, p = 0.11) for mmf plus dzb vs. control, and 1.05 (0.502.19, p = 0.83) for mmf alone vs. control. c) ratio of geometric means for mmf plus dzb vs. control groups, with 95% cis, within subgroups of subjects defined at baseline. d) likewise for mmf alone vs. control (a1c 2nd tertile upper 95% confidence limit is 28.9). during follow - up, all but eight subjects had detectable levels of c - peptide. the auc mean c - peptide fell below 0.2 pmol / ml during follow - up in 12 mmf plus dzb, 16 mmf alone, and 17 control subjects. cumulative incidence of decline of peak c - peptide below 0.2 pmol / ml did not differ between groups (fig. the geometric mean ratio for mmf plus dzb versus control subjects was 1.02 (95% ci 0.651.59) and for mmf alone versus control subjects was 1.08 (0.572.02). figure 1c and d show that these mean ratios and confidence limits within subgroups, defined by baseline characteristics, are not nominally significantly different from 1, with the exception of the effect of mmf alone within the 10 subjects in the highest tertile of baseline a1c (p = 0.042). however, comparison of ratios among the a1c tertiles, and among all other subgroups, failed to reach significance demonstrating that variation among subgroups was within the realm of chance. similar results applied to subgroups defined from the mean levels of a1c and insulin dose over 24 months. dzb reduced cd4cd25 t - cell levels maximally at 4 weeks (depletion 83.9% and blocking 97.5%) and these recovered within 612 months. the month 24 c - peptide level was not associated with either percent reduction in cd4cd25 t - cells within the mmf plus dzb group, or mmf trough levels in either the mmf plus dzb or mmf alone groups. mirroring changes in c - peptide, daily insulin dose slowly rose from below 0.5 units / kg at baseline to 0.57 units / kg with mmf plus dzb versus 0.61 units / kg among control subjects (p = 0.17) ; and to 0.65 units / kg with mmf alone versus 0.62 units / kg among control subjects (p = 0.68) (fig. there were 19 serious adverse events (aes) reported for 14 subjects (34%) in the mmf and dzb group, nine in five subjects (16%) in the mmf alone group, and three in three subjects (7%) in the control group (online appendix table 1), p < 0.01. more grade 2 or higher aes occurred in mmf plus dzb subjects (167 or 4.1 events / subject) compared with mmf alone (117, 3.8 events / subject) or control subjects (133, 3.2 events / subject), p = 0.09 (online appendix table 1). contrary to expectations, there was no difference in the occurrence of infectious or gastrointestinal events among groups. eight individuals had asymptomatic reactivation of previous ebv infection using a sensitive pcr assay (five in mmf plus dzb, one in mmf, and two in control). neutropenia and leukopenia, both side effects of mmf and dzb, occurred approximately equally among the three groups. a slight excess of elevated liver enzymes occurred in the mmf plus dzb group. major hypoglycemic events were reported for 27 subjects, with an average of two each, with no difference among groups. the aim of the present study was to arrest -cell destruction in recently diagnosed type 1 diabetic subjects when preservation of existing -cells may have a clinically meaningful effect on long - term outcomes of type 1 diabetes. we found no treatment benefit from either mmf alone or from the combination of mmf and dzb in this randomized, masked, placebo - controlled trial. although mmf has been effective in combination with other anti - rejection drugs (such as sirolimus and tacrolimus) in a number of transplant protocols (11), alone it may not have as much effect on effector cells, which can damage and kill islets without requiring cell division, the primary mode of mmf action. although several studies have reported a potential negative effect of mmf on islet cell function, we did not see any greater loss of c - peptide in the mmf alone group compared with control subjects. for both mmf and dzb, we chose the lowest known effective doses of each. dzb has been shown to reduce recurrences in multiple sclerosis (15) and uveitis (16) when given monthly. our use of two doses of dzb may not have been sufficient to affect activated effectors cells in the pancreas even with mmf, despite reasonably good depletion / coating in the peripheral circulation. cyclosporine, which also affects the il-2 signaling pathway, was shown to be effective in past trials if given at high doses and early enough in the course of disease (22). this, and the aforementioned effect of dzb in two other autoimmune diseases, suggests that the lower dose may have played a major part in the lack of effect of this therapy. although higher doses may have greater therapeutic effect, this has to be measured against the increased risk of side effects. even at the doses used in this study, there was an increase in aes when the two drugs were used together in comparison to mmf alone or placebo. cd4cd25 + regulatory t - cells play an important role in immune regulation and a potential problem with an anti - il-2 receptor antibody is worsening autoimmunity rather than reducing it if the effects of the drug on the regulatory cell population outweigh its effects on the activated - effector cell population. in this study we saw no worsening of -cell destruction or development of other autoimmune conditions with the use of dzb. while overall compliance with the mmf and dzb regimens was high, it is possible that the need to withdraw study drug, primarily mmf, for various intervals due to aes, may have affected the ability to demonstrate a beneficial effect. however, mechanistic assessments showed that mmf and dzb each were bioavailable and had the intended immunologic effects, but these effects were not associated with the c - peptide levels after 2 years. modified anti - cd3 antibodies have been shown to reduce the rate of loss of c - peptide in new - onset type 1 diabetic subjects similar to those studied in this trial (24). mmf and anti - il-2r are downstream of the important major histocompatibility complex - peptide / t - cell receptor interaction, which is the driver of the autoimmune response. therapies such as anti - cd3 and anti - cd20 as well as antigens such as gad, oral insulin, and diapep277 may have the potential to alter this critical reaction and blunt the direct activation of autoreactive t - cells rather than limit their activity and division, which is where mmf and dzb are most critical. one additional difference between this study and the cyclosporine trials (22) that had been successful was that the time to treatment was 56 days with anti - cd3 versus 76 days in this study. post hoc analysis did not reveal this to be a factor in the failure to see an effect and probably suggests that more targeted therapy at sufficient dose is necessary to arrest the diabetes process. new therapies such as diapep277 in adults (23) or gad immunization (24) have recently been shown to slow the rate of loss of c - peptide, and others are under study such as anti - cd20, abatacept, and thymoglobulin. although we were concerned with the number of adverse events that might occur from the use of immunosuppressive agents, it is clear from our analysis that the number and type we detected were for the most part within our prestudy expectations and did not prevent study subjects from continuing treatment. the finding of asymptomatic low - level pcr reactivation of ebv in both treated and untreated patients was unexpected and may reflect the differential sensitivity of our ebv viral pcr assay as well as our rigorous screening program to ensure patient safety. type 1 diabetes trialnet is an national institutes of health (nih)-sponsored multicenter trial group formed to perform intervention trials in new - onset type 1 diabetes and pre - diabetes, as well as to develop immunologic and mechanistic assays to better understand type 1 diabetes pathogenesis. proposed studies are rigorously reviewed for scientific and ethical justification, clinical feasibility and prioritization by a diverse group of clinicians, basic scientists, statisticians, and ethicists (25). trials are monitored by metabolic, infectious disease, and safety monitoring committees in addition to oversight by an independent dsmb. although this trial was unsuccessful at finding new therapies to induce clinical remission in type 1 diabetes, it showed that our network can successfully design, recruit, and conduct clinical trials of sufficient size. the use of novel agents, alone or in combination, will be facilitated by the clinical trial process developed for this first trial under the trialnet mechanism. | objectivethis trial tested whether mycophenolate mofetil (mmf) alone or with daclizumab (dzb) could arrest the loss of insulin - producing -cells in subjects with new - onset type 1 diabetes.research design and methodsa multi - center, randomized, placebo - controlled, double - masked trial was initiated by type 1 diabetes trialnet at 13 sites in north america and europe. subjects diagnosed with type 1 diabetes and with sufficient c - peptide within 3 months of diagnosis were randomized to either mmf alone, mmf plus dzb, or placebo, and then followed for 2 years. the primary outcome was the geometric mean area under the curve (auc) c - peptide from the 2-h mixed meal tolerance test.resultsone hundred and twenty - six subjects were randomized and treated during the trial. the geometric mean c - peptide auc at 2 years was unaffected by mmf alone or mmf plus dzb versus placebo. adverse events were more frequent in the active therapy groups relative to the control group, but not significantly.conclusionsneither mmf alone nor mmf in combination with dzb had an effect on the loss of c - peptide in subjects with new - onset type 1 diabetes. higher doses or more targeted immunotherapies may be needed to affect the autoimmune process. |
striae gravidarum (sg) is a common, disfiguring, gestational change that affects between 55% (chang., 2004, picard., 2015) and 90% (fitzpatrick and freedberg, 2003) of women (ghasemi. sg presents as atrophic linear scars and can cause distress, often leading to a decrease in quality of life (korgavkar and wang, 2015, park and murase, 2013). sg are often overlooked as a cosmetic concern, which does not make the patient burden any less. current research on risk factors, prevention, and management of this condition has been limited or met with little success and/or provided conflicting results (chang., 2004). throughout history, stretch marks have always been a source of distress for pregnant women. as early as 16 bc, the poet ovid alluded to women who would self - abort their pregnancies to avoid stretch marks (rayor and batstone, 1995). ancient egyptians recorded numerous preparations for the treatment of stretch marks and soranus and pliny the elder in the 1st century ad endorsed unripe olive oil and sea salt, respectively (owsei, 1991). frankincense is one of the most often recommended treatments (nwi trading co., 2016). there has been an additional wide range of therapeutics from topical medications to surgical modalities proposed for stretch marks, which reinforces the concern that is associated with this condition. sg first present as flat, pink - to - red bands (striae rubra or immature striae) that become raised, longer, wider, and violet - red (fig. 1, fig., the marks fade and become hypopigmented (striae alba or mature striae), appearing parallel to skin tension lines as scar - like, wrinkled, white, and atrophic marks (fig. 3 ; salter and kimball, 2006, sodhi and sausker, 1988, watson., 1998). sg can cause itching, burning, and discomfort and typically present on the breasts, abdomen, hips, and thighs. up to 90% of sg appear in primigravidas (chang., 2004). onset has typically been reported in the late second and early third trimester ; however, one study has demonstrated that 43% of women develop sg prior to 24 weeks of gestation the etiopathogenesis involves a combination of genetic factors (di lernia., 2001), hormonal factors (chang., 2004, murphy., 1992), and increased mechanic stress on connective tissue (fitzpatrick and wolff, 2008, ghasemi., 2007, murphy. interestingly, skin stretch has been a controversial trigger as studies have demonstrated an inconsistent association of sg with maternal weight gain and abdominal and hip girth stretch (atwal., 2006, chang., 2004, poidevin, 1959). with regard to hormonal factors, twice as many estrogen receptors and elevated androgen and glucocorticoid receptors have been observed in striae compared with those in healthy skin (cordeiro., 2010). pregnancy s distinct hormonal milieu is thought to influence connective tissue that is susceptible to sg when stretched., 1966, sheu., 1991, tsuji and sawabe, 1988), collagen fibrils (pinkus., 1966, shuster, 1979), and other extracellular membrane components (watson., 1998) are believed to underlie the pathogenesis of sg (wang., 2015). histologically, the appearance of sg is similar to striae distensae (sd) and contingent on lesion age. early on, active lesions are comprised predominantly of fine elastic fibers but aging lesions demonstrate a thinning of the dermis and decrease of collagen content in the upper dermis (watson., 1998). biopsy tissue samples of sg show a disorganization, shortening, and thinning of the elastic fiber network compared with tissue samples of normal skin (murphy., 1992, wang., 2015). although they are thin and disorganized, fibrils are tropoelastin - rich, which is likely due to uncoordinated synthesis (wang., 2015). light microscopy demonstrates a flattening of the epidermis with atrophy and loss of rete ridges and increased glycosaminoglycans (murphy., 1992, salter and kimball, 2006, watson., 1998). however, the severity and development of lesions varies among patients, which indicates a variable genetic predisposition. a systematic literature search was conducted using textbooks and pubmed and medline databases to date to identify evidence - based data on the risk factors, prevention, and management of sg. a literature search up to august 2016 revealed 28 articles available online that specifically studied sg, including cross - sectional, prospective, randomized controlled, and quasi - randomized controlled studies. the search was restricted to english - language articles with the exception of two articles that were translated from german. table 3 shows studies that are relevant to sg treatments and treatment efficacy and their adverse events. in addition to the limited number of studies on the management of sg (table 3), we incorporated a review of the most current treatments in use for nongestational sd, which may be used as a guide for future sg treatment.table 1risk factors for striae gravidarumtable 1.investigators and study typenumber of subjects and subject profilesrisk factors identifiedtreatmentspicard., 2015cross - sectional study800 primiparous examined postpartum with mean age of 26.3younger agehigher pre - pregnancy weightbody mass indexhigher weight at deliveryhigher gestational weight gainfitzpatrick skin types i and ivabsence of employmentfamily history of striae gravidarumtopical treatments to reduce the occurrence of sg were not found to be effectivekasielska - trojan., 2015cross - sectional study299 caucasian up to 6 mos after delivery, without distinguishing primiparous or multiparas.previous history of sgfamily history of sghigher bmi before pregnancylack of chronic diseaseshigher birthweightsd on the breasts increased risk (71.4% with striae on breasts vs. 28.6% without)sd on thighs decreased risk (23% with striae vs. 77% without striae)progesterone treatment was not found to be related to sgj - orh., 2008cross - sectional study280 thai who had just given birth to first child, in the immediate postpartum period.younger age (22.8 yr vs. 26.6 yr)higher pre - pregnancy bmi (21.2 kg / m2 vs. 19.8 kg / m)higher maternal bmi at pregnancy (27.3 kg / m2 vs. 25.6 kg / m)higher gestational age at delivery (39.1 wk vs. 38.6 wk)higher birth weight of baby (3,078.8 g vs. 2,895.8)alcohol drinker (91.4% vs. 8.6%)had little water intake (7.4 glasses vs. 8.3 glasses)family history of sg (82.8% vs. 17.2%)did not assessosman., 2007cross - sectional study112 primiparous lebanese assessed during the immediate postpartum periodyounger age (26.5 yr vs. 30.5)increased weight gain during pregnancy (15.6 kg vs. 38.4 kg)birth weight, gestational age at delivery, and family history of sg associated with moderate - to - severe sgdid not assessatwal., 2006cross - sectional study with questionnaire309 primiparous within 48 hours of deliverymost significant was low maternal age20% (14 of 71) of teenagers had severe striae, not seen in over 30 yr of age.pre-pregnancy bmi greater than 26maternal weight gain of more than 15 kghigh neonatal birth weightdid not assesschang., 2004cross - sectional study with anonymous survey161 who had just given birthmost significant was a history of breast or thigh striae (81% who developed sg had striae history vs. 31% without sg who had history of striae)having a mother with sgadditional family history (sisters, daughters, grandmothers, aunts, cousins) of sgnon - white had higher association with sg (odds ratio = 4.2, 95% ci 1.9, 9.6).pre - pregnancy bmi not significantly differentdid not assessersoy., 2016prospective observational study211 singleton primiparous pregnant who were hospitalized for birth and who did not have systemic diseases or other risk factors, like drug use or polyhydramnios.younger agehigher pre - conceptional bmifamily historyhaving a male babylower educational levelsmoking status, skin type, water intake, and level of financial income did not significantly predict sguse of preventive oil or drugs, did not affect development of sgfindik., 2011prospective study69 primigravidas using prophylactic iron and vitamin preparations at 36 wks gestation or greaterfamily historyreduced blood vitamin c levelsno significant relation with age, weight gain during pregnancy, abdominal / thigh circumference, or smoking statusdid not assessthomas and liston, 2004prospective observational study128 primigravid who presented in labor or for induction of laboryounger agehigher pre - delivery bmihigher baby weightdid not assessdavey, 1972prospective study76 primiparous younger agehigher weighthigher baby weightsg were less common in skin messaged with olive oilmadlon - kay, 1993retrospective cohort study48 nulliparous at 34 to 36 weeks ' estimated gestational ageyounger agemore likely to receive state medical assistancemore likely to have hip striaegreater weight gain during pregnancydiabetes tests and glycosylated hemoglobin levels were similar in with and without striae. who used oils or creams formed striae as frequently as those who did not=women ; bmi = body - mass index ; mos = months ; sg = striae gravidarum ; yrs = years.statistical significance is defined as p 0.05table 2prevention of striae gravidarumtable 2.investigators and study typenumber of subjects and subject profilespreventive methods usedresults of preventive methodsmallol., 1991randomized, double - blind, placebo - controlled study80 pregnant during their first 12 weeks of a healthy pregnancytrofolastin cream with centella asiatica extract, -tocopherol and collagen elastin hydrolysates ; applied daily from 12 week of pregnancy until deliverydevelopment of sg : 56% in placebo group vs. 34% in treatment groupintensity of sg was significantly lower in treated with cream vs. placeboin with history of striae during puberty, cream prevented sg in 89% of cases, whereas all formed sg in the placebo groupgarca hernndez., 2013randomized, double - blind, placebo - controlled study183 pregnant patients over age 18 at week 12 + /- 2cream containing hydroxyprolisilane - c, rosehip oil, centella asiatica triterpenes and vitamin e ; applied twice a day around 12 weeks of pregnancyeffective in preventing sg only in without a history of striae (6% developed sg on treatment vs. 35% on placebo)severity of previous stretch marks increased in the control group during the study, but not in the treated groupamong who developed new sg, there was increased severity in control vs. treated grouptaavoni., 2011randomized clinical study70 nulliparous aged between 20 - 30 yrs old, in 1820th week of gestation with bmi ranging between 18.5 - 25. 35 used treatment, 35 did notolive oil applied topically onto abdomen twice daily, without massaging, vs. no olive oilsg occurred at the end of the second quarter of pregnancy in 45.7% in intervention group vs. 62.9% in control group (p = 0.115)difference not statistically significantsoltanipoor., 2012randomized controlled clinical study100 nulliparous pregnant ; 50 used treatment, 50 did notolive oil applied topically onto abdomen twice daily, without massaging, vs. no olive oilfrequency of severe sg was lower in group that used olive oil.difference not statistically significantde buman., 1987randomized controlled study90 pregnant ; 30 received treatment cream, 30 received vitamin cream, 30 received placeboalphastria cream (hyaluronic acid, allantoin, vitamin a, vitamin e and calcium pantothenate) vs. vitamin cream vs. placebo cream ; messaged for a few minutes daily to the thighs, abdomen and chest, starting at the 3rd month of pregnancy and ending 3 mos after childbirthsg developed in 10% of alphastria treated group vs. 40% of vitamin treated group vs. 37% in placebo treated groupwierrani., 1992randomized controlled study50 pregnant ; 24 received treatment, 26 did not receive any treatmentverum cream (vitamin e, essential fatty acids, panthenol, hyaluronic acid, elastin and menthol) ; massaged onto the abdomen, thighs and breasts starting at the 20th week of pregnancysg developed in 29% of verum treated group vs. 62% in no treatment groupsoltanipour., 2014parallel randomized controlled clinical study150 nulliparous at their second trimester of pregnancy in iran. 50 subjects in each groupolive oil vs. saj cream that contains lanolin, stearin, triethanolamine, almond oil, and bizovax glycerin amidine vs. placebosg occurred in 72% of olive oil group vs. 64% in saj cream group vs. 60% in control group.differences, 2008randomized double - blind placebo - controlled study175 nulliparous in lebanon with singleton pregnancies between week 12 and 18 weeks of gestation. 91 with study treatment, 84 with placebococoa butter lotion vs. placebo lotion, daily from weeks 12 - 18sg developed in 45% of patients using cocoa butter cream vs. 48% using placebodifference not statistically significantbuchanan., 2010randomized, double - blind, placebo - controlled study300 pregnant ; 150 received treatment, 150 placebococoa butter lotion vs. placebo lotion, daily from 16 weeks to deliverysg developed in 44% of patients using cocoa butter cream vs. 55% using placebodifference not statistically significanttimur tahan and kafkasli, 2012posttest - only quasi - experimental design with a control group141 primiparous who visited the pregnancy unit in turkey between february 1st, 2010 and april 15th, 2011. massage, 46 in controlbitter almond oil applied with or without massage vs. control ; applied every other day in weeks 1932 of pregnancy, followed by daily until deliveryfrequency of sg : 20% among who applied oil with massage vs. 38.8% among those who applied w / o massage vs. 41.2% in control grouppoidevin, 1959prospective study116 primigravidas; 55 used treatment, 66 did notolive oil vs. no olive oil applied dailysg developed in 68% of olive oil group vs. 55% not using olive oil.difference not statistically significantdavey, 1972prospective study76 primiparous ; 35 used treatment, 41 did notolive oil massaged into abdomen daily vs. no olive oilthe prophylactic use of olive oil to massage the abdomen was associated with a lower incidence of sg=women ; bmi = body - mass index ; mos = months ; sg = striae gravidarum ; yrs = years.statistical significance is defined as p 0.05table 3treatment of striae gravidarumtable 3.investigators and study typenumber of subjects and subject profilestype of striaetreatmentefficacyadverse effectsmalekzad., 2014prospective pilot study10 aged 26 - 50, fitzpatrick skin types iii - vstriae alba1540-nm non - ablative fractional laserclinical improvement in striae ranging from 1 - 24%improvement between the 4-week treatment and the 16-week treatment was identified3 mos after final treatment, patients had observable improvement in the striae, compared with baselinemild post - inflammatory hyperpigmentation in one patient after 8-week treatment and mild acne in another patient after 4 weeks of treatmentrangel., 2001open - label, multicenter, prospective study26 with abdominal pregnancy - related striaenot reported0.1% tretinoin cream daily for 3 mos applied to sgat treatment conclusion, global improvement was achieved from baseline in all stretch markspre - selected target lesion decreased in length by 20% and width by 23%erythema and scaling were the most common adverse eventspribanich., 1994double - blind placebo controlled study11 non - pregnant who had sg, 6 received treatment and 5 placebonot reported0.025% tretinoin cream applied daily for 7 mosno statistically significant difference in treated group compared to control groupdid not assesskang., 1996double - blind, randomized, vehicle - controlled study22 healthy white with erythematous stretch marks, 10 received treatment and 12 vehiclestriae rubra0.1% tretinoin (n = 10) or vehicle (n = 12) daily for 6 mos to the affected areasat 2 mos, tretinoin patients had significant improvements in severity scores of sg vs. vehicle patientsat 6 mos, 80% of tretinoin patients had improvement vs. 8% of vehicle patientstargeted stretch marks treated with tretinoin had decrease in mean length and width of 14% and 8%, respectively, vs. an increase of 10% and 24%, respectively, in vehicle patientserythema and scaling, with itching and burningash., 1998randomized controlled study10 american nonpregnant of varying skin types, age 23 to 49 yr.striae age ranged from 8 mos to 31 yr and all were white striae.all patients had abdominal striae, 50% also had striae on thighsstriae alba20% glycolic acid + 0.05% tretinoin vs. 20% glycolic acid + 10% ascorbic acid, applied daily to abdomen or thighs for 12 weeks (each regimen was applied to half the treatment area)at treatment conclusion, 47% improvement with 0.05% tretinoin vs. 43% improvement with ascorbic acidresults not statistically significant from each other, but significant for both compared to pretreatment0.05% tretinoin increased elastin content at sites vs. untreated striae by 22% in the papillary and reticular dermis combined10% l - ascorbic acid failed to improve elastin content in either the papillary or reticular dermisboth regimens increased epidermal thickness and decreased papillary dermal thickness70% of patients experienced mild irritation at treatment initiation on both treatment sites.a single patient developed a mild irritant dermatitis=women ; mos = months ; sg = striae gravidarum ; yrs = years.statistical significance is defined as p 0.05 risk factors for striae gravidarum higher pre - pregnancy weight higher weight at delivery higher gestational weight gain fitzpatrick skin types i and iv absence of employment family history of striae gravidarum previous history of sg higher bmi before pregnancy lack of chronic diseases sd on the breasts increased risk (71.4% with striae on breasts vs. 28.6% without) sd on thighs decreased risk (23% with striae vs. 77% without striae) younger age (22.8 yr vs. 26.6 yr) higher pre - pregnancy bmi (21.2 kg / m2 vs. 19.8 kg / m) higher maternal bmi at pregnancy (27.3 kg / m2 vs. 25.6 kg / m) higher gestational age at delivery (39.1 wk vs. 38.6 wk) higher birth weight of baby (3,078.8 g vs. 2,895.8) alcohol drinker (91.4% vs. 8.6%) had little water intake (7.4 glasses vs. 8.3 glasses) family history of sg (82.8% vs. 17.2%) younger age (26.5 yr vs. 30.5) increased weight gain during pregnancy (15.6 kg vs. 38.4 kg) birth weight, gestational age at delivery, and family history of sg associated with moderate - to - severe sg most significant was low maternal age 20% (14 of 71) of teenagers had severe striae, not seen in over 30 yr of age. pre - pregnancy bmi greater than 26 maternal weight gain of more than 15 kg high neonatal birth weight most significant was a history of breast or thigh striae (81% who developed sg had striae history vs. 31% without sg who had history of striae) having a mother with sg additional family history (sisters, daughters, grandmothers, aunts, cousins) of sg non - white had higher association with sg (odds ratio = 4.2, 95% ci 1.9, 9.6). pre - pregnancy bmi not significantly different higher pre - conceptional bmi lower educational level smoking status, skin type, water intake, and level of financial income did not significantly predict sg reduced blood vitamin c levels no significant relation with age, weight gain during pregnancy, abdominal / thigh circumference, or smoking status higher pre - delivery bmi more likely to receive state medical assistance more likely to have hip striae greater weight gain during pregnancy diabetes tests and glycosylated hemoglobin levels were similar in with and without striae. =women ; bmi = body - mass index ; mos = months ; sg = striae gravidarum ; yrs = years. statistical significance is defined as p 0.05 prevention of striae gravidarum development of sg : 56% in placebo group vs. 34% in treatment group intensity of sg was significantly lower in treated with cream vs. placebo in with history of striae during puberty, cream prevented sg in 89% of cases, whereas all formed sg in the placebo group effective in preventing sg only in without a history of striae (6% developed sg on treatment vs. 35% on placebo) severity of previous stretch marks increased in the control group during the study, but not in the treated group among who developed new sg, there was increased severity in control vs. treated group sg occurred at the end of the second quarter of pregnancy in 45.7% in intervention group vs. 62.9% in control group (p = 0.115) difference not statistically significant frequency of severe sg was lower in group that used olive oil. difference not statistically significant sg developed in 10% of alphastria treated group vs. 40% of vitamin treated group vs. 37% in placebo treated group sg developed in 29% of verum treated group vs. 62% in no treatment group sg occurred in 72% of olive oil group vs. 64% in saj cream group vs. 60% in control group. differences not statistically significant sg developed in 45% of patients using cocoa butter cream vs. 48% using placebo difference not statistically significant sg developed in 44% of patients using cocoa butter cream vs. 55% using placebo difference not statistically significant frequency of sg : 20% among who applied oil with massage vs. 38.8% among those who applied w / o massage vs. 41.2% in control group sg developed in 68% of olive oil group vs. 55% not using olive oil. difference not statistically significant the prophylactic use of olive oil to massage the abdomen was associated with a lower incidence of sg =women ; bmi = body - mass index ; mos = months ; sg = striae gravidarum ; yrs = years. statistical significance is defined as p 0.05 treatment of striae gravidarum clinical improvement in striae ranging from 1 - 24% improvement between the 4-week treatment and the 16-week treatment was identified 3 mos after final treatment, patients had observable improvement in the striae, compared with baseline at treatment conclusion, global improvement was achieved from baseline in all stretch marks pre - selected target lesion decreased in length by 20% and width by 23% no statistically significant difference in treated group compared to control group at 2 mos, tretinoin patients had significant improvements in severity scores of sg vs. vehicle patients at 6 mos, 80% of tretinoin patients had improvement vs. 8% of vehicle patients targeted stretch marks treated with tretinoin had decrease in mean length and width of 14% and 8%, respectively, vs. an increase of 10% and 24%, respectively, in vehicle patients at treatment conclusion, 47% improvement with 0.05% tretinoin vs. 43% improvement with ascorbic acid results not statistically significant from each other, but significant for both compared to pretreatment 0.05% tretinoin increased elastin content at sites vs. untreated striae by 22% in the papillary and reticular dermis combined 10% l - ascorbic acid failed to improve elastin content in either the papillary or reticular dermis both regimens increased epidermal thickness and decreased papillary dermal thickness =women ; mos = months ; sg = striae gravidarum ; yrs = years. the most common risk factors for sg include younger age, maternal and family history of sg, higher pre - pregnancy and pre - delivery weight, and higher birth weight (table 1). most studies showed a statistically significant association between these risk factors and sg, although findik. (2004) did not confirm pre - pregnancy weight or maternal age as a risk factor. most studies also demonstrated that a history of striae on the breasts, hips, and thighs was associated with formation of sg ; however, a study of 299 caucasian women showed that although striae on the breasts increased risk of sg, striae on the thighs decreased the risk of sg. with regard to socioeconomic status, multiple studies showed that unemployment, receiving state medical assistance, and lower education level were also associated with sg. increased alcohol intake, decreased water consumption, decreased blood vitamin c levels, and expecting a male baby were also found to be more common among those women who developed sg in select studies. even though it has been speculated that diabetes and increased serum glucose levels could play a part in the pathogenesis of sg, the studies included here did not reveal an association with diabetes or glycosylated hemoglobin levels. creams that contain centella asiatica extract, especially trofolastin cream, are best supported by data for the prevention or reduction of the severity of sg (table 2 ; garca hernndez., 2013, mallol., 1991). centella asiatica is a medicinal herb that is thought to increase the production of collagen and elastic fibers (garca hernndez., 2013). (1991) demonstrated that trofolastin cream with centella asiatica extract, -tocopherol, and collagen elastin hydrolysates that is applied daily from gestational week 12 until delivery significantly reduced the incidence of sg compared with placebo. both mallol. (2013) found that creams that contained centella asiatica significantly reduced the intensity and/or severity of sg among women who did develop sg. 2013) also demonstrated that the severity of previous striae significantly increased in the patient group treated with placebo but did not change in the patient group treated with centella cream. the application of almond oil, olive oil, or cocoa butter consistently failed to significantly lower the incidence of sg compared with placebo group. two studies did find that when olive oil or almond oil were applied with a massage daily, they were associated with a lower incidence of sg development. however, these results may reflect the benefits of massage alone (davey, 1972, timur tahan and kafkasli, 2012). alphastria cream and verum cream, two proprietary creams that contain hyaluronic acid combined with various vitamins and fatty acids, were shown to significantly lower the incidence of sg in two studies (de buman., 1987, wierrani., 1992). hyaluronic acid, the active ingredient in both creams, is thought to increase resistance to mechanical forces and oppose atrophy through stimulation of fibroblast activity and collagen production (elsaie. the creams were applied through massage during the second trimester, which poses the question of whether the creams were truly beneficial or whether the results reflected the benefits of massage alone. although many studies that utilize topical medications or lasers for the treatment of nongestational sd have been performed, only a limited number of these studies focused specifically on sg treatment. treatment should be instituted during the early stages of sg rather than when striae have matured and permanent changes have occurred. many homeopathic and alternative therapies, including fruit and vegetable oils that hydrate the skin, are employed but limited by insufficient evidence. tretinoin cream and a combination of 20% glycolic acid + 10% ascorbic acid were shown to improve sg in clinical studies (table 3). use of tretinoin 0.05% and 0.1% creams on a daily basis for 3 to 7 months consistently resulted in overall global improvement of sg up to 47% (ash., 1998), and decreased in mean length and width up to 20% and 23% respectively (rangel., 2001), of lesions. a study by pribanich. twenty percent glycolic acid combined with either 10% ascorbic acid or 0.05% tretinoin improved the appearance of sg although there was no statistically significant difference between the two combinations (ash., 1998). tretinoin increased elastin content in the papillary and reticular dermis of the lesions but ascorbic acid and untreated areas did not show such improvement. both treatments increased epidermal thickness and decreased papillary dermal thickness in sg lesions. a 1540-nm non - ablative fractional laser demonstrated a statistically - significant clinical improvement in sg that ranged from 1 to 24% and an observable difference at 3 months post - treatment (malekzad., 2014). for nongestational sd, both fractional and non - fractional lasers have been employed with varying efficacies. among fractional lasers, both non - ablative erbium (er):glass and ablative carbon dioxide (co2) lasers have been studied. an average of 50 to 75% improvement in lesions after 2 to 6 nonablative er : glass treatments has been reported (bak., 2009, de angelis., 2011, tretti clementoni and lavagno, 2015). this laser was generally safe and treatments were well - tolerated by patients. in a study by lee. (2010), ablative co2 lasers were demonstrated to have improvements of 50 to 75%, especially in striae alba. however, other studies have shown inconsistent results (cho., 2010). co2 lasers are more painful and may have longer recovery times than non - ablative lasers. among non - fractional lasers, excimer, pulsed dye, neodymium - doped yttrium aluminum garnet (nd : yag), copper bromide, and diode have been studied in the treatment of patients with nongestational sd. the excimer 308-nm laser is used to treat mature striae alba by producing repigmentation and has achieved up to 75% increase in pigmentation ; however, results are generally temporary and pigmentation of the normal surrounding skin is an unfavorable consequence (goldberg., 2003). pulsed dye laser results in textural improvements but has shown a limited benefit to treat striae alba (mcdaniel., 1996). it may be beneficial in striae rubra by reducing erythema (aldahan., 2016). nd : yag laser, also a vascular laser, demonstrated excellent improvement of up to 70% or more, even though it is specifically for immature striae rubra (goldman. thirteen of 15 women experienced a complete resolution or modest improvement of striae for up to 2 years in a small study that used copper bromide laser (longo., 2003). the diode laser was used to treat sd in dark - skinned individuals but this laser was ineffective and 64% of patients developed undesirable hyperpigmentation (tay., 2006). light therapy modalities such as intense pulsed light (ipl), ultraviolet (uv) light, and infrared light have been employed for the treatment of nongestational sd. ipl seems to result in at least moderate improvement of striae (al - dhalimi and abo nasyria, 2013), but persistent erythema and post - inflammatory hyperpigmentation may complicate this treatment. uv light, especially a combination of uv - b and uv - a, has been shown to consistently repigment striae alba. however, the results are not permanent and maintenance treatment is required (sadick., 2007). infrared light at 800 to 1800 nm can result in 25 to 50% improvement in striae alba after only four treatment sessions (trelles., 2008). bipolar radiofrequency demonstrated clinical and histologic improvements in sd (montesi., 2007), while tripolar third generation radiofrequency (tripollar) resulted in 25 to 75% improvement at 1 week post - final treatment (manuskiatti., 2009). modalities such as microdermabrasion and microneedling have been found to be effective to improve nongestational striae in multiple studies. microdermabrasion has been especially effective for striae rubra (abdel - latif and elbendary, 2008). another study found that although microdermabrasion with sonophoresis improved striae, needling therapy yielded an even greater, statistically - significant improvement in striae compared with microdermabrasion (nassar.. needling therapy causes controlled skin injury with the goal of producing new collagen and elastin in the papillary dermis. the most common risk factors for sg include younger age, maternal and family history of sg, higher pre - pregnancy and pre - delivery weight, and higher birth weight (table 1). most studies showed a statistically significant association between these risk factors and sg, although findik. (2004) did not confirm pre - pregnancy weight or maternal age as a risk factor. most studies also demonstrated that a history of striae on the breasts, hips, and thighs was associated with formation of sg ; however, a study of 299 caucasian women showed that although striae on the breasts increased risk of sg, striae on the thighs decreased the risk of sg. with regard to socioeconomic status, multiple studies showed that unemployment, receiving state medical assistance, and lower education level were also associated with sg. increased alcohol intake, decreased water consumption, decreased blood vitamin c levels, and expecting a male baby were also found to be more common among those women who developed sg in select studies. even though it has been speculated that diabetes and increased serum glucose levels could play a part in the pathogenesis of sg, the studies included here did not reveal an association with diabetes or glycosylated hemoglobin levels. creams that contain centella asiatica extract, especially trofolastin cream, are best supported by data for the prevention or reduction of the severity of sg (table 2 ; garca hernndez., 2013, mallol., 1991). centella asiatica is a medicinal herb that is thought to increase the production of collagen and elastic fibers (garca hernndez., 2013). (1991) demonstrated that trofolastin cream with centella asiatica extract, -tocopherol, and collagen elastin hydrolysates that is applied daily from gestational week 12 until delivery significantly reduced the incidence of sg compared with placebo. both mallol. (2013) found that creams that contained centella asiatica significantly reduced the intensity and/or severity of sg among women who did develop sg. 2013) also demonstrated that the severity of previous striae significantly increased in the patient group treated with placebo but did not change in the patient group treated with centella cream. the application of almond oil, olive oil, or cocoa butter consistently failed to significantly lower the incidence of sg compared with placebo group. two studies did find that when olive oil or almond oil were applied with a massage daily, they were associated with a lower incidence of sg development. however, these results may reflect the benefits of massage alone (davey, 1972, timur tahan and kafkasli, 2012). alphastria cream and verum cream, two proprietary creams that contain hyaluronic acid combined with various vitamins and fatty acids, were shown to significantly lower the incidence of sg in two studies (de buman., 1987, wierrani., 1992). hyaluronic acid, the active ingredient in both creams, is thought to increase resistance to mechanical forces and oppose atrophy through stimulation of fibroblast activity and collagen production (elsaie. the creams were applied through massage during the second trimester, which poses the question of whether the creams were truly beneficial or whether the results reflected the benefits of massage alone. although many studies that utilize topical medications or lasers for the treatment of nongestational sd have been performed, only a limited number of these studies focused specifically on sg treatment. treatment should be instituted during the early stages of sg rather than when striae have matured and permanent changes have occurred. many homeopathic and alternative therapies, including fruit and vegetable oils that hydrate the skin, are employed but limited by insufficient evidence. tretinoin cream and a combination of 20% glycolic acid + 10% ascorbic acid were shown to improve sg in clinical studies (table 3). use of tretinoin 0.05% and 0.1% creams on a daily basis for 3 to 7 months consistently resulted in overall global improvement of sg up to 47% (ash., 1998), and decreased in mean length and width up to 20% and 23% respectively (rangel., 2001), of lesions. twenty percent glycolic acid combined with either 10% ascorbic acid or 0.05% tretinoin improved the appearance of sg although there was no statistically significant difference between the two combinations (ash., 1998). tretinoin increased elastin content in the papillary and reticular dermis of the lesions but ascorbic acid and untreated areas did not show such improvement. both treatments increased epidermal thickness and decreased papillary dermal thickness in sg lesions. a 1540-nm non - ablative fractional laser demonstrated a statistically - significant clinical improvement in sg that ranged from 1 to 24% and an observable difference at 3 months post - treatment (malekzad., 2014). for nongestational sd, both fractional and non - fractional lasers have been employed with varying efficacies. among fractional lasers, both non - ablative erbium (er):glass and ablative carbon dioxide (co2) lasers have been studied. an average of 50 to 75% improvement in lesions after 2 to 6 nonablative er : glass treatments has been reported (bak., 2009, de angelis., 2011, tretti clementoni and lavagno, 2015). this laser was generally safe and treatments were well - tolerated by patients. in a study by lee. (2010), ablative co2 lasers were demonstrated to have improvements of 50 to 75%, especially in striae alba. however, other studies have shown inconsistent results (cho., 2010). co2 lasers are more painful and may have longer recovery times than non - ablative lasers. among non - fractional lasers, excimer, pulsed dye, neodymium - doped yttrium aluminum garnet (nd : yag), copper bromide, and diode the excimer 308-nm laser is used to treat mature striae alba by producing repigmentation and has achieved up to 75% increase in pigmentation ; however, results are generally temporary and pigmentation of the normal surrounding skin is an unfavorable consequence (goldberg., 2003). pulsed dye laser results in textural improvements but has shown a limited benefit to treat striae alba (mcdaniel., 1996). it may be beneficial in striae rubra by reducing erythema (aldahan., 2016). nd : yag laser, also a vascular laser, demonstrated excellent improvement of up to 70% or more, even though it is specifically for immature striae rubra (goldman., 2008). thirteen of 15 women experienced a complete resolution or modest improvement of striae for up to 2 years in a small study that used copper bromide laser (longo. the diode laser was used to treat sd in dark - skinned individuals but this laser was ineffective and 64% of patients developed undesirable hyperpigmentation (tay., 2006). light therapy modalities such as intense pulsed light (ipl), ultraviolet (uv) light, and infrared light have been employed for the treatment of nongestational sd. ipl seems to result in at least moderate improvement of striae (al - dhalimi and abo nasyria, 2013), but persistent erythema and post - inflammatory hyperpigmentation may complicate this treatment. uv light, especially a combination of uv - b and uv - a, has been shown to consistently repigment striae alba. however, the results are not permanent and maintenance treatment is required (sadick., 2007). infrared light at 800 to 1800 nm can result in 25 to 50% improvement in striae alba after only four treatment sessions (trelles. bipolar radiofrequency demonstrated clinical and histologic improvements in sd (montesi., 2007), while tripolar third generation radiofrequency (tripollar) resulted in 25 to 75% improvement at 1 week post - final treatment (manuskiatti., 2009). modalities such as microdermabrasion and microneedling have been found to be effective to improve nongestational striae in multiple studies. microdermabrasion has been especially effective for striae rubra (abdel - latif and elbendary, 2008). another study found that although microdermabrasion with sonophoresis improved striae, needling therapy yielded an even greater, statistically - significant improvement in striae compared with microdermabrasion (nassar. needling therapy causes controlled skin injury with the goal of producing new collagen and elastin in the papillary dermis. tretinoin cream and a combination of 20% glycolic acid + 10% ascorbic acid were shown to improve sg in clinical studies (table 3). use of tretinoin 0.05% and 0.1% creams on a daily basis for 3 to 7 months consistently resulted in overall global improvement of sg up to 47% (ash., 1998), and decreased in mean length and width up to 20% and 23% respectively (rangel., 2001), of lesions. twenty percent glycolic acid combined with either 10% ascorbic acid or 0.05% tretinoin improved the appearance of sg although there was no statistically significant difference between the two combinations (ash., 1998). tretinoin increased elastin content in the papillary and reticular dermis of the lesions but ascorbic acid and untreated areas did not show such improvement. a 1540-nm non - ablative fractional laser demonstrated a statistically - significant clinical improvement in sg that ranged from 1 to 24% and an observable difference at 3 months post - treatment (malekzad., 2014). for nongestational sd, both fractional and non - fractional lasers have been employed with varying efficacies. among fractional lasers, both non - ablative erbium (er):glass and ablative carbon dioxide (co2) lasers have been studied. an average of 50 to 75% improvement in lesions after 2 to 6 nonablative er : glass treatments has been reported (bak., 2009, de angelis., 2011, tretti clementoni and lavagno, 2015). this laser was generally safe and treatments were well - tolerated by patients. in a study by lee (2010), ablative co2 lasers were demonstrated to have improvements of 50 to 75%, especially in striae alba. however, other studies have shown inconsistent results (cho., 2010). co2 lasers are more painful and may have longer recovery times than non - ablative lasers. among non - fractional lasers, excimer, pulsed dye, neodymium - doped yttrium aluminum garnet (nd : yag), copper bromide, and diode the excimer 308-nm laser is used to treat mature striae alba by producing repigmentation and has achieved up to 75% increase in pigmentation ; however, results are generally temporary and pigmentation of the normal surrounding skin is an unfavorable consequence (goldberg., 2003). pulsed dye laser results in textural improvements but has shown a limited benefit to treat striae alba (mcdaniel., 1996). it may be beneficial in striae rubra by reducing erythema (aldahan., 2016). nd : yag laser, also a vascular laser, demonstrated excellent improvement of up to 70% or more, even though it is specifically for immature striae rubra (goldman., 2008). thirteen of 15 women experienced a complete resolution or modest improvement of striae for up to 2 years in a small study that used copper bromide laser (longo., 2003). the diode laser was used to treat sd in dark - skinned individuals but this laser was ineffective and 64% of patients developed undesirable hyperpigmentation (tay., 2006). light therapy modalities such as intense pulsed light (ipl), ultraviolet (uv) light, and infrared light have been employed for the treatment of nongestational sd. ipl seems to result in at least moderate improvement of striae (al - dhalimi and abo nasyria, 2013), but persistent erythema and post - inflammatory hyperpigmentation may complicate this treatment. uv light, especially a combination of uv - b and uv - a, has been shown to consistently repigment striae alba. however, the results are not permanent and maintenance treatment is required (sadick., 2007). infrared light at 800 to 1800 nm can result in 25 to 50% improvement in striae alba after only four treatment sessions (trelles., 2008). bipolar radiofrequency demonstrated clinical and histologic improvements in sd (montesi., 2007), while tripolar third generation radiofrequency (tripollar) resulted in 25 to 75% improvement at 1 week post - final treatment (manuskiatti., 2009). modalities such as microdermabrasion and microneedling have been found to be effective to improve nongestational striae in multiple studies. microdermabrasion has been especially effective for striae rubra (abdel - latif and elbendary, 2008). another study found that although microdermabrasion with sonophoresis improved striae, needling therapy yielded an even greater, statistically - significant improvement in striae compared with microdermabrasion (nassar., 2016). needling therapy causes controlled skin injury with the goal of producing new collagen and elastin in the papillary dermis. stretch marks of pregnancy, which most commonly occur on the abdomen, breasts, hips, and thighs, have notably been a cause of distress and concern for the patient. although many attempts have been made to identify risk factors, prevention methods, and treatments, a limited number of well - conducted, randomized controlled studies exist to date. this systematic review found that more studies addressed risk factors and prevention methods for sg than they did treatments specifically for sg and many more studies evaluated treatments for nongestational sd. the most significant risk factors identified in this review are younger age, maternal and family history of sg, increased pre - pregnancy and pre - delivery weight, and increased birth weight. for prevention of sg, creams with centella asiatica extract such as trofolastin cream and a daily massage seem the most supported treatment options by the literature, but further studies are necessary. this information can be helpful for future expectant mothers who would like to try preventative treatments for sg. with regard to the management of sg, the current most effective therapies include tretinoin cream 0.05% and modalities such as nonablative fractional lasers. laser treatment appears to yield on average greater mean improvement and in a much shorter time than topical treatments, but no head - to - head studies have been conducted to date. tretinoin cream and laser treatments resulted in increased elastin content and collagen production in the treated lesions, which can partly explain the improvement observed. many new studies that test novel laser treatments, microdermabrasion, and microneedling are underway. for example, some studies observed pre - pregnancy weight as a significant risk factor (picard., 2015), but other studies did not view this but rather a genetic component as the most significant risk factor (chang., 2004). the available studies often include a small and non - randomized sample size, especially those studies that are relevant to treatment. many more studies have been conducted for nongestational sd, which brings up the concern of whether these results may be extrapolated to sg. sg are commonly regarded as a cosmetic nuisance and overlooked by practitioners as clinically insignificant. skindex-29 is a validated questionnaire on the quality of life of patients with dermatologic conditions that has been used to assess total impairment caused by sg. the questionnaire focuses on three scales : emotion (psychological effects), symptoms, and daily functioning. in a study by yamaguchi. (2012) to evaluate the quality of life of women with sg through the skindex-29 questionnaire, the authors observed significantly greater psychological and/or emotional impairment among pregnant women with sg compared with women without sg (yamaguchi., 2012). using the same questionnaire, they also noted that pregnant women with severe sg scored significantly higher in areas of psychological and/or emotional impairment as well as daily functioning impairment, compared with those without sg and those with mild sg. striae gravidarum are a common form of gestational change that can be a substantial source of distress. despite the identification of risk factors, prevention of sg further results from large, randomized - controlled studies are required to validate prevention and treatment options and their long - term efficacy data. | backgroundstriae gravidarum (sg) are atrophic linear scars that represent one of the most common connective tissue changes during pregnancy. sg can cause emotional and psychological distress for many women. research on risk factors, prevention, and management of sg has been often inconclusive.methodswe conducted a literature search using textbooks, pubmed, and medline databases to assess research performed on the risk factors, prevention, and management of sg. the search included the following key words : striae gravidarum, pregnancy stretch marks, and pregnancy stretch. we also reviewed citations within articles to identify relevant sources.resultsyounger age, maternal and family history of sg, increased pre - pregnancy and pre - delivery weight, and increased birth weight were the most significant risk factors identified for sg. although few studies have confirmed effective prevention methods, centella asiatica extract, hyaluronic acid, and daily massages showed some promise. treatment for general striae has greatly improved over the last few years. topical tretinoin 0.05% has demonstrated up to 47% improvement of sg and non - ablative fractional lasers have consistently demonstrated 50 to 75% improvement in treated lesions of striae distensae.conclusionoverall, sg has seen a resurgence in research over the last few years with promising data being released. results of recent studies provide dermatologists with new options for the many women who are affected by these disfiguring marks of pregnancy. |
intracranial dermoid cysts and colloid cysts are rare congenital lesions generally occurring along the midline. dermoid cysts are known to be ectodermal in origin while debate still exists about the embryological origin of colloid cysts. here, we report a 22-year - old young male with suprasellar dermoid cyst associated with a colloid cyst of the third ventricle and discuss the possibility of their unified embryological derivation. to best of our knowledge, a 22-year - old male patient presented with on and off frontal headaches and occasional blurring of vision for 1 year. clinical examination was normal with visual acuity of 6/6 in both eyes without evidence of any field defects on perimetry. magnetic resonance imaging (mri) revealed mainly cystic lesion in the suprasellar region with small solid component anteriorly which was hyperintense on t1-weighted sequences without any contrast enhancement. a colloid cyst of 1.2 1.5 cm was seen at the foramen of monro, mainly occupying third ventricle with evidence of mild hydrocephalus [figure 1a ]. an old bifrontal craniotomy was re - explored and complete excision of suprasellar dermoid cyst was achieved through bilateral subfrontal approach along with transcortical transventricular excision of colloid cyst in the same setting. intraoperatively, suprasellar cyst was containing a whitish fluid with fat globules with small solid vascularized fatty nodule. post - operative scans showed complete excision of both the lesions [figure 1b ]. sagittal sections of contrast - enhanced magnetic resonance images of brain showing cystic lesion in the suprasellar area with small solid part anteriorly. post - operative scans (48 hrs) scans showed complete excision of dermoid and colloid cyst with decreased mass effect on the optic chiasm (b) photomicrographs showing stratified squamous epithelium with underlying sebaceous glands (a). colloid cyst wall comprising fibrocollagenous wall and is lined by columnar epithelium (c) with high - power view of lining epithelium (d). (a, b : hande 100 ; c, d : h and e 200) intracranial dermoid cysts generally occur along the midline and are derived from the trapped somatic ectoderm during embryological development during third to fifth week of gestation. these cysts are generally associated with dermal sinus tracts and other neural tube closure defects ; however, dermal sinus tract may be absent in suprasellar and cerebello - pontine angle dermoids. these lesions mainly presents with headache, local sings of mass effect, or neural compression. colloid cysts of the third ventricle are also rare benign midline lesions presenting during early adulthood. colloid cyst generally become symptomatic in early adulthood and mainly presents with headache and hydrocephalus. although controversy exists about timing of surgery of asymptomatic colloid cysts, symptomatic ones are excised surgically at the earliest. with advent of endoscopy, complete or partial resection of colloid cyst congenital origin of these cysts is accepted universally ; however, cell of origin is highly debated. initial reports suggested their neuroepithelial origin as the derivatives of paraphysis or ventricular ependyma and these were further supported later by other studies. however, mackenzie in 1991 suggested the endodermal derivation of these cysts on the basis of ultrastructural features which was supported by other authors. until recently endodermal derivation of these cysts enjoyed the favor when nagaraju., in 2010 reignited the debate by claiming colloid cysts as paraphysis remnants in human. colloid cysts are associated with various developmental malformations like corpus callosal agenesis, anterior cranial fossa encephalocels, cavum veli interpositi, and multiple neuroepithelial cysts and aqueductal stenosis. these malformations represent a spectrum of neural tube closure defects and associated anomalies. although isolated sporadic form of colloid cyst is most common occurrence, these associations suggest disordered embryogenesis as a possible mechanism in their formation. cheng., in 1999 reported a case of nasal dermoid sinus cyst associated with colloid cyst of third ventricle and proposed a fascinating theory of anterior neuropore corridor defects to explain the spectrum of these associated rare midline lesions. according to this theory, defective closure of situs neuroporicus promotes the neuroepithelium adjacent to commissural plate derivatives to form a vacuolated paraphysis growing slowly as colloid cyst of the third ventricle. similar derivation from paraphysial remnants was also reported by nagaraju., in 2010 supporting cheng 's hypothesis. although lack of substantial evidence remained main drawback of this theory, disordered embryogenesis appears to be the most plausible explanation in our case rather than mere coincidence. suprasellar dermoid cyst associated with colloid cyst of the third ventricle is a rare presentation. this association appears more than coincidence and can be partially explained by anterior neuropore corridor defect theory. | intracranial dermoid cyst and colloid cysts of the third ventricle are rare benign congenital lesions of early adulthood. both lesions are thought to be congenital in origin however association is rare. only one case of this association has been reported. we report a 22-year - old male with suprasellar dermoid cyst and colloid cyst of the third ventricle presenting simultaneously. embryogenesis of this association has been discussed. |
it has been widely accepted that the application of coronary computed tomographic angiography (ccta) is most likely to benefit patients who are at intermediate risk of having stable coronary artery disease (scad). as a result, in 2013 european society of cardiology guidelines on the management of scad (2013 g), importance was given to systematical testing with the consideration of pretest probability and the updated diamond - forrester model (udfm) was recommended as the clinical algorithm. according to 2013 g, ccta should be considered as an alternative to stress test in patients with ejection fraction (ef) 50% and pretest probability between 15% and 50%, or a complement to stress tests in patients with ef 50% and pretest probability between 50% and 85% or with ef 120 mol / l), new york heart association class iii or iv heart failure, atrial fibrillation, aortic disease, or > 90 years old. patients were considered to have diabetes mellitus, hyperlipidemia, or hypertension if they were taking corresponding treatment or had been diagnosed as such according to their medical records. typical chest pain was defined as having (1) substernal chest pain or discomfort, which was (2) provoked by exertion or emotional stress and (3) relieved by rest and/or nitroglycerin. if one or none of the criteria was present, the patient was classified as having nonangina associated chest pain. all scans were performed with a second - generation dual - source ccta scanner (somatom definition flash, siemens medical solutions, forchheim, germany). patients received heart rate control, as well as sublingual nitroglycerin before ccta, and underwent prospectively electrocardiogram (ecg)-triggered high - pitch spiral scan, step - on sequence, or retrospective spiral scan as appropriate. two experienced observers, a radiologist and a cardiologist, evaluated the ccta data on a syngo multimodality workstation (siemens medical solutions, forchheim, germany) using volume rendering, multiplanar reformation and maximum intensity projection. each segment was assessed for diameter, location, presence of atherosclerotic changes (calcified and noncalcified), and concomitant coronary artery stenosis. a significant stenosis was defined as an obstructive luminal reduction (50%) compared with normal reference segment, while nonsignificance referred to mild stenosis (120 mol / l), new york heart association class iii or iv heart failure, atrial fibrillation, aortic disease, or > 90 years old. patients were considered to have diabetes mellitus, hyperlipidemia, or hypertension if they were taking corresponding treatment or had been diagnosed as such according to their medical records. typical chest pain was defined as having (1) substernal chest pain or discomfort, which was (2) provoked by exertion or emotional stress and (3) relieved by rest and/or nitroglycerin. if one or none of the criteria was present, the patient was classified as having nonangina associated chest pain. all scans were performed with a second - generation dual - source ccta scanner (somatom definition flash, siemens medical solutions, forchheim, germany). patients received heart rate control, as well as sublingual nitroglycerin before ccta, and underwent prospectively electrocardiogram (ecg)-triggered high - pitch spiral scan, step - on sequence, or retrospective spiral scan as appropriate. two experienced observers, a radiologist and a cardiologist, evaluated the ccta data on a syngo multimodality workstation (siemens medical solutions, forchheim, germany) using volume rendering, multiplanar reformation and maximum intensity projection. each segment was assessed for diameter, location, presence of atherosclerotic changes (calcified and noncalcified), and concomitant coronary artery stenosis. a significant stenosis was defined as an obstructive luminal reduction (50%) compared with normal reference segment, while nonsignificance referred to mild stenosis (2 vessels : 6.6%). this proportion did not change over time (p = 0.3451), but a significant (p 2 vessels : 6.6%). this proportion did not change over time (p = 0.3451), but a significant (p < 0.05) decline was observed in complications, as well as radiation exposure. ccta procedural characteristics before and after the publication of 2013 g values are presented as mean sd or n (%).p<0.05, p<0.01;. lm : left main ; lad : left anterior descending ; other lad included ramus intermedius, diagonal branch and middle and distal of lad ; lcx : left circumflex ; other lcx included posterior descending artery from lcx, posterior - lateral branch from lcx and middle and distal of lcx ; rca : right coronary artery ; other rca included posterior descending artery from rca, posterior - lateral branch from rca and middle and distal of rca. to investigate the rate of inappropriate ccta application, enrolled patients were sorted and compared according to different clinical risk levels [figure 1 ]. patients with ef 50% and pretest probability 15%, who were devoid of other complications, received more inappropriate cctas after 2013 g in comparison to those before 2013 g (16.3% vs. 13.9%, p = 0.0243). we observed a decrease in inappropriate use in patients with ef < 50% and without typical angina who should receive stress tests before ccta (2% vs. 3.3%, p = 0.0047) although there was no significant difference between these 2 time periods (p = 0.0821). (a) patients with ejection fraction 50% and pretest probability 15% should have other diseases excluded. (b) patients with ejection fraction 50% and pretest probability between 50% and 85% should take stress tests before coronary computed tomographic angiography. (c) patients with ejection fraction < 50% and without typical angina should take stress tests before coronary computed tomographic angiography. (d) for patients with ejection fraction < 50% and typical angina or ejection fraction 50% and pretest probability 85%, invasive coronary angiography was necessary. the bars indicate the 95% confidence interval. in an analysis of appropriate ccta applications continuously, a nadir was found in the rate of appropriate ccta in may 2013, 3 months prior to the publication of the 2013 g [figure 2 ]. to avoid influencing the examination of trends in ccta within the time periods, the first time period was divided into two stages at the nadir (march 1, 2013 to may 1, 2013 and may 1, 2013 to september 1, 2013) the rate of appropriate coronary computed tomographic angiography over the study period is shown for the overall study population. furthermore, a significant decline was demonstrated in the adjusted rate of appropriate ccta [figure 3 ] from the nadir on may 1, 2013 to september 1, 2014 (odds ratio [or ] for appropriate ccta per 15-day increase in time, 0.974 ; 95% confidence interval [ci ], 0.9600.987 ; p = 0.0002). however, there was no significant change before (or, 0.989 ; 95% ci, 0.9581.021 ; p = 0.9683) or after (or, 1.002 ; 95% ci, 0.9821.021 ; p = 0.8678) the publication of the 2013 g. the ors referred to the adjusted odds of appropriate ccta in the interval of 15 days. when subgroups were analyzed on the basis of gender, age, other risk factors, patients (in - patient or out - patient), or symptom, no significant change was found in the adjusted rate of appropriate ccta following the publication of the 2013 g. odds ratios for change in the rate of appropriate coronary computed tomographic angiography in different subgroups, per 15-day period. the final model was adjusted for gender, age, number of other risk factors, pretest probability, ejection fraction, appropriate stress test, patients (admitted and out), and symptom. in this study, in patients subjected to ccta due to suspected scad, the rate of appropriate ccta was slightly more than 60%, and no significant change was found in this rate following the publication of the 2013 g. our study indicated that there was a trend toward a decrease over time in the rate of ccta in some subgroups. despite the follow - up for 12 months following the publication of the 2013 g, inappropriate ccta continued to be performed in a considerable proportion of patients suspected to have scad. these findings suggested that the evidence provided by recent studies and the guideline recommendations have not been widely incorporated into clinical practice. although reasons for the lack of impact of the 2013 g on clinical practice are multifactorial, the following one should emerge as a particularly strong candidate. it has been investigated extensively that a pretest probability model developed in one population may overestimate or underestimate the probability in another population with different characteristics. for example, several observations indicated a superior predictive accuracy of duke clinical score, but significant overestimations were observed by others. udfm was the most efficient and operational model in the study of jensen., only revealing a moderate predictive accuracy. taking the limitations of the models into consideration, physicians were mainly concerned regarding the overestimation, which might lead to overuse of downstream tests and therapies. to improve acceptability of the pretest probability models in clinical practice, more rigorous external validations for the existing models what is more, a standardized, reliable and easily usable model is more likely to contribute to promoting the application of guidelines in clinical practice. except for the belief that the model may overestimate the pretest probability, actually, nearly one in two inappropriate cctas resulted from the lack of appropriate stress tests, regardless of whether they were before or after the publication of 2013 g. neither the class i recommendation (should be considered) for stress tests as an initial test nor the class iia recommendation (is recommended / is indicated) for ccta as a complement to stress tests in patients (1) with ef 50% and pretest probability between 50% and 85% or (2) with ef < 50% and without typical angina was widely incorporated into clinical practice. although there is a general consensus that stress tests are more cost - effective than ccta in patients with a higher rate of intermediate risk, several speculative reasons accounted for the unsatisfactory use of stress tests, such as the lack of awareness, resources and reimbursement, and fear about the increase in malpractice liability. the other major reason for inappropriate ccta was the overuse of ccta in patients with ef 50% and pretest probability 15%. according to the class iii recommendation (is not recommendeds) in 2013 g however, physicians are not inclined to alter their practice based on a negative recommendation, especially when it contradicts traditional beliefs and external influences, such as conflicting patient expectations, financial incentives, and fear of missed diagnosis. all of these may play an important role in the development of the barriers between evidence and practice, but future studies may provide a more valuable explanation. it is noteworthy that there are two different types of barriers : overuse and underuse. this study only retrospectively included patients referred for ccta and focused on the overuse of ccta. according to the guideline recommendation, some patients who should have undergone ccta after an inconclusive stress test were referred directly to invasive angiography. more investigations are required to determine the underuse of ccta, which lead to overuse of invasive angiography in actual clinical practice. first, while we strived to define a population that reflected the inclusion and exclusion criteria of udfm and 2013 g, there are considerable differences between the cohort in this study and others previously described. the population in this study consisted of asymptomatic patients at risk for scad, such as patients with palpitation (caused by arrhythmia rather than scad according to ecg) and dizziness. instead of estimation of pretest probability, assessment of cardiovascular risk using screening tests (such as ecg, carotid ultrasound, and coronary calcium) was recommended (iia or iib) and led to ccta overuse for these patients. however, after sending them into the estimation of pretest probability, it resulted in an increase in the amount of appropriate ccta. then, as a complement to stress tests in patients with a higher rate of intermediate risk, ccta was recommended after an inconclusive stress test. in this subgroup, we defined an appropriate ccta as one with a prior stress test, regardless of the conclusion. as a result, an unnecessary ccta after a conclusive stress test was defined as an appropriate one. finally, the influence of coronary artery calcification on ccta was not taken into consideration in this study. although all of the limitations may lead to overestimation of the actual rate of appropriate ccta, they would have had no influence on the examination of trends in the rate of appropriate ccta. although we collected data from a large, complex, and professional center, the impact of 2013 g in different centers, regions, and countries with different models of health care delivery and practice environments has not been assessed. thus, a multicenter study with a large sample size will be needed in the future. in conclusion, this study found very little evidence that 2013 g has influenced practice in the subsequent 12 months. the use of ccta as a screening test or an inappropriate alternative to a stress test remains commonplace despite little evidence to support it and new clinical practice guidelines recommending against it. this study causes two main concerns : on one hand, it demonstrated that many patients suspected of scad continue to undergo a costly and ineffective ccta. on another hand, a huge investment in the generation of clinical evidence has yet to effectively influence clinical practice. according to this study, a widely accepted method to estimate pretest probability and interventions in education, media campaigns, technical innovation, finance, multidisciplinary collaboration, and quality management may fundamentally improve this incomplete knowledge transfer. this work was supported by grants from the national natural science foundation of china (no. this work was supported by grants from the national natural science foundation of china (no. | background : coronary computed tomographic angiography (ccta) has been widely used in patients who are at intermediate risk for having stable coronary artery disease (scad), and 2013 european society of cardiology guidelines on the management of scad (2013 g) recommended the appropriate application of ccta. however, 2013 g has not been subjected to systematic analyses for subsequent impact on clinical practice.methods:a total of 5320 patients suspected with scad were enrolled and scheduled for ccta from march 2013 to september 2014. for each patient, pretest probability of scad was calculated according to updated diamond - forrester model (udfm). appropriate ccta or appropriate stress test was determined as described in the 2013 g. a generalized estimating equation model was used to determine the trends in the half - monthly rate of appropriate ccta.results:overall, only 61.37% of patients received appropriate ccta, and there was insignificant change over time (p = 0.8701). the application of ccta in patients who should have had a stress test accounted for most of the inappropriate ccta before (22.29%) or after (19.98%) the publication of the 2013 g. in all patients or any subgroup, no significant change in the adjusted half - monthly rate of appropriate ccta was found after the publication of the 2013 g (odds ratio, 1.002 ; 95% confidence interval, 0.9821.021 ; p = 0.8678).conclusions : these findings suggest that the 2013 g have not, to date, been fully incorporated into clinical practice, and the clinical utilization of ccta remains unreasonable to some extent. |
as reported in henschen, a petrous ridge meningioma was first described by rokitansky in 18556). in 1938, only one did well and, accordingly, he emphasized a high surgical risk in dealing with infratentorial meningiomas25). because of its deep location, narrow visual field of operation and proximity to cranial nerves, brain stem and important blood vessels, surgical intervention for this pathology have a high lethal and crippled rate16). clinical presentation and microsurgical anatomy, particularly in regard to cranial nerve dislocation, critically depend on the tumor 's dural origin22). infratentorial meningiomas include cerebellar convexity, tentorial, cerebellopontine angle, jugular foramen, petroclival, peritorcula, and foramen magnum. however, infratentorial meningiomas have been a challenge for operation, because of its close proximity to critical vascular and neurologic structures and slow growth25). in the early series, the surgical try to remove these lesions was associated with high rates of morbidity and mortality9). as developing radiologic tools, microsurgical techniques and surgical approaches of these tumors, surgical management has been evolving and shown good outcomes along with a decrease in surgical morbidity and pamortality2,11,20,28). despite these improvements, surgery for infratentorial meningiomas, we analyzed surgical outcomes of patients with infratentorial meningiomas surgically treated at our institution to postulate the predictors for surgical resection, recurrence, complication, and survival. the study is in compliance with the declaration of helsinki (sixth revision, 2008). this study fulfils all the requirements for patient anonymity was approved by the institutional review board. between april 1993 and may 2013, 782 case of intracranial meningioma were surgically resected at our institution. among these cases, 158 patients (20.2%) were diagnosed with infratentorial meningioma by means of radiological studies [non - enhanced and enhanced computed tomographic (ct) scanning and magnetic resonance imaging (mri) ] and their surgical records. recurred cases were excluded from this study. to define the clinical and radiological characteristics of the patients, several variables such as age, sex, presenting symptoms, pathology, tumor features (size, location, peritumoral edema), preoperative and postoperative kps, postoperative transient / permanent complications, recurrence and survival rate were evaluated. the patients consisted of 32 males and 126 females (female predominance, 79.7%), with a median age of 57.1 years (range, 16 - 77 years). the most common presenting symptoms were headache, followed by dizziness and disturbance of gait. the exact location of the patients was decided by radiological study and operation records, with regard to the origin of the mass, including tentorial meningioma in 39 patients, cerebellopontine angle (cpa) in 75 patietns, petroclival in 21 patients, peritorcular in 8 patients, cerebellar convexity in 7 patients, foramen magnum in 6 patients, and jugular foramen in 2 patients. tumor locations were classified into 3 groups according to their origin site (group i, cerebellopontine angle & cerebellar convexity ; group ii, tentorial & peritorcular ; group iii, petroclival & other skull base location). based on t1-weighted enhanced & t2-weighted non - enhanced mri, the maximal size of the tumor and peri - tumoral edema was evaluated. four centimeter was cut - off value as the size parameter (< 4 cm vs. 4 cm). according to the peri - tumoral edema, the patients were divided into two groups [none or minimal (< 5 mm in edema thickness) vs. moderate or severe (5 mm in edema thickness) ]. gross total resection was defined as simpson 's grades i and ii resection without any visible tumor remnant on surgical finding and follow - up mri. all tumors were graded pathologically according to the world health organization (who) classification system. according to the pamortality had been classified by who grade that grade i in 148 patients, grade ii & iii in 10 (9 patients in grade ii & 1 in iii). on follow - up mri, new enhancing mass in completely resected case or re - growing symptomatic mass in non - completely resected case was regarded as a recurred lesion. surgery - related complication was defined as a new developed neurological deficit or aggravated pre - existing deficit. the transient complication was defined as the new or aggravated postoperative problem resolved generally within 3 - 6 months after the operation. if the problem persisted or needed another operation, it was regarded as the permanent complication. the time to measure of post - operative kps was 6 months after the operation. all surviving patients were monitored ; the mean duration of follow - up was 48.4 months with a range of 0.8 to 242.2 months. recurrence - free survival (rfs) was calculated as the time from surgery to the date of recurrence. the probability of rfs was analyzed according to the kaplan - meier method and compared with the log - rank test. overall survival was also calculated as the time from surgery to the date of death or last follow - up visit. for the multivariate analysis, independent prognostic factors were determined using the cox 's proportional hazards model. the relation between resection degree / permanent complication occurrence and categorical variables was compared by using a chi - square test or fisher - exact probability test. all statistical analyses were performed using spss version 20.0 software program for windows (chicago, il, usa). the study is in compliance with the declaration of helsinki (sixth revision, 2008). this study fulfils all the requirements for patient anonymity was approved by the institutional review board. between april 1993 and may 2013, 782 case of intracranial meningioma were surgically resected at our institution. among these cases, 158 patients (20.2%) were diagnosed with infratentorial meningioma by means of radiological studies [non - enhanced and enhanced computed tomographic (ct) scanning and magnetic resonance imaging (mri) ] and their surgical records. to define the clinical and radiological characteristics of the patients, several variables such as age, sex, presenting symptoms, pathology, tumor features (size, location, peritumoral edema), preoperative and postoperative kps, postoperative transient / permanent complications, recurrence and survival rate were evaluated. the patients consisted of 32 males and 126 females (female predominance, 79.7%), with a median age of 57.1 years (range, 16 - 77 years). the most common presenting symptoms were headache, followed by dizziness and disturbance of gait. the exact location of the patients was decided by radiological study and operation records, with regard to the origin of the mass, including tentorial meningioma in 39 patients, cerebellopontine angle (cpa) in 75 patietns, petroclival in 21 patients, peritorcular in 8 patients, cerebellar convexity in 7 patients, foramen magnum in 6 patients, and jugular foramen in 2 patients. tumor locations were classified into 3 groups according to their origin site (group i, cerebellopontine angle & cerebellar convexity ; group ii, tentorial & peritorcular ; group iii, petroclival & other skull base location). based on t1-weighted enhanced & t2-weighted non - enhanced mri, the maximal size of the tumor and peri - tumoral edema was evaluated. four centimeter was cut - off value as the size parameter (< 4 cm vs. 4 cm). according to the peri - tumoral edema, the patients were divided into two groups [none or minimal (< 5 mm in edema thickness) vs. moderate or severe (5 mm in edema thickness) ]. gross total resection was defined as simpson 's grades i and ii resection without any visible tumor remnant on surgical finding and follow - up mri. all tumors were graded pathologically according to the world health organization (who) classification system. according to the pamortality had been classified by who grade that grade i in 148 patients, grade ii & iii in 10 (9 patients in grade ii & 1 in iii). on follow - up mri, new enhancing mass in completely resected case or re - growing symptomatic mass in non - completely resected case was regarded as a recurred lesion. surgery - related complication was defined as a new developed neurological deficit or aggravated pre - existing deficit. the transient complication was defined as the new or aggravated postoperative problem resolved generally within 3 - 6 months after the operation. if the problem persisted or needed another operation, it was regarded as the permanent complication. the time to measure of post - operative kps was 6 months after the operation. all surviving patients were monitored ; the mean duration of follow - up was 48.4 months with a range of 0.8 to 242.2 months. recurrence - free survival (rfs) was calculated as the time from surgery to the date of recurrence. the probability of rfs was analyzed according to the kaplan - meier method and compared with the log - rank test. overall survival was also calculated as the time from surgery to the date of death or last follow - up visit. for the multivariate analysis the relation between resection degree / permanent complication occurrence and categorical variables was compared by using a chi - square test or fisher - exact probability test. all statistical analyses were performed using spss version 20.0 software program for windows (chicago, il, usa). for some cases, far lateral, subtemporal, or orbitozygomatic route was selected to resect the lesion located on skull base region. in our series, 129 patients (81.6%, 129/158) were classified into simpson 's grade i and ii, 28 patients (17.7%) into grade iii and iv and 1 patient (0.6%) into grade v. on univariate analysis, female (p=0.019), non - skull base location (p<0.001) and postoperative kps over than 90 (p=0.012) were significantly related with the possibility of complete resection (table 2). however, only non - skull base location was associated with complete resection, on multivariate analysis [hazard ratio (hr) : 0.162, 95% confidence index (ci) : 0.065 - 0.405, p<0.001 ]. in this study, recurrence rate was 13.3% (21/158) and the 5-, 10-, and 15-year recurrence rates were 8.2%, 12.0%, and 13.3%, respectively (fig. the results of analyses of the variables that could be correlated with recurrence are shown in fig. 2 and table 3. on univariate analysis, age, pathology, postoperative kps, postoperative complication, edema, size, preoperative symptom and simpson 's grade showed statistical significance. the patient 's age at the operation was important factor for rfs [mean value, 36.55.2 months (group < 40 years) vs. 155.512.9 months (40 group < 60 years), 107.66.5 months (group 60 years), p=0.004 ]. the younger - aged group showed shorter rfs than the older - aged group (mean value, 36.55.2 months vs. 153.411.1 months, p=0.001). the more aggressive pathology group (who grade ii and iii) showed the recurrence in earlier postoperative period than the benign group (mean value, 45.612.5 months vs. 155.711.1 months, p<0.001). the size of tumor was also an important factor [mean value, 175.29.8 months (group < 4 cm) vs. 107.712.9 months (4 cm group < 6 cm), 106.322.8 months (group 6 cm), p=0.026 ]. in the group with large - size tumor (4 cm), the rfs was shorter than small - size group (mean value, 175.29.8 months vs. 113.311.1 months, p=0.007). the patient with brain stem or cranial nerve sign showed a shorter rfs time, compared to the patient with other minor symptoms (value, 157.912.8 months vs. 113.811.4 months, p=0.038). in regard to the peritumoral edema, higher edema group (moderate or severe) was significantly associated with shorter rfs (mean value, 168.111.4 months vs. 73.08.6 months, p<0.001). not surprisingly, non - complete resection group (simpson 's grade iii - v) demonstrated a shorter rfs compared to complete resection group (mean value, 154.012.1 months vs. 114.021.9 months, p=0.002). the absence of postoperative complication (mean value, 157.611.3 months vs. 70.412.4 months, p=0.001) and postoperative kps over 90 (mean value, 163.212.0 months vs. 71.59.7 months, p<0.001) were significantly related with recurrence in a longer follow - up period. on multivariate analysis, pathologic grade, postoperative kps, peritumoral edema and resection degree were independent predictable factors for tumor recurrence. benign pathology (hr 0.109, 95% ci 0.026 - 0.453, p=0.002), postoperative kps over 90 (hr 0.078, 95% ci 0.025 - 0.243, p<0.001), low peritumoral edema (hr 0.134, 95% ci 0.046 - 0.392, p<0.001), and complete resection (hr 0.226, 95% ci 0.077 - 0.669, p=0.007) were significantly associated with longer rfs time. the incidence of death rate was 5.0% over follow - up period (8/158). the surgery - related mortality which the death occurred within one month after resection was 3.16% in this study (5/158). the 5-, 10-, and 15-year survival rates were 96.2%, 94.9%, and 94.9%, respectively (fig. the results of analyses of the variables that could be correlated with survival time are shown in fig. 4 and table 4. on univariate analysis, pathology, location, size, postoperative complication and kps, and simpson 's grade showed statistical significance. the more aggressive pathology group (who grade ii and iii) showed a shorter survival time than the benign group (mean value, 40.116.2 months vs. 227.46.2 months, p<0.001). the tumor location was important factor for survival time [mean value, 167.16.7 months (group i) vs. 236.65.5 months (group ii), 98.08.6 months (group iii), p=0.009 ]. the group located at skull base showed shorter survival time than the non - skull base group (mean value, 98.08.6 months vs. 232.36.3 months, p=0.002). the size of tumor is also important variable in survival time [mean value, 200.52.6 months (group < 4 cm) vs. 192.120.8 months (4 cm group < 6 cm), 152.711.2 months (group 6 cm), p=0.023 ]. in larger - sized group (4 cm), the survival time was shorter compared that of small - size group (mean value, 200.52.6 months vs. 200.815.9 months, p=0.008). interestingly, non - complete resection group (simpson 's grade iii - v) demonstrated a shorter survival time than the complete resection group (mean value, 232.85.7 months vs. 139.117.2 months, p<0.001). with regard of permanent complication after surgical resection, the presence of complication was significantly related with shorter survival time (mean value, 233.14.5 months vs. 85.912.9 months, p=0.001). the patient with low level of postoperative kps (< 90) also showed shorter survival time, compared to the patient with higher score (mean value, 239.42.8 months vs. 89.79.2 months, p<0.001). our study showed that the patient lesser than 90 in preoperative kps survived shorter than the patients of high level kps, with a marginal statistical significance (mean value, 234.54.4 months vs. 132.910.2 months, p=0.051). on multivariate analysis, pathology, resection degree and postoperative kps were independent predictable factors for survival rate. benign pathology (hr 0.041, 95% ci 0.005 - 0.343, p=0.003), complete resection (hr 0.150, 95% ci 0.029 - 0.769, p=0.023) and postoperative kps over 90 (hr 0.041, 95% ci 0.005 - 0.351, p=0.004) were significantly associated with a longer survival rate. most common permanent complication is cranial nerve injury (42.9%) followed by operation - related hemorrhage or infarction, motor weakness, hydrocephalus, and infection. the results of analyses of the variables that could be correlated with permanent complications are shown in table 5. on univariate analysis, location, preoperative symptom, and preoperative kps showed statistical significance. the tumor location was important factor for development of permanent complication (8.1% in group i vs. 2.3% in group ii, 28.0% in group iii, p=0.002). the group located at skull base showed higher possibility of permanent complication than the non - skull base group (28.0% vs. 6.0%, p=0.001). the patient with brain stem or cranial nerve sign showed higher possibility for postoperative complication, compared to the patient with other minor symptoms (19.0% vs. 7.4%, p=0.043). the patient with low level of postoperative kps (< 90) was also significantly associated with permanent complication (37.1% vs. 3.5%, p<0.001). on multivariate analysis, location and postoperative kps were independent predictable factors for development of permanent complication. non - skull base location (hr 0.206, 95% ci 0.058 - 0.727, p=0.014) and postoperative kps over 90 (hr 0.048, 95% ci 0.012 - 0.197, p<0.001) were significantly related with a higher complication rate. for some cases, far lateral, subtemporal, or orbitozygomatic route was selected to resect the lesion located on skull base region. in our series, 129 patients (81.6%, 129/158) were classified into simpson 's grade i and ii, 28 patients (17.7%) into grade iii and iv and 1 patient (0.6%) into grade v. on univariate analysis, female (p=0.019), non - skull base location (p<0.001) and postoperative kps over than 90 (p=0.012) were significantly related with the possibility of complete resection (table 2). however, only non - skull base location was associated with complete resection, on multivariate analysis [hazard ratio (hr) : 0.162, 95% confidence index (ci) : 0.065 - 0.405, p<0.001 ]. in this study, recurrence rate was 13.3% (21/158) and the 5-, 10-, and 15-year recurrence rates were 8.2%, 12.0%, and 13.3%, respectively (fig. the results of analyses of the variables that could be correlated with recurrence are shown in fig. 2 and table 3. on univariate analysis, age, pathology, postoperative kps, postoperative complication, edema, size, preoperative symptom and simpson 's grade showed statistical significance. the patient 's age at the operation was important factor for rfs [mean value, 36.55.2 months (group < 40 years) vs. 155.512.9 months (40 group < 60 years), 107.66.5 months (group 60 years), p=0.004 ]. the younger - aged group showed shorter rfs than the older - aged group (mean value, 36.55.2 months vs. 153.411.1 months, p=0.001). the more aggressive pathology group (who grade ii and iii) showed the recurrence in earlier postoperative period than the benign group (mean value, 45.612.5 months vs. 155.711.1 months, p<0.001). the size of tumor was also an important factor [mean value, 175.29.8 months (group < 4 cm) vs. 107.712.9 months (4 cm group < 6 cm), 106.322.8 months (group 6 cm), p=0.026 ]. in the group with large - size tumor (4 cm), the rfs was shorter than small - size group (mean value, 175.29.8 months vs. 113.311.1 months, p=0.007). the patient with brain stem or cranial nerve sign showed a shorter rfs time, compared to the patient with other minor symptoms (value, 157.912.8 months vs. 113.811.4 months, p=0.038). in regard to the peritumoral edema, higher edema group (moderate or severe) was significantly associated with shorter rfs (mean value, 168.111.4 months vs. 73.08.6 months, p<0.001). not surprisingly, non - complete resection group (simpson 's grade iii - v) demonstrated a shorter rfs compared to complete resection group (mean value, 154.012.1 months vs. 114.021.9 months, p=0.002). the absence of postoperative complication (mean value, 157.611.3 months vs. 70.412.4 months, p=0.001) and postoperative kps over 90 (mean value, 163.212.0 months vs. 71.59.7 months, p<0.001) were significantly related with recurrence in a longer follow - up period. on multivariate analysis, pathologic grade, postoperative kps, peritumoral edema and resection degree were independent predictable factors for tumor recurrence. benign pathology (hr 0.109, 95% ci 0.026 - 0.453, p=0.002), postoperative kps over 90 (hr 0.078, 95% ci 0.025 - 0.243, p<0.001), low peritumoral edema (hr 0.134, 95% ci 0.046 - 0.392, p<0.001), and complete resection (hr 0.226, 95% ci 0.077 - 0.669, p=0.007) were significantly associated with longer rfs time. the incidence of death rate was 5.0% over follow - up period (8/158). the surgery - related mortality which the death occurred within one month after resection was 3.16% in this study (5/158). the 5-, 10-, and 15-year survival rates were 96.2%, 94.9%, and 94.9%, respectively (fig. the results of analyses of the variables that could be correlated with survival time are shown in fig. 4 and table 4. on univariate analysis, pathology, location, size, postoperative complication and kps, and simpson 's grade showed statistical significance. the more aggressive pathology group (who grade ii and iii) showed a shorter survival time than the benign group (mean value, 40.116.2 months vs. 227.46.2 months, p<0.001). the tumor location was important factor for survival time [mean value, 167.16.7 months (group i) vs. 236.65.5 months (group ii), 98.08.6 months (group iii), p=0.009 ]. the group located at skull base showed shorter survival time than the non - skull base group (mean value, 98.08.6 months vs. 232.36.3 months, p=0.002). the size of tumor is also important variable in survival time [mean value, 200.52.6 months (group < 4 cm) vs. 192.120.8 months (4 cm group < 6 cm), 152.711.2 months (group 6 cm), p=0.023 ]. in larger - sized group (4 cm), the survival time was shorter compared that of small - size group (mean value, 200.52.6 months vs. 200.815.9 months, p=0.008). interestingly, non - complete resection group (simpson 's grade iii - v) demonstrated a shorter survival time than the complete resection group (mean value, 232.85.7 months vs. 139.117.2 months, p<0.001). with regard of permanent complication after surgical resection, the presence of complication was significantly related with shorter survival time (mean value, 233.14.5 months vs. 85.912.9 months, p=0.001). the patient with low level of postoperative kps (< 90) also showed shorter survival time, compared to the patient with higher score (mean value, 239.42.8 months vs. 89.79.2 months, p<0.001). our study showed that the patient lesser than 90 in preoperative kps survived shorter than the patients of high level kps, with a marginal statistical significance (mean value, 234.54.4 months vs. 132.910.2 months, p=0.051). on multivariate analysis, pathology, resection degree and postoperative kps were independent predictable factors for survival rate. benign pathology (hr 0.041, 95% ci 0.005 - 0.343, p=0.003), complete resection (hr 0.150, 95% ci 0.029 - 0.769, p=0.023) and postoperative kps over 90 (hr 0.041, 95% ci 0.005 - 0.351, p=0.004) were significantly associated with a longer survival rate. most common permanent complication is cranial nerve injury (42.9%) followed by operation - related hemorrhage or infarction, motor weakness, hydrocephalus, and infection. the results of analyses of the variables that could be correlated with permanent complications are shown in table 5. on univariate analysis, location, preoperative symptom, and preoperative kps showed statistical significance. the tumor location was important factor for development of permanent complication (8.1% in group i vs. 2.3% in group ii, 28.0% in group iii, p=0.002). the group located at skull base showed higher possibility of permanent complication than the non - skull base group (28.0% vs. 6.0%, p=0.001). the patient with brain stem or cranial nerve sign showed higher possibility for postoperative complication, compared to the patient with other minor symptoms (19.0% vs. 7.4%, p=0.043). the patient with low level of postoperative kps (< 90) was also significantly associated with permanent complication (37.1% vs. 3.5%, p<0.001). on multivariate analysis, location and postoperative kps were independent predictable factors for development of permanent complication. non - skull base location (hr 0.206, 95% ci 0.058 - 0.727, p=0.014) and postoperative kps over 90 (hr 0.048, 95% ci 0.012 - 0.197, p<0.001) were significantly related with a higher complication rate. a rising number of patients with intracranial meningiomas have been operated on in recent years and a few surgical series were reported in the literature22). and, surgical results with infratentorial meningiomas have much improved during the last two decades, but radical removal continues producing a high morbidity rate and still occasional mortality19). in intracranial meningioma resection, surgical outcome and its predictive factors were not fully identified. some authors mentioned the presence of peritumoral edema as a predictor of poor outcome in elderly patients3,8,13), whereas others did not find any relationship between edema and unfavorable outcome10,23). tumors size has been considered as a predictive factor for postoperative outcome by some authors3,8). in other reports, however, tumor size did not have any significant influence on surgical morbidity10,15,24). some authors have pointed out that outcome is less favorable in elderly patients with meningiomas located at the base of the skull, especially the posterior fossa1,2,4,5,20). in consideration of previous studies, surgery for infratentorial meningioma is still associated with significant postoperative deficits, thus confirming that these lesions represent a great challenge even for contemporary neurosurgeons25). since the first classification of infratentorial meningiomas was proposed by cushing and eisenhardt in 1938, castellano and ruggiero subdivided these tumors into five groups ; cerebellar convexity, tentorium, posterior petrous, clivus and foramen magnum meningiomas9). based on this classification, total 158 patients with infratentorial meningiomas underwent surgery during 20 years in out hospital (20.2%, in total 782 patients with intracranial meningiomas). although a radical resection of these histologically benign tumors is the ideal objective of surgical therapy, this is not always possible25). during surgical planning, it should be kept in mind that meningiomas show a marked tendency to invade dura, nerves, and surrounding bone21). the dura surrounding the tumor should be resected and the bony invasion should be drilling until normal bone is seen27). and vascular encasement, although representing an additional surgical problem, by itself did not generally prevent the pursuance of gross total removal25). in fact, as noted by sekhar.28), blood vessels are surrounded by an arachnoid plane that can be carefully dissected by experienced surgeons. subtotal resection carries a lower risk of morbidity than radical excision, but residual tumor may lead some patients to the initial clinical scenario sooner or later, and reoperation is usually less successful and more risky than initial surgery, particularly when the patient is given radiotherapy after the initial operation18,28). at the present moment the majority of authors recommend subtotal resection for old patients or when there are factors defying complete removal7,12,17). the critical factors influencing the possibility of radical and safe resection of meningiomas are tumor - vascular relationships and the integrity of the arachnoid plane between the tumor and the brainstem19). in our series, the recurrence rate of infratentorial meningiomas is particularly difficult to estimate because of differences in the reported series regarding the rates of radical excision, the methods of assessment of complete removal, the follow - up periods, the average age of the patients and the percentage of patients receiving postoperative radiotherapy19). some studies have shown that meningiomas may recur after an apparently radical excision, but it is clear that completeness of resection is the main factor preventing regrowth in all locations1,4,17). others indicated that frozen section analysis for intraoperative pathological sample is appropriate since a diagnosis of meningioma will direct that bone adjacent to the tumor site of origin be removed to reduce recurrence rate30). in our series, non - benign pathology, non - complete resection, moderate or severe degree of peritumoral edema, and low postoperative kps (< 90) were independent factors for short rfs in multivariate analysis. it is not difficult to anticipate that meningioma with high pathological grade or non - complete resection frequently recurred after the surgery. it means, in our opinion, that the group of postoperative kps under 80 tend to have subtotal resection because of intraoperative events, such as accidental injuries or poor dissection plane of cranial nerves, surrounding en - passing vessels, or brain stem, even though initial attempt for gross total resection. therefore, these groups seem to have lower postoperative kps, have higher recurrence rate. the more have edema, the more have recurrence rate. this is probably because the patients have moderate to severe edema tend to have high - grade pathology, large size, or poor dissection plane, leading to eventual recurrence of tumor. considering that the goal of surgery of meningiomas is gross total resection with minimal morbidity and low mortality14), single - stage total removal should be attempted27). but the attempt for aggressive surgical resection continues producing a high morbidity rate and still occasional mortality19). the perioperative mortality rate in the surgical management of infratentorial meningiomas has varied from 0 - 15.7%25). in our series, the total death rate during the follow - up period was 5.0% (8/158) of case. the postoperative mortality rate, as defined patients ware expired within 30 days after initial operation, was 3.16% (5/158). non - benign pathology, non - complete resection, and low postoperative kps (< 90) were independent factors for short survival in multivariate analysis. in our study, as previously reported by several authors, the most common postoperative complication was found to be cranial nerve paresis or palsy2,11,20,26). in our study, the permanent neurological deficits occurred in 12% (19/158) of case and the most common type is cranial nerve injury (47%, 9/19). in multivariate analysis, skull base location and low postoperative kps were independent factors for the occurrence of permanent complication. because of more difficult anatomical factor in petroclival, foramen magnum or jugular foramen, surgical resection for these lesions have high risk of permanent complication, even though experienced neurosurgeon. although a radical resection of these histologically benign tumors is the ideal objective of surgical therapy, this is not always possible25). during surgical planning, it should be kept in mind that meningiomas show a marked tendency to invade dura, nerves, and surrounding bone21). the dura surrounding the tumor should be resected and the bony invasion should be drilling until normal bone is seen27). and vascular encasement, although representing an additional surgical problem, by itself did not generally prevent the pursuance of gross total removal25). in fact, as noted by sekhar.28), blood vessels are surrounded by an arachnoid plane that can be carefully dissected by experienced surgeons. subtotal resection carries a lower risk of morbidity than radical excision, but residual tumor may lead some patients to the initial clinical scenario sooner or later, and reoperation is usually less successful and more risky than initial surgery, particularly when the patient is given radiotherapy after the initial operation18,28). at the present moment the majority of authors recommend subtotal resection for old patients or when there are factors defying complete removal7,12,17). the critical factors influencing the possibility of radical and safe resection of meningiomas are tumor - vascular relationships and the integrity of the arachnoid plane between the tumor and the brainstem19). in our series, the recurrence rate of infratentorial meningiomas is particularly difficult to estimate because of differences in the reported series regarding the rates of radical excision, the methods of assessment of complete removal, the follow - up periods, the average age of the patients and the percentage of patients receiving postoperative radiotherapy19). some studies have shown that meningiomas may recur after an apparently radical excision, but it is clear that completeness of resection is the main factor preventing regrowth in all locations1,4,17). others indicated that frozen section analysis for intraoperative pathological sample is appropriate since a diagnosis of meningioma will direct that bone adjacent to the tumor site of origin be removed to reduce recurrence rate30). in our series, non - benign pathology, non - complete resection, moderate or severe degree of peritumoral edema, and low postoperative kps (< 90) were independent factors for short rfs in multivariate analysis. it is not difficult to anticipate that meningioma with high pathological grade or non - complete resection frequently recurred after the surgery. it means, in our opinion, that the group of postoperative kps under 80 tend to have subtotal resection because of intraoperative events, such as accidental injuries or poor dissection plane of cranial nerves, surrounding en - passing vessels, or brain stem, even though initial attempt for gross total resection. therefore, these groups seem to have lower postoperative kps, have higher recurrence rate. this is probably because the patients have moderate to severe edema tend to have high - grade pathology, large size, or poor dissection plane, leading to eventual recurrence of tumor. considering that the goal of surgery of meningiomas is gross total resection with minimal morbidity and low mortality14), single - stage total removal should be attempted27). but the attempt for aggressive surgical resection continues producing a high morbidity rate and still occasional mortality19). the perioperative mortality rate in the surgical management of infratentorial meningiomas has varied from 0 - 15.7%25). in our series, the total death rate during the follow - up period was 5.0% (8/158) of case. the postoperative mortality rate, as defined patients ware expired within 30 days after initial operation, was 3.16% (5/158). non - benign pathology, non - complete resection, and low postoperative kps (< 90) were independent factors for short survival in multivariate analysis. in our study, patients with high postoperative kps represented lower complication rate and good postoperative condition. for this reason, as previously reported by several authors, the most common postoperative complication was found to be cranial nerve paresis or palsy2,11,20,26). in our study, the permanent neurological deficits occurred in 12% (19/158) of case and the most common type is cranial nerve injury (47%, 9/19). in multivariate analysis, skull base location and low postoperative kps were independent factors for the occurrence of permanent complication. because of more difficult anatomical factor in petroclival, foramen magnum or jugular foramen, surgical resection for these lesions have high risk of permanent complication, even though experienced neurosurgeon. in this study, gross - total resection was achieved in 81.6% of the cases and related with non - skull base location. the recurrence rate was 13.3% and longer rfs was associated with benign pathology, postoperative kps over than 90, low peritumoral edema, and complete resection. the 5-, 10-, and 15-year overall survival rates were 96.2%, 94.9%, and 94.9%, respectively. benign pathology, postoperative kps over than 90 and complete resection were independent factor for a longer survival. the permanent complication was developed in 13% of the patients and related with skull base location and postoperative kps less than 90. our results show relatively low morbidity and mortality rates, and good surgical resection and low recurrence rate, compare to the results of past, due to significant advances contemporary neurosurgical techniques, neuromonitoring and neuroanestehsia. however, infratentorial meningioma still has been a challenge for neurosurgeon because of their close to critical vascular and nerves structure. to achieve the goal that a little more lower morbidity and mortality, the surgeon need to consider of these factors and try to appropriate surgical planning of each case. | objectivebased on surgical outcomes of patients with infratentorial meningiomas surgically treated at our institution, we analyzed the predictors for surgical resection, recurrence, complication, and survival.methodsof surgically treated 782 patients with intracranial meningioma, 158 (20.2%) consecutive cases of infratentorial location operated on between april 1993 and may 2013 at out institute were reviewed retrospectively. the patients had a median age of 57.1 years (range, 16 - -77 years), a female predominance of 79.7%, and a mean follow - up duration of 48.4 months (range, 0.8 - -242.2 months).resultsgross total resection (simpson 's grade i & ii) was achieved in 81.6% (129/158) of patients. non - skull base location was an independent factor for complete resection. the recurrence rate was 13.3% (21/158) and the 5-, 10-, and 15-year recurrence rates were 8.2%, 12.0%, and 13.3%, respectively. benign pathology, postoperative kps over than 90, low peritumoral edema, and complete resection were significantly associated with longer recurrence - free survival rate. the 5-, 10-, and 15-year survival rates were 96.2%, 94.9%, and 94.9%, respectively. benign pathology, postoperative kps over than 90 and complete resection were significantly associated with a longer survival rate. the permanent complication rate was 13% (21/158). skull base location and postoperative kps less than 90 were independent factors for the occurrence of permanent complication.conclusionour experience shows that infratentorial meningiomas represent a continuing challenge for contemporary neurosurgeons. various factors are related with resection degree, complications, recurrence and survival. |
main determinants in the clinical care of patients with urolithiasis are the location, size, and chemical composition of the calculi, the latter being particularly important in the presence of uric acid (ua) stones, since ua stones may be dissolved by urinary alkalinization. the imaging modality of choice for the detection of urinary stone disease is unenhanced computed tomography (ct), offering high specificity and sensitivity. however, despite promising in vitro results, the transferability of results to an in vivo setting was hampered by misregistration problems. symptomatic urinary stone disease affects approximately 900,000 persons in the united states each year, resulting in annual medical costs of $ 5.3 billion. the morbidity associated with urolitiasis includes colic pain and kidney obstruction, which can lead to renal failure and severe urinary tract infections such as pyonephrosis and septic shock. moreover, the institution of further prophylactic measures to prevent recurrences is of utmost importance. this necessitates a thorough metabolic workup and an accurate quantitative stone analysis. without an appropriate workup, stone analysis and proper follow up, the recurrence rates may be as high as 1023%/year and may reach to 50% within 5 years. among all types of urinary stones, the frequency of calcium stone is 7080%, struvite stone 510%, uric acid stone 510%, and cystine stone 1%. in general, stones composed of ua are broken up easily by shock waves, whereas stones of calcium oxalate monohydrate (com), brushite, or cystine are difficult to break. previous attempts [911 ] to predict stone composition using spiral ct were based on the analysis of ct attenuations.. demonstrated that high resolution spiral ct yields unique ct attenuations for common types of stones if proper window settings are used to localize homogeneous regions within the stones. currently, the following methods are available for stone analysis : (1) chemical analysis, (2) emission spectroscopy, (3) polarizing spectroscopy, (4) x ray diffraction, (5) x ray coherent scatter / crystallography, (6) thermogravimetry, (7) scanning electron microscopy, and (8) infrared spectroscopy [6, 2 ]. chemical analysis was traditionally used most widely due to its ease and low cost. however, this is time consuming, necessitates large stone samples and can not distinguish between the two commonly occurring calcium stones (monohydrate / dihydrate). with the exception of infrared spectroscopy contradictory findings were published in literature regarding the ability of helical ct to accurately assess the chemical composition of urinary stones. two in vivo studies [9, 10 ], both conducted at 120 kv with 35 mm collimation, concluded that ct density (attenuation / stone size) was the best predictor of stone composition and could differentiate ua from calcium oxalate stones. most in vitro studies [14, 15 ] placed human calculi in a water bath to evaluate ct attenuation values as parameters predicting stone composition ; they could distinguish ua stones from other stones. the aim of the present study was to investigate retrospectively the impact of urinary stone volume on computed tomography stone attenuations measured in hounsfield units in 253 patients with urolithiasis using postoperative in vitro infrared spectroscopy (100 ftir, perkinelmer). this retrospective study was approved by the institutional ethics board of our university. from 2008 to 2010, 253 consecutive patients (189 men, 64 women) with urolithiasis were included into our analysis. children and pregnant women were excluded from the study. in patients with acute flank pain, suspected of having urinary stones, we performed so called stone ct, which is an unenhanced low participants were scanned using a 64slice computed tomography unit (siemens ag, somatom, sensation 64, erlangen, germany). one unenhanced scan (acquisition : 64x0.6 mm, 120 kv, 150 mas) of the abdomen and the pelvis with a slice thickness of 3 mm was made. this protocol is routinely used at our institution to evaluate patients with acute flank pain suspected to have renal colic. a region of interest (roi) was drawn on each stone, and the attenuation was measured. the sizes and positions of the regions of interest were validated by an experienced abdominal radiologist (a.l) by using a conventional soft tissue window and narrow bone windows. when the calculi had irregular contours, special attention was given not to include any surrounding soft tissue, which has much lower ct attenuation values than calculi. the average, highest, and lowest ct attenuation values in hounsfield units were calculated. our abdominal radiologist, who was blinded to the chemical composition of the stones, retrospectively reviewed the imaged and analyzed data to determine the hu of the calculi. spss software package version 13 (spss, ibm, chicago, illinois) was used. hereby, the p values were based on levene 's test of equality of means. all statistical tests were two sided. according to the european association of urology the size of a concrement (stone burden) the most common way of expressing size in the literature is to use the largest diameter, i.e. the length of the stone as measured on a plain film. the stone surface area (sa) can be estimated for most stones from the length (l) and width (w) of the stone using the following formula sa = l. w.. 0.25 (=3.14159) with the more common use of ct examinations, it is possible to get an even better estimate of the stone volume (sv) by combining measures of length (l), width (w) and depth (d) using the formula : sv = l. w. d.. 0.167 (= 3.14159). the biochemical composition of the stone was established after spontaneous passage, operation or shock wave lithotripsy. stone analysis was based on infrared spectroscopy, which determines the molecular and crystalline composition of the stone. infrared spectroscopy is the spectroscopy that deals with the infrared region of the electromagnetic spectrum, that is light with a longer wavelength and lower frequency than visible light. it covers a range of techniques, mostly based on absorption spectroscopy. as with all spectroscopic techniques, it can be used to identify and study chemicals. a common laboratory instrument that uses we used perkinelmer spectrum 100 ftir spectrometer (perkinelmer, shelton, usa) to analyse urinary stones after passage or operation. this retrospective study was approved by the institutional ethics board of our university. from 2008 to 2010, 253 consecutive patients (189 men, 64 women) with urolithiasis were included into our analysis. children and pregnant women were excluded from the study. in patients with acute flank pain, suspected of having urinary stones, we performed so called stone ct, which is an unenhanced low participants were scanned using a 64slice computed tomography unit (siemens ag, somatom, sensation 64, erlangen, germany). one unenhanced scan (acquisition : 64x0.6 mm, 120 kv, 150 mas) of the abdomen and the pelvis with a slice thickness of 3 mm was made. this protocol is routinely used at our institution to evaluate patients with acute flank pain suspected to have renal colic. a region of interest (roi) was drawn on each stone, and the attenuation was measured. the sizes and positions of the regions of interest were validated by an experienced abdominal radiologist (a.l) by using a conventional soft tissue window and narrow bone windows. when the calculi had irregular contours, special attention was given not to include any surrounding soft tissue, which has much lower ct attenuation values than calculi. the average, highest, and lowest ct attenuation values in hounsfield units were calculated. our abdominal radiologist, who was blinded to the chemical composition of the stones, retrospectively reviewed the imaged and analyzed data to determine the hu of the calculi. spss software package version 13 (spss, ibm, chicago, illinois) was used. variables were compared by anova. hereby, the p values were based on levene 's test of equality of means. all statistical tests were two sided. according to the european association of urology the size of a concrement (stone burden) the most common way of expressing size in the literature is to use the largest diameter, i.e. the length of the stone as measured on a plain film. the stone surface area (sa) can be estimated for most stones from the length (l) and width (w) of the stone using the following formula sa = l. w.. 0.25 (=3.14159) with the more common use of ct examinations, it is possible to get an even better estimate of the stone volume (sv) by combining measures of length (l), width (w) and depth (d) using the formula : sv = l. w. d.. 0.167 (= 3.14159). the biochemical composition of the stone was established after spontaneous passage, operation or shock wave lithotripsy. stone analysis was based on infrared spectroscopy, which determines the molecular and crystalline composition of the stone. infrared spectroscopy is the spectroscopy that deals with the infrared region of the electromagnetic spectrum, that is light with a longer wavelength and lower frequency than visible light. it covers a range of techniques, mostly based on absorption spectroscopy. as with all spectroscopic techniques, it can be used to identify and study chemicals. a common laboratory instrument that uses this technique we used perkinelmer spectrum 100 ftir spectrometer (perkinelmer, shelton, usa) to analyse urinary stones after passage or operation. we included 253 patients from 2008 to 2010 into our analysis (189 men and 64 women) with the mean age being 51.2 years and the median age 49.6 years (see table 1). according to urinary stone incidence in relation to gender, we found a male predominance (2.9 higher risk). patients and study objects there were 134 pure calcium oxalate monohydrate (whewelit, com) calculi, 29 stones with 50% uric acid and 50% whewelit, 29 stones with 80% whewelit and 20% cystine and 12 pure calcium oxalate dihydrate (whedelit, cod). there were 49 mixed calculi according to their chemical compositions : struvite, carbonate, and xanthine, as well as protein. the calculi were divided into 4 groups according to their clinical relevance : calcium oxalate monohydrate and dihydrate, uric acid stones, struvite, cystine. calcium oxalate monohydrate and dihydrate, the overall differences between the densities of the stones was not statistically significant (table 2) (p = 0.05) and there was a cross over in densities among all studied stones ; a sharp demarcation among different types could not be found. hu of calcium and uric acid as well as for cystine and struvite stones to study the effect of stone volume on density, we divided the stones into 2 groups according to their median stone volume values : group 1. 126 stones with stone size > 37.78 mm and volume > 4.3 mm group 2. the smallest stone had a size of 0.5 mm and a volume of 0.9 mm. the largest stone had a size of 3620 mm and a volume of 19 mm. when the calculi were compared with each other, levene 's test was used. groups of uric acid and non ua according to stones volume we compared groups 1 and 2 with each other according to levene 's test. there was significant relationship between stone volume and ct attenuation only in stones with a volume 4.3 mm or more (p 4.3 mm on x axes and their hounsfield units on y axes. group 2 stones with volume 3 mm with a lower dose single energy ct. according to ascenti, characterization of stones <3 mm is often clinically less important because a high percentage of stone spontaneous passage. we failed to distinguish between ua and non ua stones because our analyzed stones were not pure stones, which are not uncommon in a clinical setting and most stones are not pure stones. contradictory results were documented by matlaga. who found no significant differences in stones size among ua and calcium oxalate groups. medical prophylaxis to prevent recurrence is still at the core of the treatment of urinary stone disease. stone analysis complements, but does not replace, urine and serum studies to assess for metabolic stone disease. however, stone analysis can present useful information, as it represents a biochemical patient history, documenting the urinary environment over time through type and conformation of crystal deposition. quality control studies conducted in europe from 1980 to 2001 demonstrated that the overall accuracy of stone analysis was improving with time and experience. ir and x ray diffraction methods were most reliable for artificial stone substances, but wet chemical methods produced an error rate from 6.594%. with existing technology, several steps could increase the reliability of stone analysis. due to incomplete sampling leading to errors, all stones obtained by the patient or the surgeon should be submitted for analysis ; this gives the analysis laboratory the best chance to detect all components that are present. according to singh, infrared spectroscopy is a sensitive, reliable, accurate, safe, and quick method of accurate stone analysis suitable for use in a clinical laboratory. measurements of specific absorption peaks of the spectrum of each sample, mean error rate being 22.5%. the quantitative stone analysis by infrared spectroscopy allows accurate separate zone wise analysis of the stone nucleus, external and internal layers not possible by other methods. limitations of our study : results should be interpreted with caution because the retrospective design of our study. we failed to show the effect of stone volume on its attenuations, and could not distinguish ua from non ua stones because most of our analyzed stones were not pure stones. | introductionto investigate retrospectively the impact of urinary stone volume on computed tomography stone attenuations measured in hounsfield units in 253 patients with urolithiasis.material and methodsct scans were performed in 253 patients with suspected urinary stone disease from 2008 to 2010 using ct scanner siemens, somatom, sensation 64. one experienced radiologist (a.l) who was blinded to the chemical composition of the stones retrospectively reviewed images and analyzed data to determine the composition of the stones. the results were compared with the biochemical analysis results obtained by infrared spectroscopy (100 ftir, perkinelmer).results253 consecutive patients from 2008 to 2010 were included into analysis : 189 males, and 64 females. mean age was 51.2. according to stone volume, stones were divided into 2 groups : 126 stones with volume of 4.3 mm or more, 127 stones with volume less than 4.3 mm. there was a significant relationship between stone volume and its ct attenuation only in stones with a volume 4.3 mm or more (p < 0.05).conclusionswe failed to show a significant relationship between stone volume and its attenuations in hounsfield units. we could not distinguish uric acid stones from non uric acid stones. |
deranged hematological parameters, including thrombocytopenia, are features of human immunodeficiency virus (hiv) infection.1 thrombocytopenia, defined by platelet cell count of less than 15010/l,2,3 occurs in about 4%24% of hiv - infected cases.4,5 mechanisms for thrombocytopenia, such as platelet involvement in immune responses, cytopathic effect of antiretroviral therapy (art) regimens, and antigenic mimicry, are key in a setting of hiv.6,7 thrombocytopenia has been linked to adverse sequelae and is regarded as an independent predictor of morbidity and mortality among the hiv - infected group, owing to increased risk of bleeding, which may occur in the mucous membranes, skin, soft tissue, and intracranial sites.8,9 the associated bleeding may cause death if it involves critical sites.10 to avert the risk of bleeding, platelet transfusion may be indicated ; however, in uganda, component transfusion is not readily available,11 which limits success of clinical interventions in an immunologically compromised population. although cytopenias have been widely reported in hiv infection, there is little data regarding prevalence, correlates, and etiologic association of hiv - related thrombocytopenia in uganda. this study sought to determine hiv - related thrombocytopenia in a high - hiv / acquired immune deficiency syndrome (aids) study population (southwest uganda). the findings from this study will form a basis for management of complications that arise from thrombocytopenia among hiv clients in this setting. these were hiv - positive adult males and females who had been enrolled for care at the immune suppressed syndrome (iss) clinic in mbarara, uganda. we sought informed consent of patients with thrombocytopenia as found in their previous full blood count (fbc) results, and investigated the presence of antiplatelet antibodies. for each sample, 3 ml of blood was collected into a plain vacutainer, allowed to clot, and centrifuged, to obtain hemolysis - free serum that was kept frozen at 80c at the epicentre mbarara research centre, mbarara, uganda. we performed indirect monoclonal antibody - specific immobilization of platelet antigens (maipa) for 40 serum samples from thrombocytopenic hiv clients, to screen and identify antiplatelet antibodies. antibody screening was done using platelets from a pool of six group o donors selected for their platelet genotype (advanced practical diagnostics bvba, turnhout, belgium) ; these were incubated with serum, and mouse monoclonal antibodies specific for platelet glycoproteins ia / iia, ib / ix, iib / iiia, and anti -2-microglobulin. lysates were cleared by centrifugation and transferred to microplate wells precoated with goat anti - mouse immunoglobulin g (igg). the bound complex was detected using goat peroxidase - coupled anti - human igg and revealed by peroxidase substrate o - phenylenediamine. the reaction was stopped using sulfuric acid, and absorbance was read at 492 nm. all positive antibody screens were identified, using a standard six - cell genotyped panel, by similar or additional methods. a participant was regarded to have antiplatelet antibodies if one or more platelet antiglycoproteins were identified. retrospective data from the iss database and data from laboratory analyses were entered into a statistical software package of excel 5.0 (microsoft corp, redmond, wa, usa) and transferred to stata 12 (statacorp lp, college station, tx, usa) to carry out data management and analysis, respectively. we sought clients informed consent, and the study was approved by the institutional review committee of mbarara university of science and technology, and the uganda national council for science and technology. these were hiv - positive adult males and females who had been enrolled for care at the immune suppressed syndrome (iss) clinic in mbarara, uganda. we sought informed consent of patients with thrombocytopenia as found in their previous full blood count (fbc) results, and investigated the presence of antiplatelet antibodies. blood was drawn, with minimal stasis, from the antecubital vein. for each sample, 3 ml of blood was collected into a plain vacutainer, allowed to clot, and centrifuged, to obtain hemolysis - free serum that was kept frozen at 80c at the epicentre mbarara research centre, mbarara, uganda. we performed indirect monoclonal antibody - specific immobilization of platelet antigens (maipa) for 40 serum samples from thrombocytopenic hiv clients, to screen and identify antiplatelet antibodies. antibody screening was done using platelets from a pool of six group o donors selected for their platelet genotype (advanced practical diagnostics bvba, turnhout, belgium) ; these were incubated with serum, and mouse monoclonal antibodies specific for platelet glycoproteins ia / iia, ib / ix, iib / iiia, and anti -2-microglobulin. lysates were cleared by centrifugation and transferred to microplate wells precoated with goat anti - mouse immunoglobulin g (igg). the bound complex was detected using goat peroxidase - coupled anti - human igg and revealed by peroxidase substrate o - phenylenediamine. the reaction was stopped using sulfuric acid, and absorbance was read at 492 nm. all positive antibody screens were identified, using a standard six - cell genotyped panel, by similar or additional methods. a participant was regarded to have antiplatelet antibodies if one or more platelet antiglycoproteins were identified. retrospective data from the iss database and data from laboratory analyses were entered into a statistical software package of excel 5.0 (microsoft corp, redmond, wa, usa) and transferred to stata 12 (statacorp lp, college station, tx, usa) to carry out data management and analysis, respectively. we sought clients informed consent, and the study was approved by the institutional review committee of mbarara university of science and technology, and the uganda national council for science and technology. records for 15,030 hiv clients at enrollment into care at the mbarara regional referral hospital iss clinic were used. participants had a median age of 35.0 (range 1878 ; iqr 2842) years. out of 15,030 participants, 2,617 had thrombocytopenia, giving an overall prevalence of 17.4% (95% confidence interval [ci ] : 16.8%18.0%). the prevalence of thrombocytopenia among hiv clients who were art - nave (n=2,675) was 17.8% (95% ci : 17.1%18.4%), while that for clients who were on art for up to 6 months (n=6) was 13.0% (95% ci : 0.3%21.9%). we used univariate and multivariate analyses to determine association of thrombocytopenia with clinical stage, cd4 count, art, anemia, and leucopenia, which we found significant (p<0.05) (tables 1 and 2). of the 40 thrombocytopenic samples tested for anti - platelet antibodies, two (5.0%) had glycoprotein iib / iiia and iia / ia. in this study, the overall prevalence of hiv - related thrombocytopenia was 17.4% (95% ci : 16.8%18.0%) ; this is comparable to the 20% reported in iran12 and 12.7% in ethiopia.13 this is attributed to hiv - induced hemopoiesis dysfunction and platelet immunologic involvement, which aggravates their destruction.8 the prevalence of thrombocytopenia among hiv - infected, art - nave clients was 17.8% (95% ci : 17.1%18.4%) ; this is similar to the 16.1% reported in nigeria14 and 13.5% in rwanda15 but higher than the 8.3% reported in kampala, uganda16 and the 5.9% reported in ethiopia.17 the high prevalence of thrombocytopenia in this group is probably attributed to already established mechanisms of hiv - related thrombocytopenia.18,19 this study showed a positive effect of regimens containing the nucleoside analog reverse - transcriptase inhibitor, zidovudine (azt), which is similar to other reports;17 thus exclusive use of prophylactic co - trimoxazole among the art - nave hiv clients may be an independent predictor of accelerated thrombocytopenia. since we detected antiglycoproteins in one of the art - nave participants, it is likely that such antibodies cause thrombocytopenia.20 the prevalence of thrombocytopenia among hiv - infected clients who were on art for up to 6 months was 13.0% (95% ci : 0.3%21.9%) ; this is much higher than the 4.1% reported in ethiopia.21 this is probably due to diminished use of azt regimens, in our study population, due to associated anemia.22 we analyzed for art combinations as a predictor of thrombocytopenia, and from this, we elucidate reduced thrombocytopenia in participants on azt regimens. the observed prevalence may as well be attributed to factors linked to induced cytopenias that vary according to the participant s profile, the etiologic basis, and art option.23 the occurrence of antiglycoprotein in one of the participants in this group shows antiplatelet antibody involvement in the etiology.8 using univariate and multivariate analyses to relate the independent predictors of thrombocytopenia, diminished cd4 counts, other cytopenias, and enrollment on art showed significant association with the occurrence of thrombocytopenia in our study participants (p<0.05), which is consistent with findings reported by other studies in the region.16,21 this may be due to defective hemopoiesis and the effect of art, as previously reported.21,24 the study found a significant association of thrombocytopenia with other cytopenias in all hiv stages (refer to table 3). this may be due to defective bone marrow, accelerated platelet destruction, and to azt- or stavudine - containing regimens.25 there was an increasing risk of thrombocytopenia with hiv infection progression, and this is similar to findings from other studies.17,26 this may be because thrombocytopenia is greater in advanced hiv infection.15 the antiplatelet antibody prevalence of 5.0% is comparable with the 9.4% found among hiv chilean population27 but lower than the 17.0% in the italian population.28 the higher anti - human platelet antigens (hpas) against iib / iiia than ia / iia is due to their close association with the chronic thrombocytopenic states associated with hiv.29 anti - hpa-5, which targets ib / ix, was missing in our study participants, as these are linked to thrombocytopenia due to neonatal alloimmunization.30 antiplatelet antibodies were seen in mild and moderate thrombocytopenic clients, which implies that severe thrombocytopenia is not associated with the production of antiplatelet antibodies, as already reported.28 the key limitation of study was the small sample size of participants enrolled for antiplatelet antibody assay owing to time and financial constraints. the authors recommend a larger study to be carried out and that as a basis of critical care and management, immune thrombocytopenia should be considered in our study population. the key limitation of study was the small sample size of participants enrolled for antiplatelet antibody assay owing to time and financial constraints. the authors recommend a larger study to be carried out and that as a basis of critical care and management, immune thrombocytopenia should be considered in our study population. this study has observed a high prevalence of thrombocytopenia, especially in the art - nave population, and the occurrence of antiglycoproteins in our sample population. these results indicate that there is a need to monitor platelet counts and to initiate platelet component transfusion among hiv thrombocytopenic clients, in our current blood banking practice. | aims / objectiveswe aimed to determine the prevalence and correlates of thrombocytopenia among people living with human immunodeficiency virus (hiv)/acquired immune deficiency syndrome (aids) and to assess occurrence of antiplatelet antibodies, among thrombocytopenic hiv clients at mbarara regional referral hospital, southwestern uganda.materials and methodsthis was a retrospective review of hematologic results at enrollment to hiv care from 2005 to 2013. the prevalence and correlates of thrombocytopenia were estimated based on the immune suppressed syndrome (iss) clinic electronic database. a cross - sectional study determined the occurrence of antiplatelet antibodies, using the monoclonal antibody - specific immobilization of platelet antigens (maipa) technique.resultswe reviewed 15,030 client records. the median age was 35.0 (range 1878 ; interquartile range [iqr ] 2842) years, and there were 63.2% (n=9,500) females. the overall prevalence of thrombocytopenia was 17.4% (95% confidence interval [ci ] : 16.8%18.0%). the prevalence of thrombocytopenia was 17.8% (95% ci : 17.1%18.4%) among antiretroviral therapy (art)-nave clients (n=2,675) and was 13.0% (95% ci : 0.3%21.9%) for clients who were on art (n=6). the study found a significant association between thrombocytopenia and other cytopenias, cd4 counts, art, and deteriorating hiv stage (p<0.05). two of the 40 participants (5.0%) had antiplatelet antibodies.conclusionthis study has showed a high prevalence of hiv - related thrombocytopenia. antiplatelet antibodies were found in 5.0% of hiv - infected thrombocytopenic participants. our study shows a significant association of thrombocytopenia burden in a high - hiv study population (southwest uganda) ; therefore, there is need to monitor platelet counts and initiate platelet transfusion in our blood banking practices, to avert possible risks of bleeding. |
the increase of ageing, obesity, and diabetes in conjunction with inappropriate healthcare programs have emphasized the problem of having to treat almost 1.5 billion people affected by skin and mucosal infections due to bacteria, viruses, protozoa, and dysmetabolism. pathologies range from the diabetic foot (ulcer with necrosis), bed sores, ulcers after a trauma or burns, chronic viral infections due to either herpes virus i and ii, or human papylloma viruses, vaginal infections now frequent also in young girls due to candida, trichomonas, and chlamidia, rectal mucosa infections such as anal ragadis, abscesses with fistula to end with mouth aphthous ulcers. these infections are rarely deadly but are considerably distressing because many patients often suffer of diabetes or vascular diseases with tissue hypoxia, other patients are immunosuppressed drugaddicts, or with concomitant hiv infection. official medicine provides a variety of drugs that are expensive and often poorly efficacious because infections in hypoxic tissue contain methicillin - resistant staphylococcus aureus and pseudomonas aeruginosa. patients are suffering not only because they become uncompliant to frequent medications but they are discouraged by observing a lack of healing. wound healing is a multiphase process involving blood clotting, inflammation, tissue proliferation, and remodelling, but both innate and adoptive immune systems are too often hindered by the chronic infection naturally difficult to overcome. this is also the reason explaining the failure of growth factors in heavily contaminated ulcers [3, 4 ]. the judicious use of ozone (o3) appears providential because first of all eliminates the pathogens and then, by releasing oxygen (o2), activates the proliferation of fibroblasts, hence the building of intercellular matrix with consequent proliferation of keratinoblasts and successive healing. in section 2, we propose to briefly review the physical chemistry of oil ozonation and all the basic analyses necessary for demonstrating the quality of the obtained products. in section 3, it appears useful to inform readers that both skin and mucosae are sensitive to excessive amounts of gaseous o3 as there are clear demonstrations of a variety of alterations linked to a prolonged exposure. in section 4, we will then clarify the various procedures devised to enhance the disinfectant and healing - promoting properties of o3. finally, after an extensive analysis of a cornucopia of proposals, we will try to suggest guidelines for the future medical application of topical ozone and its derivatives (section 5). unsaturated lipid substrates react with insufflated gaseous o2/o3 mixture leading to therapeutically active ozonated derivatives (figure 1). briefly, the postulated mechanism known as criegee reaction provides that ozone combines with an unsaturated bond to form an initial, unstable primary ozonide which readily decomposes to form a zwitterions and a carbonyl fragment. in anhydrous environment these substrates combine to give the typical cyclic trioxolane derivative. however, the word ozonated is itself without scientific meaning if it is not associated with how much in fact, from a therapeutic point of view, the ozonide compositions have the capacity to deliver active o2 and/or other useful species deep within the lesion without causing primary skin irritation. the few studies concerned with the therapeutic effects of ozonated oils on acute cutaneous wound healing in animal models do not investigate the dose / behaviour response, expressed as the amount of peroxides existing in the ozonated derivative used. recently, a quantitative evaluation of the therapeutic effect of topically applied ozonated sesame oil on acute cutaneous wound healing in mice as animal model has been developed. the results indicate that both low (3000), as expressed in terms of peroxide value (see the corresponding section in this paper), delay cutaneous wound healing. such an evidence is reinforced by a number of results between groups where the middle concentration (about 1500) has the most beneficial effect in accelerating the wound closure ratio. from an industrial applicative viewpoint, the overall quality of ozonated derivatives depends upon several parameters, such as : (i) the type and the quality of ozone generators ; (ii) the ozonation conditions, in terms of reactors and time, material type and amount, presence of water and/or catalyzers ; (iii) the efficacy of the ozonizer, in terms of o3 concentration output, gas flow, gas carrier. as for the latter, the use of medical grade o2 instead of air is an important point to be considered ; in fact, air feedstock (containing about 78% of nitrogen) used for the ozonation of unsaturated substrates could lead to the production of potentially toxic nitrated by - products, and to a significant decrease of the ozonation efficiency. another important feature is that ozonated oil has to be unequivocally characterized in terms of the species contents as well as the reaction kinetics. for these purposes, the knowledge of the physicochemical properties of ozonated vegetable oils during production has a great importance for their characterization and identification. for determining the quality of ozonated products, spectroscopic techniques, as fourier - transformed infrared (ft - ir) and h and c - nmr, together with analytical methods as peroxide, acidity, and iodine values as well as viscometric determination ft - ir spectroscopy is used to highlight differences in the functional groups during the oil ozonation, in particular the decrease of the bands corresponding to both c = c and = c h stretching (e.g., sesame oil at 1654 cm and 3009 cm, respe), and the increase of the band corresponding to ozonide co stretching (e.g., sesame oil at 1105 cm). an adequate aliquot (usually about 2 l) of sample is deposited between two disks of kbr, avoiding air bubble formation, then the percentage transmittance or other suitable parameters are measured in the range 4000800 cm. spectra are obtained setting the appropriate scan summations and minimal resolution (generally, 16 at 4 cm, resp.). an adequate aliquot (usually about 2 l) of sample is dissolved in a suitable solvent (preferably chloroform) and then the solution is settled in the sample holder avoiding air bubble formation, then the transmittance (expressed as a percentage) or other suitable parameters are measured in the range 4000800 cm. spectra are obtained setting the appropriate scan summations and minimal resolution (generally, 16 at 4 cm, resp.). h and c nmr spectroscopies are performed to obtain more information about the variation of the functional groups involved in the reaction of ozonation. both the disappearance of the signals relative to protons and carbons on the double bond (e.g., in sesame oil 5.29 ppm, and various signals in the range 127.8130.0 ppm, resp.) and the parallel appearance of a signal on the proton and carbon of 1,2,4-trioxolane (e.g., in sesame oil in the 5.115.08 ppm range, and 103.4104.3 ppm range, resp.) are evidenced. quantitative analysis can be performed by spectra normalized with respect to the integral areas of the och2 protons (glycerol) that remain constant during the whole process. spectra will be obtained using suitable instruments by solubilizing the ozonated sample in a proper solvent (preferably cdcl3). particularly, an adequate aliquot (usually about 100 l) of sample is solubilised with 750 l of cdcl3 in a 5 mm nmr tube, then the analysis will be performed. to obtain quantitative data, it is sufficient to perform a h - nmr, while c - nmr essentially provides qualitative informations. the iodine value (iv) represents the quantity of iodine (in grams) that will react with the double bonds in 100 grams of sample. the iv is calculated by means of the following equation : (1)iv=1.269(n1n2)m, where n1 is the volume in ml of thiosulphate solution (0.1 m) used for carry out a blank test, n2 is the volume in ml of thiosulphate solution (0.1 m) used for the titration and m the quantity, in grams, of substance. it is, therefore, a measure of the total number of double bonds present in the sample and for such a reason it is a chemical analysis useful for evaluating the decrease of double bonds during the oil ozonation process, giving information about the 1,2,4-trioxolane formation. the acid value (av) is an index that expresses, in mg, the quantity of potassium hydroxide required to neutralise the free acids presents in 1 g of the substance. the av is calculated by means of the following equation : (2)av=5.610nm, where n is the volume in ml of titrant and m the quantity, in grams, of substance. it is representative of the acidity level of the product and it represents an index of the degradation by - products that could be formed during the ozonation process. peroxide value, (pv), is usually used as an indicator of the advancement and/or the control of the ozonation process because of its simplicity, rapidity, and low cost. moreover, the pv may be adequate for the stability evaluation of vegetable oil ozonides and it appears to be very important for commercial distribution as well as for the determination of the better storage modalities. however, it had been necessary to standardize the methodology for a validated pv. in the present paper, a detailed analysis of pv assessments of ozonated lipid derivatives based on both literature data and our laboratory experiments such a report allows an in - depth acquaintance of the ozonation process of vegetable oils as well as of the related products obtained, allowing to define the quality parameters useful for industrial purposes. specifically, the peroxide value (pv) represents the quantity of peroxide expressing in milliequivalents of active o2 contained in 1000 g of the sample. for the pv evaluation, first official monograph described in pharmacopoeia (e.g., european pharmacopoeia, british pharmacopoeia, united states pharmacopoeia), which provides the solubilization of sample in 30 ml of chloroform / glacial acetic acid (2 : 3), the addition of saturated potassium iodide solution (0.5 ml) and the titration after 1 minute with a solution of sodium thiosulphate. second method described by martinez tellez., which always provides the solubilization of sample in 30 ml of chloroform / glacial acetic acid (2 : 3) and the addition of saturated potassium iodide solution (0.5 ml), but the titration is done after 24 hours. briefly, 2 g of so were weighed in a 250 ml conical flask and 30 ml of chloroform / glacial acetic acid (2 : 3) were added. the flask was stirred at reflux temperature (60c) for various times (5180 minutes). after this time, the solution was cooled and 25 ml of water were added. solutions of sodium thiosulphate at the appropriate concentration (0.00010.1 m) were used for the titration. in all determinations the pv was calculated by means of the following equation : (3)pv=1000(v1v0)cm, where v1 is the volume in ml of thiosulphate solution used for the titration, v0 is the volume in ml of thiosulphate solution used for carry out a blank, c the thiosulphate concentration and m the sample quantity (grams). the ozonation efficiency (expressed as a percentage) represents ratio of the amount of peroxidation due to ozonation process, as estimated by pv value, to the o3 total amount applied to the system. it was calculated by means of the following equation : (4)oe=(pvspv0)100024oad100, where pvs is the ozonated sample pv, pv0 is the pv of untreated sample, and oad stands for the o3 applied dose (mg / g). viscosity evaluation is a useful technique because it is fast and it could be online, giving an estimation of the double bonds present in the sample. in fact, the greater the ozonation time the higher the product viscosity because of the disappearance of the double bonds. moreover, its typical trend can be a useful tool in providing a rapid quality control assessment during the entire ozonation process, as well as to decide on the process time for obtaining the desired ozonation level of the sample. the skin, along with the respiratory tract, is directly exposed to environmental pollutants including o3, an important constituent of photochemical smog. although numerous studies have documented effects of o3 on the respiratory tract in animals and humans [1215 ], only recently some studies characterizing its effect on cutaneous tissue have been published [1620 ]. the skin consists of two main layers, the inner dermis, mainly composed of fibroblasts and connective tissue matrix, and the outer epidermis, which contains keratinocytes that, by progressively differentiating to form enucleate corneocytes, become imbedded in a lipid matrix and together comprise the outermost part of the epidermis, the stratum corneum (sc) [21, 22 ]. previous studies have shown that exposure to o3 results in the depletion of both water soluble and lipophilic antioxidants such as uric acid, ascorbic acid, and tocopherol, and this was accompanied by increase in parameters of both lipid peroxidation and protein modification, primarily in the outermost skin layers [16, 17, 23 ]. in further studies, we were also able to show that the exposure of hairless mice to o3 will not only deplete the antioxidant levels and increase oxidative markers but these molecules are able to induce active cell responses. these effects can be briefly summarized as follows. (1) induction of redox sensitive transcription factorsozone, like many others environmental challenges, is able to activate transcriptional factors redox sensitive such as nuclear factor k b (nfkb). this transcriptional factor acts as an activator for a multitude of proinflammatory genes (il-8, tnf, tgf) and adhesion molecules (icam and vcam). it has been assessed that o3 is able to activate nfkb using both in vitro and in vivo systems. thiele., using an immortalized human keratinocytes (hacat cells), were able to show that o3 induced the activation of nfkb by electrophoretic mobility shift assay (emsa). this effect was likely to be mediated by ros, particularly h2o2, because it was inhibited by the incubation of the cells with lipid soluble antioxidants (tocopherol). ozone, like many others environmental challenges, is able to activate transcriptional factors redox sensitive such as nuclear factor k b (nfkb). this transcriptional factor acts as an activator for a multitude of proinflammatory genes (il-8, tnf, tgf) and adhesion molecules (icam and vcam). it has been assessed that o3 is able to activate nfkb using both in vitro and in vivo systems. thiele., using an immortalized human keratinocytes (hacat cells), were able to show that o3 induced the activation of nfkb by electrophoretic mobility shift assay (emsa). this effect was likely to be mediated by ros, particularly h2o2, because it was inhibited by the incubation of the cells with lipid soluble antioxidants (tocopherol). (2) induction of heat shock protein (hsp) and inflammatory markersas a consequence of the induction of transcription factors, o3 exposure (6 days to 0.8 g / ml for 6 hours / day) induced the expression of proinflammatory markers in skin homogenates such as cyclooxygenase-2 (cox-2). this induction was accompanied by an increase level of heat shock protein (hsp) 32, also known as heme oxygenase-1 (ho-1). in this paper, we were the first to demonstrate the upregulation of hsps 27, 32 and 70 in homogenized murine skin upon o3 exposure. hsp27 showed the earliest (2 hours) and highest (20-fold) response to o3 compared with the delayed induction (12 hours) of hsp70 and ho-1. hsp27 is expressed predominantly in the suprabasal epidermis in human skin, whereas hsp70 predominates in the dermis compared with the epidermis. these differences in location between hsp27 and hsp70 might explain the different time course of induction of these stress proteins upon o3 exposure. it is therefore possible that the generated bioactive compounds may be responsible for the induction of hsps as was also shown after uv irradiation. as a consequence of the induction of transcription factors, o3 exposure (6 days to 0.8 g / ml for 6 hours / day) induced the expression of proinflammatory markers in skin homogenates such as cyclooxygenase-2 (cox-2). this induction was accompanied by an increase level of heat shock protein (hsp) 32, also known as heme oxygenase-1 (ho-1). in this paper, we were the first to demonstrate the upregulation of hsps 27, 32 and 70 in homogenized murine skin upon o3 exposure. hsp27 showed the earliest (2 hours) and highest (20-fold) response to o3 compared with the delayed induction (12 hours) of hsp70 and ho-1. hsp27 is expressed predominantly in the suprabasal epidermis in human skin, whereas hsp70 predominates in the dermis compared with the epidermis. these differences in location between hsp27 and hsp70 might explain the different time course of induction of these stress proteins upon o3 exposure. it is therefore possible that the generated bioactive compounds may be responsible for the induction of hsps as was also shown after uv irradiation. (3) induction of matrix metalloproteinases (mmps)among the multiple systems altered in the skin by environmental pollutants, mmps are among the major targets. indeed, o3 exposure is able to affect their synthesis and/or activity with logical consequences on tissue remodeling and wound healing [23, 24 ]. within the mmp family, mmp-2 and mmp-9 are the only members able to degrade type - iv collagen of the basal membranes. mmp-2 is involved in pathological processes such as photoageing and precancerous / cancerous skin lesions after uv exposure ; moreover, mmp-2 is capable of cleaving other substrates, in addition to type - iv collagen, including other mmps and therefore can (indirectly) control extracellular matrix degradation and remodelling.mmp-9, like mmp-2, plays a role in human skin ageing tumor development, as well as in other cutaneous lesions such as psoriasis and dermatitis [28, 29 ]. in a recent study, we were able to demonstrate that o3, was able to affect mmp activity. it has been also demonstrated that o3 is able to induce no production via the activation of inos in cutaneous tissues. when produced in excess, no, may combine with superoxide to form peroxinitrite (derived from other sources) that can activated mmps especially mmp-9. thus, the increase of oxidative stress after o3 exposure, plus the interaction between o2 and nitrogen active molecules might be the main mechanism that leads to the enhanced mmps activities in skin tissues. this can be also a result from an imbalance between mmps and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (timps).in fact, the activities of mmps are regulated by timps, which can be produced by a multitude of cell types present in the cutaneous tissue. while mmp activity is altered by the o3, neither timp-1 nor timp-2 level expression is affected. the lack of changes in timp-1 and 2 levels, combined with the increased activity of mmps suggest that o3 can cause a net increase in matrix degradation. on the other hand, in a comparative study where normal skin has been exposed for two hours to environmentally realistic levels of ozone, only a moderate state of oxidative stress at level of the stratum corneum has been induced, without producing a visible clinical response. among the multiple systems altered in the skin by environmental pollutants, mmps are among the major targets. indeed, o3 exposure is able to affect their synthesis and/or activity with logical consequences on tissue remodeling and wound healing [23, 24 ]. within the mmp family, mmp-2 and mmp-9 are the only members able to degrade type - iv collagen of the basal membranes. mmp-2 is involved in pathological processes such as photoageing and precancerous / cancerous skin lesions after uv exposure ; moreover, mmp-2 is capable of cleaving other substrates, in addition to type - iv collagen, including other mmps and therefore can (indirectly) control extracellular matrix degradation and remodelling. mmp-9, like mmp-2, plays a role in human skin ageing tumor development, as well as in other cutaneous lesions such as psoriasis and dermatitis [28, 29 ]. in a recent study, we were able to demonstrate that o3, was able to affect mmp activity. it has been also demonstrated that o3 is able to induce no production via the activation of inos in cutaneous tissues. when produced in excess, no, may combine with superoxide to form peroxinitrite (derived from other sources) that can activated mmps especially mmp-9. thus, the increase of oxidative stress after o3 exposure, plus the interaction between o2 and nitrogen active molecules might be the main mechanism that leads to the enhanced mmps activities in skin tissues. this can be also a result from an imbalance between mmps and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (timps). in fact, the activities of mmps are regulated by timps, which can be produced by a multitude of cell types present in the cutaneous tissue. while mmp activity is altered by the o3, neither timp-1 nor timp-2 level expression is affected. the lack of changes in timp-1 and 2 levels, combined with the increased activity of mmps suggest that o3 can cause a net increase in matrix degradation. on the other hand, in a comparative study where normal skin has been exposed for two hours to environmentally realistic levels of ozone, only a moderate state of oxidative stress at level of the stratum corneum has been induced, without producing a visible clinical response. wound healing is a critical process in the skin and it has known to be affected by oxidative stress and also to decline with increasing age. although the exact sequence of wound healing is complex, cutaneous wound healing begins with wounding induced signaling factor - based transformation of stationary keratinocytes into cells capable of both replication and migration. upon transformation, these cells express a host of molecules that promote the invasion of the injured epithelial matrix and reepithelialisation of the wound surface. as mentioned above, o3 exposure is also associated with activation of transcription factor nfkb, which is important to regulate inflammatory responses and eventually entire wound healing. o3 exposure increased levels of transforming growth factor (tgf-) that is a critical factor in tissue remodeling [35, 36 ]. we can summarize that while o3 as an oxidant, might stimulate wound healing, it would be detrimental in an aging environment due to the increased concentration - dependent oxidative stress. therefore, these aspects have biological as well as practical implications and needed further investigations. in a recent study, we demonstrated the detrimental effects of o3 on cutaneous wound healing in the aged animals. in fact, when hairless young (8-week - old) and aged mice (18-months - old) with after full thickness excisional wounds were exposed to 0.5 g / ml o3 for 6 hours per day we also showed induction of protein and lipid oxidation assessed as changes in protein oxidation (carbonyls) and lipid peroxidation (4-hydroxynonenal, hne adducts) in the old mice compared to the young mice during the later stage of cutaneous wound healing. in fact, it significantly delayed wound closure in old mice, while in young mice, it led to accelerated trend during the first few days of the exposure. this might be attributed to the antibacterial properties of o3, as it has been shown that application of hydropressive ozonation provides fast cleansing of wound surface from pyonecrotic masses, promotes elimination of infection and thus substantially reduces the period of treatment of the patients. recently, clinical treatments using hyperbaric oxygen therapy demonstrated that increased o2 tension at the wound site increases the formation of granulation tissue, enhances accelerated wound closure and ameliorates impaired dermal wound healing ; therefore, accelerated trend of wound closure shown in young population may be due to decreased bacterial infection and/or increased o2 tension by o3 exposure in wound area. one of the possible driving processes of the effect of o3 on wound healing can be also in this case the modulation of the transcription factor nfkb. effect relationship between level of oxidative stress and nfkb exhibits a biphasic profile : while moderate levels of oxidative stress activate nfkb through an ikb kinase independent mechanism, extremely high levels of oxidative stress have been shown to inhibit nfkb activation by blocking ikb phosphorylation. one potential explanation for the differential effect in the older animals is that the level of oxidative stress generated by o3 exposure combined with aging causes levels of oxidative stress that inhibits ikb phosphorylation, thereby resulting in a decline in nfkb activation. this finding is consistent with what mentioned previously that o3 exposure induced skin antioxidants depletion. this interpretation is also bolstered by data on tgf- a crucial modulator of tissue remodeling and is linked to both nfkb status as well as to levels of oxidative stress during entire wound healing process. the reduced tgf levels in both air and o3 exposed old mice as well as the lower induction of tgf by o3 exposure in the old animals suggests that the noted delays in wound closure might be related to defects in oxidative stress - dependent nfkb status as well as levels of oxidative stress and tgf signaling in aged mice during later stage of wound healing. to the best of our knowledge, the first application of gaseous o3 was performed during world war i for treating german soldiers affected by gaseous gangrene due to clostridium anaerobic infections very sensitive to o3 [41, 42 ]. in 1936, dr p. aubourg, by using a metal cannula, was the first to propose the insufflations of gaseous o2/o3 in the rectum to treat chronic colitis, anal ragadis and fistulae. this approach is very empirical and unprecise and today it is mostly used by cuban physicians. in 1937, a swiss dentist, e. a. fisch (18991966) had the idea to use it in his practice and, by a twist of fate, he treated dr. payr was so enthusiastic of the o3 effect to use it in his surgical practice with great advantage. later on, werkmeister mastered the use of gaseous o3 in several skin ulcers due to atherosclerosis, diabetes and radiotherapy by either enclosing a leg in a polythene - bag (the so - called bagging system) or using an ozone - resistant plastic cup applied in other areas. in the former application the o3 concentrations varied between a high 80 g / ml in very purulent ulcers and progressively lower concentrations down to 10 g / ml as the ulcers improved because excessive o3 would be deleterious for healing. as the cup system had an inlet and an outlet, werkmeister could realize a continuous gas flow with a modest depression that enhanced the vasodilation of the ulcer 's area. with both systems he treated many extensive and otherwise incurable lesions within 50200 days. it is noteworthy that gaseous o3 works well only in a water vapour - saturated bag because it must dissolve into superficial water or in the exudate to react proficiently. the normal skin does not undergo any damage during the treatment. today these procedures are still in use but they are somewhat cumbersome and great care must be exercised to prevent air contamination. probably, when the stable triozonide comes into contact with the warm exudate of the wound, it slowly decomposes into different peroxides, which readily dissolves in water, probably generating hydrogen peroxide that can explain the prolonged disinfectant and stimulatory activity. if it is correct, this reasoning implies that we should have titrated preparations with high, medium, or low ozonide concentrations to be used during the inflammatory septic phase i, regenerating phase ii or remodelling phase iii, respectively. these phases have been related to the rapidly changing cell types and to the release of cytokines and growth factors that modulate the complex healing process. an alternative method for treating diabetic foot ulcers is the use of hyperbaric oxygen therapy (hot) but in such a case one disadvantage is the use of only hyperbaric o2 and another is the need to close the patient in the chamber for two hours. therapeutic results are far more modest than topical o3 application, particularly when it is contained in a close cabinet with thermostatically - controlled temperature. however this procedure requires considerable idle times and, if an aspirating pump is unavailable, it may contaminate the operating room. for these reason today for cleaning and disinfecting cutaneous and mucosal infections and lesions due to many causes (like, e.g., trauma, ischemia, burns), it appears preferable to use at once freshly ozonated water and then ozonated oil, particularly during the night or at rest conditions. the process of water ozonation needs of double distilled water and o3 concentrations ranging from 20 up to 100 g / ml of gas to have a final yield of 5 up to 25 g / ml, respectively. o3 is directly bubbled into the water and the gas in excess is passed through a dehydrating device and finally through a destructor. depending upon the water volume and the gas flow, a period of ozonation between 520 minutes is sufficient to saturate the water with gaseous o3. in fact, if the water is ultrapure, o3 physically dissolves in the absence of chemical reactions and if kept in a glass bottle closed with a teflon cap, the concentration halves only after 300 hours at 0c. however, at 20c the half - life is about 10 hours. it must be noted that monodistilled water allows a much faster o3 decomposition and it is not practical. it is adviced to maintain the bottle at 4c and to quickly close the bottle at any time, or better to have a valve system to prevent gas losses. it would be useful to device a procedure for maintaining the o3 concentration for longer times and we are investigating a possible procedure. on the other hand, ozonation of either olive or sunflower oils requires a much longer time and the procedure needs to be well - standardized in terms of gas - flow, o3 concentration, oil volume, and temperature. as recently reviewed, at least twenty different vegetable oils have been patented but so far it remains impossible to define their relative cost / benefit. at this stage, after evaluating several physicochemical criteria, stability, efficacy, and cost, it seems that sesame oil has several advantages in comparison to other oils. chronic wounds range from diabetic foot to putrid and deep ulcers due to limb atherosclerosis, or trauma and burns. moreover, both immunosuppressive chemotherapy and/or malnutrition cause abscesses, anal fissures and fistulae, bed sores, furunculosis, and osteomyelitis which are difficult to treat and often fail after prolonged treatments. about 7 million patients in the united states are affected with a cost over us$ 25 billion annually. various types of disinfectants, antibiotics, antifungal, antiprotozoal, and growth factors are scarcely effective because the deranged metabolism and local hypoxia are not modified. several other approaches such as vacuum therapy [47, 48 ], maggot therapy and devices for providing topical oxygen therapy in a clinical setting have been proposed and variably used. this last approach has a rationale in the sense that enhanced oxygenation is useful for activating the metabolism and cell proliferation of ischemic tissues [5052 ]. however, it has also considerable limitations because it is a cumbersome therapy, with minimal disinfectant activity and modifications of the fundamental pathogenetic mechanisms. although rarely deadly (as the toxic shock syndrome due to a forgotten absorbent tampon), a majority of women physically and psychologically frequently suffer from a number of infections due to several pathogens such as neisseria gonorrhoeae, trichomonas vaginalis, candida albicans, chlamidia trachomatis, herpes virus type - ii (hv - ii), human papilloma viruses (hpv), human immunodeficiency virus (hiv), often due to unprotected sexual intercourses, stress, change in sexual partners and also physiological hormonal changes during menopausa. about 20 million americans are affected by the distressing hv - ii and as many 40 million have the genital hpv with warts and the impending risk of cervix cancer. moreover it is unfortunate that orthodox medications are expensive and not so useful because of drug - resistant pathogens and side effects limiting the compliance. so far official medicine has not yet entertained the topical use of o3 and derivatives in therapy because they are not profitable and no extensive clinical trials have been published in peer - reviewed journals : the therapy has remained in practitioners ' hands and the results remain anecdotal. moreover, the parenteral use of ozone, also known as ozone therapy, is very useful as adjuvant : it is reasonably ease to perform in terms of classical ozonated major and minor autohemotherapy. the latter modality has been successfully used for eliminating recurrences of hv - i and ii infections. however, topical therapy is essential and it is carried out by using vaginal irrigation of fresh ozonated water and application of vaginal ozonated oil pessaries for the night. during prolonged treatment the ozonated compounds creams containing ozonated oils can be used 3 - 4 times daily for external genital areas and also for several anorectal affections. as for the oral infections (aphthae, hv - i, opportunistic superinfections, or acne) the earliest as possible application of ozonated ointments, by minimizing pathogen diffusion and enhancing microcirculation, reduces the swelling, last but not least, clinical trials in tinea pedis as well as onychomycosis [54, 55 ] have been recently published and have shown the usefulness of ozonated sunflower oil. at the present, especially in young people, venereal infections are increasingly frequent and therefore a suitable, effective medication with ozonated compounds will be a huge economical and social value. also, elderly people are burdened with a variety of wounds and ulcers, some of which never heal, making life miserable. it is hoped that the present paper will inform official medicine for this advance and will incite to programme suitable clinical trials to show the full efficacy of ozone therapy by evidence - based medicine. | although orthodox medicine has provided a variety of topical anti - infective agents, some of them have become scarcely effective owing to antibiotic- and chemotherapeutic - resistant pathogens. for more than a century, ozone has been known to be an excellent disinfectant that nevertheless had to be used with caution for its oxidizing properties. only during the last decade it has been learned how to tame its great reactivity by precisely dosing its concentration and permanently incorporating the gas into triglycerides where gaseous ozone chemically reacts with unsaturated substrates leading to therapeutically active ozonated derivatives. today the stability and efficacy of the ozonated oils have been already demonstrated, but owing to a plethora of commercial products, the present paper aims to analyze these derivatives suggesting the strategy to obtain products with the best characteristics. |
more than 50 years ago, owen. (1954) made the remarkable discovery that most twin cattle were born with a stable mixture of each other s red cells. (1988) later found that human leukocyte antigen (hla) broadly sensitized patients commonly failed to produce antibodies against mismatched non - inherited maternal antigens (nima), but were fully capable of producing anti - non - inherited paternal antigens (nipa). the definition of nima or nipa is based on an offspring - based hla haplotype that is not inherited from the mother or father, respectively. (1953) then showed that injection of allogeneic splenocytes from murine fetuses enabled the acceptance of later skin grafts from the same donor. this phenomenon is now referred to as fetomaternal tolerance, and suggests that perinatal exposure to nima may affect the developing immune system of neonates. these phenomena have been clinically utilized in organ transplantation and allogeneic hematopoietic stem cell transplantation (hsct ; burlingham., 1998 ; van rood., 2002). (1998) showed the superior graft survival rate in nima- compared to nipa - mismatched renal transplant recipients from sibling donors. furthermore, van rood. (2002) demonstrated that hsct from nima - mismatched sibling donors showed a lower incidence of severe acute graft - versus - host disease (gvhd) compared with that from the other family donors. (2004) have demonstrated the feasibility of hla - haploidentical hsct from nima - mismatched relatives without t cell depletion. these clinical studies have been performed based on the presence of fetomaternal microchimerism as a result of fetomaternal immunological tolerance. nevertheless, some cases developed severe acute gvhd despite the existence of microchimeric cells (kanda., 2009). we recently reported that nima effects directed toward the major histocompatibility complex (mhc) antigen were divided into immunogenic and tolerogenic reactivities (araki., 2010). the reactivities were predictable by an mlr - elispot (mixed lymphocyte reaction ; enzyme linked immunospot) assay. we found that non - t cell - depleted (tcd) nima - mismatched haploidentical hsct could be performed safely by evaluating the reaction of ifn--producing cells of the donors against nima before transplantation. alloantigens can be divided into mhc antigen and minor histocompatibility antigen (miha), the former being responsible for eliciting the strongest immune responses to allogeneic tissues. the mhc is referred to as the hla complex in humans and as the h-2 complex in mice (table 1). the genes related to the hla system encode a complex array of histocompatibility molecules that play a central role in immune responsiveness and in determining the outcome of hsct in humans (beatty. the primary goal of histocompatibility testing for patients who are undergoing hsct is the identification of a suitable hla - matched donor to reduce the risk of post - transplant complications, which may result from hla incompatibility. the mhc identity of the donor and host is not the sole factor determining the immunological reactivity in hsct. when transplantation is performed in an unrelated setting, even if the mhc antigens of donor are identical to those of recipient, considerable transplant reactions may occur because of differences at various minor histocompatibility loci. mihas, peptides derived from polymorphic proteins, are capable of eliciting cellular alloimmune responses in vitro and in vivo. their immunogenicity arises as a result of their presentation in the context of mhc class i or ii, where they are recognized by alloreactive mhc - restricted t cells. the most important immune reactions elicited by in vivo alloreactivity to miha are graft rejection and acute gvhd. to date, human mihas have not been fully characterized, although some murine mihas have been compared with the human counterparts (table 1). immunological targeting of hy proteins results in a relatively high incidence of acute gvhd when male recipients receive hsct from female donors (stern., while approximately one - third of the known mihas are encoded on the y chromosome, many mihas are located on autosomal chromosomes. a genetic linkage analysis has been used to define the genomic regions encoding the mihas (akatsuka., 2003 ; de rijke., 2005). with the recent introduction of more advanced analytical techniques, more human miha epitopes have been identified (van bergen., 2010 ; sellami., 2011) graft survival in hsct is optimal when the donor and recipient are hla - identical. however, in some situations, if this is not possible, haploidentical siblings, parents, and offspring are considered as potential donors. contact between the mother and child during pregnancy can lead to tolerization, and subsequently have an additional benefit on the transplant outcome. a new nomenclature was proposed to assign the haplotypes of a family in which one of the siblings is a potential transplant donor (van rood and claas, 2000) as depicted in figure 1. the parents or siblings that share one haplotype with the recipient and differ for the other haplotype are potential donors. the patient inherits the inherited maternal hla antigens (i m a) haplotype from the mother, and the inherited paternal hla antigens (ipa) from the father. when the patient is transplanted from one of the parents or from a haploidentical sibling, the nima or nipa is the mismatched haplotype. this nomenclature scheme can also be used in cases where the mother or father is the potential donor (figure 1). because of the existence of fetomaternal tolerance, nipa is more immunogenic than nima. therefore, the order of donor eligibility is ima / ipa, nima / ipa followed by ima / nipa. siblings of the patient share one haplotype with the donor, and the other haplotype is the non - inherited haplotype. the hla haplotypes in parentheses are shown as representative examples. when the patient is transplanted with donor from one of the parents or from a haploidentical sibling, the non - inherited maternal hla antigens (nima) or non - inherited paternal hla antigens (nipa) are the mismatched haplotype. several studies have been performed to investigate the influence of non - inherited and inherited parental antigens on transplantation, and tolerizing effects (a nima effect) have been described. in hsct, van rood. (2002) and ichinohe. (2004) showed that the patients who received non - tcd bmt from a nima - mismatched donor had a significantly lower incidence of acute gvhd than a nipa - mismatched donor. however, even in non - tcd bmt from a nima - mismatched donor, 10% of patients still experienced severe acute gvhd (ichinohe., 2004). furthermore, graft rejection and hyperacute gvhd after hsct from nima - mismatched siblings have been observed in spite of the fact that maternal microchimerism was detected (okumura., 2007). (2009) described that a substantial proportion of long - term survivors after nima - mismatched hsct could discontinue the administration of immunosuppressive agents, despite the frequent occurrence of moderate to severe chronic gvhd. there have been several investigations of nima in murine models (burlingham., 1998 ; andrassy., the immunological effects of developmental exposure to nima are heterogeneous (mold., 2008 ; molitor - dart., 2008 ; verhasselt., 2008). not only in the mhc - identical, but also under mhc - haploidentical conditions, miha alloreactivities may be induced upon transplantation (verdijk., 2004). therefore, focusing on the nima effect separated by the mhc (h-2) and miha responses is clinically relevant. the mouse miha loci confer a wide range of immunogenicity, ranging from weakly to strongly immunogenic (table 1 ; mendoza., 1997 ; choi., 2001 recent studies have provided evidence that gvhd could be caused by a limited number of miha, including h4, h7, h13, h28, h60, and h - y (eden., 1999 ; choi., 2002 ; yang., 2003). the immunodominance of these miha was manifested on genetically varied backgrounds among b10, balb / c, and dba/2 strains (sanderson and frost, 1974 ; mendoza., 1997 ; malarkannan., 2000 so far, there has been no report distinguishing h-2 from miha with regard to nima. we have classified mouse models of nima based on the major and minor histocompatibility antigens to nima (hirayama and azuma, 2011). in our study, b10 congenic mice were used as nima models and the miha matched entirely in this system (figure 2b). on the other hand, in the conventional model (figure 2a), the nima includes not only non - inherited h-2, but also non - inherited miha. therefore, our nima model, but not the conventional nima model, did not affect the immunogenicity of miha. we examined the tolerogenic potential of nima - exposure for h-2 of class i and ii disparities without any influences of the miha (araki., 2010). 2009), we found no evidence of the nima effect (araki., 2010). the reason for the difference remains to be determined, but it could be due to the abrogation of the miha effect in our system (figure 2b). (a) left, c57bl/6 (b6) males (h-2) were mated with (b6 dba/2) f1 females (h-2), thus exposing the h-2 offspring in utero and via breastfeeding to nima antigens. right, (b6 dba/2) f1 males were mated with b6 females, creating h-2 backcross offspring that had not been exposed to d, as reported by andrassy. (b) left, b10.br males (h-2) were mated with (b10.d2 b10) f1 females (h-2), thus exposing the h-2 type offspring to nima, and h-2 type offspring to nima. right, (b10.d2 b10) f1 males (h-2) were mated with b10.br females (h-2), creating the controls ; both h-2 and h-2 type offspring that had not been exposed to b and d, respectively. these mice have a b10 background, in other words, their miha are matched, and the h-2 antigens are mismatched for both class i and ii. the bidirectional exchange of cells, both mature and progenitor types, at the maternal fetal interface is a common feature of mammalian reproduction (lo., 1996). the presence of semiallogeneic cells in a host can have significant immunological effects on transplantation tolerance and rejection. maternal cells and dna were detected for a long time after parturition in the peripheral blood and lymphoid organs of offspring (maloney. breastfeeding during the neonatal period also might contribute to building - up maternal microchimerism in the offspring, because breast milk is rich in soluble maternal mhc antigens (verhasselt. maternal microchimerism may cause tolerance, resulting in the acceptance of an allograft bearing antigens shared by the microchimeric cells. however, microchimerism may also cause sensitization, thus resulting in rejection. distinguishing which of these effects is likely to occur prior to the transplant may revolutionize the field of living - related renal transplantation, wherein microchimerism can exert a powerful influence on graft outcome (van rood., long - term maternal microchimerism is easily detected from the peripheral blood or various tissues, including the skin, liver, and thyroid gland, by using highly sensitive polymerase chain reaction - based techniques (ichinohe., 2002). although many investigators have suggested the association of long - term maternal and fetal microchimerism with the development of autoimmune diseases, including systemic sclerosis, primary biliary cirrhosis, juvenile inflammatory myopathies, and biliary atresia (nelson, 2003 ; suskind., 2004), it is difficult to establish a precise etiological link, because maternal microchimerism is frequently found in healthy females with a history of uncomplicated pregnancy, and in more than two - thirds of immunocompetent individuals without any manifestations of autoimmune attacks (kodera., 2005). (1999) suggested that passenger leukocytes were involved in the induction phase of allograft acceptance and microchimerism in the thymus, but not in the blood, and were also associated with allograft survival. these results suggest that microchimerism may play a role in allograft survival, but that the persistence of peripheral microchimerism is not required. (2005) described partial deletion of b cells having high affinity for the nima. (1998) showed central and peripheral deletion of donor - specific t cells after establishing mixed chimerism in the recipient with a high dose of bone marrow cells. low doses of bone marrow cells induced a form of tolerance that was regulatory t cells (treg)-dependent, consistent with the establishment of microchimerism rather than mixed chimerism. bonilla. (2006) described that deletion of effector t cells due to a form of suppressive microchimerism in antigen - presenting cells was a consequence of the establishment of a dominant treg - population in the host. tsang. (2008) described the possibility of inducing nima - specific treg in the direct and indirect presentation of maternal microchimerism. on the other hand, since oral tolerance is known to generate tgf--producing treg (gonnella., 2003), oral exposure to maternal mhc antigens present in breast milk (molitor., 2004) may generate nima - specific treg, which may prevent the deletion of maternal cells by nima - specific effector t cells, resulting in a high level of microchimerism. (2009) reported that exposure to nima both in utero and by breastfeeding appears to generate higher levels of maternal microchimerism than in utero exposure alone, and that the degree of microchimerism, correlates with a prolonged survival in maternal heart grafts. predicting acute gvhd in vitro before transplantation has been tried in an hla - mismatched setting, but satisfactory methods had not been established. the frequencies of cytotoxic t lymphocyte precursor (ctlp) and helper t lymphocyte precursor (htlp) cells, as well as mlr, were reported for the methods that had been evaluated to detect an individual s reactivity to nima in vitro (falkenburg., 1996 ; moretta., 1999 ; (1999) described that the frequency of nima - specific ctlp in cord blood samples could be measured in order to better define the phenomenon of nima tolerance. nima - reactive cord blood cells were detectable, but the authors of that study could not show a difference in the ctlp frequency toward nima and nipa. neither the ctlp nor htlp frequencies against nipa were not significantly different from those against nima. indeed, kircher. (2004) showed that the ctlp and htlp frequencies were not predictive for the risk of acute gvhd in patients who received allogeneic hsct. collectively, established test systems are not available for predicting an alloreaction and the outcome after hsct. ctlp reflects the alloreactivity of class i mismatch, and mlr and htlp reflect alloreactivity of class ii mismatch. they generated carboxyfluorescein diacetate succinimidyl ester - labeled cd4cd25 foxp3 cells in mlr, which they called treg mlr, with varying hla disparities and cell components. thus, all of the above - mentioned methods can detect mhc class i or class ii separately, but it is difficult to detect them simultaneously (table 2). we recently, reported a novel method, mlr - elispot assay, that overcomes these disadvantages, as shown in figure 3a. (a) the elispot assay combined with mlr (mlr - elispot) is a sensitive functional assay to detect alloreactivity for both major and minor histocompatibility antigens in mice. (b) the mice were classified into two groups based on their reactivity to nima ; the high responders (hr mean 1 sd in nima - non - exposed) or the low responders (lr < mean 1 sd) group by using mlr (araki., 2010). the ifn--producing ability before the induction of gvhd was presented by the mlr - elispot assay. peripheral blood mononuclear cells from nima - exposed lr mice (n = 8), nima - exposed hr mice (n = 7), and non - exposed mice (n = 6) were stimulated with b10 mouse peripheral blood mononuclear cells. the data are expressed as the means sd of individual animals. p < 0.05. the alloreactivities of nima - exposed mice and nima - non - exposed mice were evaluated by mlr, and we found a wide range of reactivity (araki., 2010). this indicates that the fetomaternal interaction acts on both tolerance (low - responder, lr) and sensitization (high - responder, hr ; molitor - dart., 2008 ; van halteren., 2009). (2000) detected a reactivity to nima by mlr, and ctlp and htlp, respectively. interestingly, when we scrutinized the figures in their articles, the individual reactivities of the nima - exposed group showed a wider range than the control group, and those reactivities seem to be divided into low and high reactions, although the authors of those studies did not discuss these observations. this was a further indication that reactivity to nima could be detected in vitro, and that the fetomaternal interaction promoted either tolerance or sensitization. recently, we demonstrated that the number of cells producing ifn- was significantly lower in the nima - exposed lr group than the hr group by using an mlr - elispot assay in a murine model (figure 3b). thus, the capacity for an individual to produce ifn- against allogeneic antigens or nima could differentiate lr from hr. this assay is easily applicable in humans, and is a versatile method to detect reactivities to mhc class i, as well as class ii. in other words, this assay might be useful to predict the total immunological reaction of donor t cells to the recipient in hla - mismatched hsct. non - inherited maternal antigens - mismatched haploidentical hsct has been progressing, and now can lead to sustained engraftment, lower early treatment - related mortality, and acceptable rates of acute gvhd. however, it is difficult to predict severe acute gvhd prior to transplantation. our recent report addressed this issue (araki., 2010). the nima effect directed toward mhc antigens there was an unevenness in the acquisition and maintenance of microchimerism in offspring, which was not due solely to differences in mhc gene inheritance. although t cell replete haploidentical transplantation is performed only when there is positive microchimerism, the individual reactivity of the donor is not evaluated at present. therefore, our study is clinically relevant, and t cell replete nima - mismatched haploidentical transplantation can be performed more safely in the future by evaluating the responses of the ifn--producing cells of the donor against nima. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | major histocompatibility complex antigens that provoke severe transplant reactions are referred to as the human leukocyte antigen (hla) in human and as the h-2 in mice. even if the donor and recipient are hla - identical siblings, graft - versus - host reactions have been linked to differences in the minor histocompatibility antigen. as the chance of finding an hla - identical sibling donor is only 25%, attention has been focused on using alternative donors. an hla - mismatched donor with non - inherited maternal antigens (nima) is less immunogenic than that with non - inherited paternal antigens, because the contact between the immune systems of the mother and child during pregnancy affects the immune response of the child against nima. however, the immunologic effects of developmental exposure to nima are heterogeneous, and can be either tolerogenic or immunogenic. we recently have devised a novel method for predicting the tolerogenic effect of nima. in this review, we overview the evidence for the existence of the nima tolerogenic effect, the possible cellular and molecular basis of the phenomenon, and its utilization in hematopoietic stem cell transplantation. we suggest a future direction for the safe clinical use of this phenomenon, fetomaternal tolerance, in the transplantation field. |
these disorders are caused by mutations in genes coding for enzymes involved in degradation of glycosaminoglycans (gags). storage of gags in cells of patients results in progressive damage to the affected tissues, including the heart, respiratory system, bones, joints and, in most mps types and subtypes, the central nervous system (cns). mps are usually fatal diseases, with average expected life span of 1 or 2 decades, although patients with milder forms can survive into adulthood. there are 11 known types and subtypes of mps, and each type is variable in severity of particular symptoms. prediction of severity and clinical progression of mps is difficult, even when biochemical and genetic parameters are determined, but recent studies indicate that considering 2 or more parameters may give significantly better results than attempting to make conclusions based on a single biomarker. mps type iii (mps iii, called also sanfilippo disease) is a group of 4 conditions (mps iiia, b, c and d), revealing similar clinical symptoms and characterized by lysosomal storage of heparan sulfate (hs), one of gags. particular subtypes are classified according to gene coding for an enzyme involved in degradation of hs (heparan n - sulfatase in mps iiia, -n - acetylglucosaminidase in mps iiib, acetyl - coa : -glycosaminide acetyltransferase in mps iiic and n - acetylglucosamine 6-sulfatase in mps iiid) which is defective. although other mps types are characterized by severe somatic symptoms and either some (sometimes also severe) or no neurological and behavioral problems, mps iii is specifically associated with severe learning difficulties and behavioral disturbances, accompanied with relatively mild somatic involvement. in most affected patients the progressive nature of the disease leads to death in the second (or rarely third) decade of life. as this disorder primarily affects the brain and nervous system, attempts to cure patients have been especially difficult, and aside from experimental treatments (see below), the best care that can be offered is palliative or symptomatic. enzyme replacement therapy (ert), based on an intravenous infusion of an active, recombinant form of a deficient enzyme, is currently the only officially approved treatment of mps, and is used in clinical practice for mps types i, ii and vi. unfortunately, neurological symptoms due to hs accumulation in cns can not be managed by ert, due to an inefficient delivery of proteins, including recombinant enzymes, through the blood - brain barrier. substrate reduction therapy (srt), called also substrate deprivation therapy (sdt), is an alternative approach for treatment of patients with lysosomal storage diseases. this therapy is based on the action of small molecules that may, directly or indirectly, impair synthesis of compounds that can not be efficiently degraded in cells of patients. since such small molecules may likely cross the blood - brain - barrier, it is hoped that they could be used to manage neurological symptoms of these diseases. previous studies with rhodamine b ([9-(2-carboxyphenyl)-6-diethylamino-3-xanthenylidene]-diethylammonium chloride) and genistein (5, 7-dihydroxy-3-(4-hydroxyphenyl) -4h-1-benzopyran-4 - 1) revealed that each of these compounds can reduce the efficiency of gag synthesis in cells of various mps types. importantly, these studies demonstrated not only a lack of further gags accumulation, but also a reduction in lysosomal storage of these compounds. in fact, recently reported experiments indicated that rna interference - mediated silencing of genes coding for various gag synthetases causes a decrease in the amount of the storage material in cell lines of patients with sanfilippo disease. tests on animal models of sanfilippo disease demonstrated efficacy of rhodamine b and genistein in vivo, including behavior of affected and treated mice. although it is unlikely that rhodamine b can be used in clinical practice due to its potential toxicity to humans, genistein is a non - toxic and safe compound. moreover, unlike rhodamine b, the mechanism of genistein - mediated inhibition of gag synthesis has been demonstrated. this isoflavone inhibits phsophorylation of the epidermal growth factor receptor (egfr), which in turn causes an impairment of expression of genes coding for enzymes necessary for gag synthesis. gene expression - targeted isoflavone therapy (abbreviated as get it), and an open - label, pilot study in 10 pediatric mps iii patients, treated with a genistein - rich isoflavone extract for 12 months, was performed. after 1 year of treatment, statistically significant improvement in all measured parameters (urinary gag excretion, hair morphology and cognitive functions) was demonstrated. since longer - term effects of get it for mps iii are not known, we performed a follow - up study with genistein - treated mps iii patients. considering learning difficulties and behavioral disturbances to be the most severe clinical problems of sanfilippo disease, we focused on monitoring cognitive functions and general status of patients, including their behavior. the patients were diagnosed for sanfilippo disease (either mps iiia, mckusick s omim no. 252900, or mps iiib, mckusick s omim 252920) by estimation of urinary gag levels (highly elevated gag levels, observed in hemi - quantitative electrophoretic analysis, gave a preliminary indication for mps) and measurement of activities of particular lysosomal hydrolases in leukocytes. deficiency in activity of heparan n - sulfatase (control value : 4.11.4 nmoles / mg of protein/18h) or -n - aceltyl glucosaminidase (control value : 9034 nmoles / mg of protein/42 h) was considered as a diagnosis for mps iiia or mps iiib, respectively. mutations in genes coding for the above - mentioned enzymes were determined for 5 patients, and 1 mutation was determined for 1 patient. eight patients (6 with mps iiia and 2 with mps iiib) were enrolled into the study. all patients were of caucasian origin ; 2 males and 6 females. among the 8 patients, 5 (patients : iiia-2, iiia-4, iiia-5, iiib-2 and iiib-5) were previously enrolled into the pilot study. the mps iii patients characterized in table 1 were treated for 36 months with a genistein - rich soy isoflavone extract (called se-2000), provided by the manufacturer (biofarm, pozna, poland) in the form of tablets (product name soyfem). the extract consisted of genistin and genistein (26.90%), daidzin and daidzein (13.37%), glycitin and glycitein (1.98%), and soy proteins, carbohydrates and lipids (remaining amount). this extract was administered orally (in the form of whole tablets or tablets crushed into powder) at the dose corresponding to 5 mg of genistin and genistein (genistin is a glycan that can be converted to genistien by either acid environment or intestinal bacterial flora) per 1 kg of body weight daily. the extract was administered twice a day, with equal amounts at morning and evening. two types of evaluation (the modified bae test and parents questionnaire, both described in the next subsections) were performed at baseline and after 12 and 36 months of treatment. the monitoring of adverse effects was based on reports of parents, who were appropriately instructed to signal any such effects immediately (by either phone or email, with confirmation of receipt of the information), and who provided an assessment of such effects in writing every 3 months (even if no adverse effects were observed). this experimental treatment was approved by the independent bioethics committee of the medical university of gdask, poland (approval no. parents of the children involved in this study signed the informed consent form. to assess cognitive functions and behavioral changes, a modified version of the brief assessment examination this test estimated the following parameters : (i) alertness / activity, (ii) obeying commands, (iii) pointing at objects, (iv) pointing at colors, (v) matching shapes, (vi) speech, (vii) auditory digit span, and (viii) mathematics. parents of patients were asked to evaluate 18 different parameters concerning changes in conditions of their children from a year before the onset of the treatment to the start of the therapy, from the onset of the treatment to a year after the start of the therapy, and from the onset of the treatment to 3 years after the start of therapy. some parents reported they depended also on the opinion of other, uninitiated, people (eg, teachers). the patients were diagnosed for sanfilippo disease (either mps iiia, mckusick s omim no. 252900, or mps iiib, mckusick s omim 252920) by estimation of urinary gag levels (highly elevated gag levels, observed in hemi - quantitative electrophoretic analysis, gave a preliminary indication for mps) and measurement of activities of particular lysosomal hydrolases in leukocytes. deficiency in activity of heparan n - sulfatase (control value : 4.11.4 nmoles / mg of protein/18h) or -n - aceltyl glucosaminidase (control value : 9034 nmoles / mg of protein/42 h) was considered as a diagnosis for mps iiia or mps iiib, respectively. mutations in genes coding for the above - mentioned enzymes were determined for 5 patients, and 1 mutation was determined for 1 patient. eight patients (6 with mps iiia and 2 with mps iiib) were enrolled into the study. all patients were of caucasian origin ; 2 males and 6 females. among the 8 patients, 5 (patients : iiia-2, iiia-4, iiia-5, iiib-2 and iiib-5) were previously enrolled into the pilot study. the mps iii patients characterized in table 1 were treated for 36 months with a genistein - rich soy isoflavone extract (called se-2000), provided by the manufacturer (biofarm, pozna, poland) in the form of tablets (product name soyfem). the extract consisted of genistin and genistein (26.90%), daidzin and daidzein (13.37%), glycitin and glycitein (1.98%), and soy proteins, carbohydrates and lipids (remaining amount). this extract was administered orally (in the form of whole tablets or tablets crushed into powder) at the dose corresponding to 5 mg of genistin and genistein (genistin is a glycan that can be converted to genistien by either acid environment or intestinal bacterial flora) per 1 kg of body weight daily. the extract was administered twice a day, with equal amounts at morning and evening. two types of evaluation (the modified bae test and parents questionnaire, both described in the next subsections) were performed at baseline and after 12 and 36 months of treatment. the monitoring of adverse effects was based on reports of parents, who were appropriately instructed to signal any such effects immediately (by either phone or email, with confirmation of receipt of the information), and who provided an assessment of such effects in writing every 3 months (even if no adverse effects were observed). this experimental treatment was approved by the independent bioethics committee of the medical university of gdask, poland (approval no. to assess cognitive functions and behavioral changes, a modified version of the brief assessment examination was used. this test estimated the following parameters : (i) alertness / activity, (ii) obeying commands, (iii) pointing at objects, (iv) pointing at colors, (v) matching shapes, (vi) speech, (vii) auditory digit span, and (viii) mathematics. parents of patients were asked to evaluate 18 different parameters concerning changes in conditions of their children from a year before the onset of the treatment to the start of the therapy, from the onset of the treatment to a year after the start of the therapy, and from the onset of the treatment to 3 years after the start of therapy. some parents reported they depended also on the opinion of other, uninitiated, people (eg, teachers). eight patients suffering from sanfilippo disease (mps iiia and iiib) were monitored during an experimental therapy consisting of a genistein - rich soy isoflavone extract (soyfem) at the dose of 5 mg / kg / day for 24 months. five of these patients were enrolled in a 1-year pilot study (patients iiia-2, iiia-4, iiia-5, iiib-2, iiib-5), described previously, and the treatment (called get it) was continued for the next 2 years (parents of these patients collaborated with researchers for the next 2 years and sent back their questionnaires on time ; although parents of the other 5 patients also agreed to collaborate, they failed to return completed questionnaires on time). urinary gag levels and hair morphology are relatively simple assays that can be used for monitoring of mps treatment ; however, they do not reflect the behavioral and cognitive status of examined patients. moreover, a statistically significant improvement in gag excretion and normalization of hair morphology were reported after 1 year of get it ; hence, one would not expect further improvement in these parameters during the next 24 months. since cognitive and behavioral dysfunctions are the most severe symptoms in sanfilippo disease, we decided to monitor cognitive ability and behavior of patients by using a specific psychological test and a questionnaire for patients parents, as described previously. this corroborates the conclusion that get it is a safe treatment, as described previously. to assess cognitive functions of the patients, a modified bae psychological test (see appendix for details) was performed at baseline, and at 12 and 36 months after the start of treatment. during the first year of the treatment, an improvement in the score in 7 patients and stabilization in 1 patient (iiia-7) were observed (table 2), which corroborates previous results. after the third year (2-year follow - up) further improvement (relative to results after the first year) was observed in 2 patients (iiia-2, iiia-5), stabilization was observed in 3 patients (iiia-4, iiia-7, iiib-5), and deterioration was observed in 3 patients (iiia-6, iiia-8, iiib-2). in this last - mentioned group of patients, the scores after 36 months of treatment were equal or very similar to those achieved at baseline (table 2). to estimate the efficacy of the treatment, patients parents were asked to evaluate 18 different parameters concerning changes in conditions of their children from a year before the start of the treatment to the start of the therapy, from the onset of the treatment to a year after the start of the therapy, and from the start of the treatment to 3 years after the start of the therapy. as expected, parents reported deterioration of patients in many parameters in the period from 1 year before the treatment to the start of the trial, which confirmed that the children were in the clinically progressive stage of the disease (table 3). to estimate status of the patients, we calculated a general score for each patient on the basis of answers to the questionnaires. this general score was calculated by summing all the answers and giving the following weights to particular types of answers : (2) considerably deteriorated, (1) somewhat deteriorated, (0) no changes, (+ 1) somewhat improved, and (+ 2) considerably improved. the results of questionnaires revealed improvement in all patients after the first year of treatment, further improvement in 5 patients (iiia-2, iiia-5, iiia-8, iiib-2, iiib-5), and deterioration in 3 patients (iiia-4, iiia-6, iiia-7), during the next 2 years (table 2). after unsuccessful trials with bone marrow transplantation and with treatments employing small molecules, either miglustat or glucosamine, to our knowledge, this pilot study on a specific substrate deprivation therapy, called get it, demonstrates the first treatment of patients with sanfilippo disease that demonstrates a statistically significant improvement in biochemical and morphological parameters, as well as improvement in clinical status of patients. since get it appeared to be a promising treatment, in the absence of information on clinical effects of longer treatment, a follow - up of treated patients was necessary. in this report we present a 2-year follow - up of sanfilippo disease patients treated with a genistein - rich soy isoflavone extract (soyfem) at the dose corresponding to the amount of genistein equal to 5 mg / kg / day. when monitoring cognitive abilities and general status of the patients after the third year of the treatment, we observed either further improvement / stabilization of the patients, or their slow deterioration. this is in contrast to rapid deterioration of untreated mps iii patients presented in the literature and evident from questionnaire responses in this report (table 3). interestingly, such a picture of the clinical manifestation of symptoms in treated mps iii patients is similar to that observed during ert of mps i, although only effects on somatic tissues could be considered in the latter case. namely, mps i patients treated with laronidase improve relatively rapidly during the first several months of the treatment, and then slow improvement, stabilization or slow deterioration occurs. it is plausible that during the first period of both ert and get it, the reversible effects of diseases are corrected, and thereafter it is only possible to either prevent further progression or to slow the irreversible changes. the main mechanism of genistein - mediated impairment of gag synthesis is inhibition of phosphorylation of egfr. thus, one might predict that when using higher doses of genistein it should be possible to slow the gag production even more effectively. the dose of the se-2000 extract used in both the previous trial and this study corresponded to the amount of genistein equal to 5 mg / kg / day. since it was recently reported that a dose as high as 160 mg / kg / day is safe (with no adverse effects) for mps iiib mice, it is likely that significantly (at least 200%) higher doses than that used in this study may be safe for humans. therefore, there is great need for a clinical trial evaluating get it for sanfilippo disease. oxidative stress, inflammation, cytotoxicity and apoptosis may be involved in the mechanism of neurodegeneration in sanfilippo disease [1820 ]. moreover, accumulation of hyperphosphorylated tau protein (p - tau), which forms aggregates characteristic of alzheimer s disease, has been detected in brains of mps iiib mice. thus, it is intriguing that genistein has been described as a factor attenuating oxidative stress in the brain, having a neuroprotective effect against beta amyloid - induced neurotoxicity, inhibiting apoptosis in primary neuronal cell cultures, preventing alzheimer s disease - associated inflammation, and revealing a general neuroprotective effect. genistein, apart from its action as an indirect inhibitor of gag synthesis, may also be beneficial for mps iii patients due to its neuroprotective functions. although some of these functions were observed in vitro at genistein concentrations as low as 100 nm, in most in vivo studies some effects were evident only at 1015 mg / kg [2227 ]. these facts strengthen the need for results of clinical studies in which patients with sanfilippo disease are treated with genistein at doses of 1015 mg / kg / day or higher. results of recent studies on animals suggest that get it may be effective in treatment of other types of mps. since improvement in cns was observed in genistein - treated mice with mps ii, an x chromosome - linked disorder, get it could be considered as a potential treatment for mps ii - associated mental retardation. the treatment of sanfilippo patients with a genistein - rich soy isoflavone extract (get it), at the dose corresponding to 5 mg / kg / day, may be effective in either inhibition (in some patients) or slowing (in other patients) of the cognitive dysfunction, behavioral problems and general deterioration caused by the disease, over a period of 3 years. an increase in the dose of genistein is suggested to improve the efficacy of the treatment. general excitement 0 points short - term goal directed behaviour 2 points short - term goal directed activity 4 points repeated goal directed activity 6 points full adequate activity 8 points fulfilling simple commands none fulfilled 0 points none pointed 0 points none pointed 0 points none matched 0 points individual echoic words 2 points individual words 4 points simple sentences 6 points expanded statements 8 points none reproduced 0 points 1 reproduced 4 points 2 reproduced 6 points 3 reproduced 8 points does not count 0 points counts to 10 8 points general excitement kinetic excitement, no goal - oriented activity, lack of even short - term concentration on a single stimulus. short - term goal directed behaviour temporary concentration, e.g. watching somebody or something. short - term goal directed activity conscious, purposeful manipulation of an item, visual examination, intentional attempts to socialize. full adequate activity goal directed activity dominates in child s behaviour. fulfilling simple commands commands like give mammy the toy, put the book on the shelve, put the ball into the bag may be supported by the gesture but without presentation. pointing as a response to a verbal request, e.g. show me, where the flower is pointing items in a named colour, e.g. show the blue block (among several basic colours : red, yellow, blue, green or white). matching the shapes in a big puzzle into the holes in a desk : individual echoic words : any attempts to direct towards anybody vocal communication, any repeated voice reactions to the stimuli, etc. repeating by heart digits in a line of two or three elements (e.g. 74, 582). does not count, has no idea of numbers counts to 5 : counts items in the limit of five counts to 10 : counts items in the limit of 10. | summarybackgroundmucopolysaccharidoses (mps) are inherited metabolic disorders caused by deficiencies in enzymes involved in degradation of glycosaminoglycans. mps type iii (sanfilippo disease) is clinically characterized mainly by progressive and severe behavioral disturbances and cognitive dysfunction. recent 1-year experimental treatment of 10 patients with a genistein (4, 5, 7-trihydroxyisoflavone)-rich extract resulted in improvement of tested parameters, including cognitive and behavioral functions.material/methodseight pediatric patients with sanfilippo disease were enrolled into the study. the modified version of the brief assessment examination was used to assess cognitive functions. moreover, 18 different parameters concerning changes in conditions of patients were assessed by their parents.resultsduring the first year of the treatment, an improvement of cognitive functions in 7 patients and stabilization in 1 patient were assessed, while after the third year (2-year follow - up) further improvement was observed in 2 patients, stabilization in 3 patients and some deterioration in 3 patients. monitoring of general and behavioral symptoms revealed improvement in all patients after the first year of the treatment, further improvement in 5 patients, and deterioration in 3 patients during the next 2 years.conclusionswe conclude that the treatment of sanfilippo patients with a genistein - rich soy isoflavone extract (called gene expression - targeted isoflavone therapy [get it ]) may be effective in either inhibition (in some patients) or slowing down (in other patients) of behavioral and cognitive problems over a longer period. an increased dose of genistein may improve the efficacy of the treatment. |
henoch schnlein purpura (hsp) belongs to the group of nongranulomatous small vessel vasculitis. intussusception, gangrene of the bowel, bowel perforation, and massive hemorrhage are the most common gastrointestinal complications of hsp. a comprehensive search of pubmed, embase, and web of science was performed for all relevant papers published before july 1, 2015. because the full text of six articles was unavailable, only 13 cases described in 12 full - text articles were included in this study. then, we investigated the clinical features, treatments, and prognoses of hsp - related pancreatitis cases. the main clinical characteristics of the 13 patients (six males, seven females) are summarized in table 1. the patients ranged in age from 3 to 70 years, half of cases were 520 years old. pancreatitis presented as the initial manifestation of hsp in eight cases. in addition, pancreatitis and typical purpura occurred at the same time in the two cases. from the onset of pancreatitis to the diagnosis of hsp, the time elapsed ranged from 1 day to 75 days, and 62.5% (5/8) of the patients began to have typical purpura within 7 days of the onset of pancreatitis. in addition to abdominal pain in all cases, seven patients presented with vomiting, one patient had hematemesis, one patient had hematochezia, and one patient had poor appetite. iu / l) in all cases. abdominal computed tomography (ct) revealed pancreatic edema, ascites, and a wide range of intestinal wall edema in seven cases. a pancreatic cyst was found in one patient, it appeared over the course of 35 days and disappeared in 55 days ; however, five patients did not demonstrate any morphological changes in the pancreas. all patients experienced relief through treatment with fasting, gastrointestinal decompression, nutritional support, antiacid drug, glucocorticoid, and somatostatin. the abdominal pain relief time (144 days) was noted in six cases. patient prognoses were described in three cases, and these patients were cured without recurrence through follow - up. main clinical characteristics of 12 previously reported cases of henoch - schnlein purpura - related pancreatitis hsp occurs about twice as often in boys as in girls, half of affected patients are younger than 10 years of age. however, our study suggested that hsp - related pancreatitis usually occurs in adolescent girls. the reason remains unclear ; it may be associated with the immune state of adolescent girls. compared with other types of pancreatitis, hsp - related pancreatitis has the following characteristics. in our study, before the onset of hsp - related pancreatitis, there was no prominent cause, such as biliary tract disease, overeating, drinking, hyperlipidemia, viral infection, or drugs. we speculate that hsp - related pancreatitis may be associated with small blood vessel thrombosis, vasculitis, and intimal thickening. although the clinical manifestation was relatively mild, acute pancreatitis may be the initial manifestation of hsp. hsp - related pancreatitis is diagnosed clinically but requires ct evaluation or ultrasound imaging to differentiate mild acute pancreatitis from severe necrotic pancreatitis. in our study, approximately 41.7% (5/12) of the patients with pancreatic morphology were normal, and only 41.7% of the patients presented with pancreas swelling. the imaging changes in hsp - related pancreatitis were atypical, and pancreatic necrosis or pseudocysts and other local complications were relatively rare. in our study, all patients had elevated serum amylase and urine amylase levels, with an increase of at least three times the upper limits in blood and urine, but the level was not positively correlated with the disease severity. some researchers believe that elevated amylase creatinine clearance and serum lipase levels are appropriate for the early diagnosis of pancreatitis. it has been reported that the measurement of plasma factor xiii could be useful for the early diagnosis of hsp, particularly when the typical purpura is preceded by abdominal pain. in addition to the conventional treatment for pancreatitis, more attention should be paid to hsp. in our study, the symptom of pancreatitis was improved after steroid treatment in all patients. somatostatin can inhibit gastric and pancreatic secretions, thereby reducing enzymatic activity ; it can also reduce capillary permeability, open the sphincter of oddi, and promote the excretion of pancreatic enzyme. after a diagnosis of hsp - related pancreatitis in addition to the symptomatic and supportive treatment, the use of corticosteroids to control hsp helps to alleviate hsp - related pancreatitis. | to summarize the experience of diagnosing and treating patients with henoch schnlein purpura (hsp)-related pancreatitis, a systematic review of previously published cases was conducted. among 13 reported cases, there were six males and seven females whose age from 3 to 70 years. the clinical features of these patients indicated that acute pancreatitis could be the initial manifestation of hsp, the radiological change was atypical, and most cases were alleviated with steroidal treatment. good outcomes can be achieved in patients who are diagnosed early with hsp - related pancreatitis, and it is vital to begin timely treatment of hsp - related pancreatitis with corticosteroid. |
patients with angioid streaks are prone to develop a subretinal hemorrhage after ocular injury, due to fragility of bruch s membrane. the purpose of this study was to report a patient with angioid streaks in whom subfoveal choroidal neovascularization (cnv) developed after blunt ocular trauma. a 60-year - old man was accidentally struck in the left eye with a crowbar handle while engaged in the demolition of wooden building materials in may 2011 and was initially evaluated at our hospital. corrected visual acuity was 0.3 in the right and 1.2 in the left eye, and relative afferent pupillary defect was negative. funduscopy revealed choroidal atrophy around the optic papillae and angioid streaks radiating from around the optic discs in both eyes. in the right eye fluorescein angiography of the left eye showed a blockage due to subretinal hemorrhage and increasing hyperfluorescent spots superior to the fovea, suggestive of extrafoveal cnv. spectral - domain optical coherence tomography showed macular thinning in the right eye and no obvious abnormalities near the subfoveal region in the left eye. two months later, the patient noticed decreased visual acuity in the left eye and was reevaluated. visual acuity had decreased to 0.7, and well - defined cnv, one disc diameter in size, was presenting slightly superior to the macula, including the subfoveal region. two weeks later, anti vascular endothelial growth factor (anti - vegf) antibody (bevacizumab) was injected intravitreally ; the cnv then regressed, and visual acuity improved to 1.2. marked improvement in visual acuity with early treatment has not been reported in angioid streaks with subfoveal cnv after ocular injury. intravitreal injection of an anti - vegf antibody should be considered early after the diagnosis of cnv. angioid streaks (as) are characterized by linear irregularities radiating from around the optic disc due to a weakening or rupture of bruch s membrane, elastic fiber degeneration, or calcium deposition at the same site. if these streaks reach the macular region, choroidal neovascularization (cnv) will occur under the macular region, resulting in severe visual impairment. cnv occurs in 72%86% of cases of as,1,2 and the gass type ii cnv seen in exudative age - related macular degeneration is common. significant visual impairment reportedly develops in 15% of patients with as who suffer head trauma,3 and regular examination is required to assess changes before and after injury. as far as we have been able to determine, few reports have described the development of cnv after blunt trauma in as patients. we report herein a case in which an as patient who developed cnv after blunt trauma was treated with intravitreal injection of bevacizumab, an anti vascular endothelial growth factor (anti - vegf) antibody, soon after onset of cnv, resulting in improved visual acuity. in may 2011, a 60-year - old man was accidentally hit in the left eye with a crowbar handle while engaged in the demolition of wooden building materials and underwent initial examination in our department on the same day. his medical history included a fundal hemorrhage after having been hit in the right eye 34 years earlier. his best corrected visual acuity (bcva) was 0.3 (with 1.50 diopters) in the right eye and 1.5 (with 3.00 diopters) in the left eye. the intraocular pressure was 15 mmhg on the right and 22 mmhg on the left. light reflex was rapid in both eyes, and relative afferent pupillary defect was negative. examination of the anterior segments showed a slight presence of inflammatory cells only in the left anterior chamber. fundal examination revealed choroidal atrophy around the optic papillae and as radiating from around the optic discs in both eyes. atrophic changes to the macular region were evident in the right eye, and a subretinal hemorrhage was apparent around the macular region in the left eye (figure 1a and b). two weeks later, fluorescein angiography (fa) (visucam ; carl zeiss meditec ag, jena, germany) was performed. the middle phase fa image of the right eye showed tissue staining and a window defect in the macular region (figure 1c). the early phase fa image of the left eye revealed signs of blocking due to subretinal hemorrhage and hyperfluorescent spots due to early leakage superior to the fovea (figure 1d). additionally, the fa images of the middle (figure 1e) and late (figure 1f) phases showed increasing hyperfluorescence due to the leakage, suggestive of extrafoveal cnv. spectral - domain optical coherence tomography (oct) (cirrus hd - oct model 4000 ; carl zeiss meditec ag) using the hd 5-line raster scan protocol (horizontal scan of 6 mm) revealed thinning of the central retina with subretinal tissue in the right eye (figure 1 g), but no obvious abnormality was seen in the neighborhood of the fovea in the left eye (figure 1h). examination of the skin revealed characteristic skin lesions on the neck (figure 2a) and axillae on both the right (figure 2b) and left side, suggestive of pseudoxanthoma elasticum. the subretinal hemorrhage in the left eye was subsequently absorbed, but 2 months after the injury, the patient became aware of metamorphopsia in the left eye. one month later (3 months after the injury), bcva in the patient s left eye had decreased to 0.7, and well - defined cnv, one disc diameter in size, was evident somewhat above the macular region (figure 3a). the extrafoveal cnv, which had developed superior to the fovea, progressed to a subfoveal location. oct demonstrated that cnv was clearly located in the juxtafoveal (figure 3b) and subfoveal (figure 3c) regions, extending across the retinal pigment epithelial line into the retina, resulting in gass type ii cnv. fa revealed well - defined, predominantly classic cnv in the early phase (figure 3d) and a high level of fluorescein leakage coincident with cnv in the late phase (figure 3e). cnv was also visualized on indocyanine green angiography (visucam) from the early phase (figure 3f) to the late phase (figure 3 g). two weeks later (3.5 months after injury), as visual impairment persisted in the left eye, intravitreal injection of 1.25 mg/0.05 ml bevacizumab (avastin ; genentech, south san francisco, ca, usa) was administered. the offlabel use of bevacizumab was approved by the institutional review board of the jikei university. visual acuity gradually improved and by approximately 2 months after starting intravitreal bevacizumab (ivb) had recovered to 1.2 in the left eye, with pronounced regression of the cnv (figure 4a) and disappearance of the subfoveal cnv (figure 4b). as of 1 year later, no further visual impairment has appeared. in the present patient, who had both as and pseudoxanthoma elasticum, although visual acuity was excellent immediately after the blunt trauma, extrafoveal cnv appeared within 2 weeks after injury, and visual acuity decreased. no previous case reports have described improvements in visual acuity due to early administration of an anti - vegf agent for cnv occurring after blunt injury in an as patient. pandolfo reported a case in which the use of laser photocoagulation was attempted to treat extrafoveal cnv that appeared 4 months after blunt trauma, but subfoveal cnv recurred, and a fibrous scar developed. alpay and caliskan5 reported that pronounced visual impairment, due to a fibrous lesion in the fovea, occurred 3 weeks after injury in an as patient who had a macular subretinal hemorrhage and optic disc edema after blunt trauma, despite treatment with oral corticosteroids. de benedetto described an as patient with a loss of visual acuity and extrafoveal cnv originating from choroidal rupture in the left eye, who had suffered from ocular blunt trauma 1 year earlier, and reported that visual acuity in this patient remained unchanged after ivb injection. although some reports have described the occurrence of subretinal hemorrhage following blunt trauma in as patients,4,5,7,8 the subsequent incidence of cnv is not necessarily high. reported treatment for cnv, as a complication of as that is unrelated to ocular injury, include photodynamic therapy911 and intravitreal injection of the anti - vegf antibodies bevacizumab1216 and ranibizumab.17,18 photodynamic therapy has a short - term effect and may help prevent visual impairment9,10 but has also been reported to cause enlargement of scarring and loss of vision over the long term.11 according to the majority of reports,1218 bevacizumab and ranibizumab are effective in maintaining and improving vision over both the short and long terms, but often, repeated injections are needed. in the present case, we administered a single injection of ivb. cnv regressed after the injection, and marked improvements in visual acuity became evident, indicating that a single ivb injection was useful. in the present case, a subretinal hemorrhage was evident immediately after injury, but extrafoveal cnv, which developed 2 weeks after injury, progressed to a subretinal location within 3 months after injury. it is important that construction workers who suffer from as always wear goggles or other eye protection to protect their eyes while working because cnv may develop after blunt ocular trauma. when cnv develops, the administration of intravitreal anti - vegf antibody as soon as possible should be considered. | backgroundpatients with angioid streaks are prone to develop a subretinal hemorrhage after ocular injury, due to fragility of bruch s membrane.objectivethe purpose of this study was to report a patient with angioid streaks in whom subfoveal choroidal neovascularization (cnv) developed after blunt ocular trauma.case reporta 60-year - old man was accidentally struck in the left eye with a crowbar handle while engaged in the demolition of wooden building materials in may 2011 and was initially evaluated at our hospital. corrected visual acuity was 0.3 in the right and 1.2 in the left eye, and relative afferent pupillary defect was negative. funduscopy revealed choroidal atrophy around the optic papillae and angioid streaks radiating from around the optic discs in both eyes. in the right eye, there was macular atrophy. in the left eye, there was a subretinal hemorrhage around the macular region. fluorescein angiography of the left eye showed a blockage due to subretinal hemorrhage and increasing hyperfluorescent spots superior to the fovea, suggestive of extrafoveal cnv. spectral - domain optical coherence tomography showed macular thinning in the right eye and no obvious abnormalities near the subfoveal region in the left eye. two months later, the patient noticed decreased visual acuity in the left eye and was reevaluated. visual acuity had decreased to 0.7, and well - defined cnv, one disc diameter in size, was presenting slightly superior to the macula, including the subfoveal region. two weeks later, anti vascular endothelial growth factor (anti - vegf) antibody (bevacizumab) was injected intravitreally ; the cnv then regressed, and visual acuity improved to 1.2.conclusionmarked improvement in visual acuity with early treatment has not been reported in angioid streaks with subfoveal cnv after ocular injury. intravitreal injection of an anti - vegf antibody should be considered early after the diagnosis of cnv. |
this report is a result of a collaborative effort between the national microbiology laboratory (nml), health canada, and the provincial public health laboratories (pphls) in canada. the pphls represent every province in canada and also include the yukon and north west territories. all s. paratyphi b dt identified at the pphls were forwarded to the nml for additional characterization. between 1998 and 2002, 252 s. paratyphi b dt strains were submitted to the nml, of which 246 were from a human source. incidence of s. paratyphi b dt has generally increased since 1998 ; the spike in s. paratyphi b dt in the third and fourth quarters of 1999 can be attributed to an outbreak in british columbia, alberta, and saskatchewan caused by contaminated alfalfa sprouts (7). in addition to this large outbreak, additional outbreaks were reported between 1998 and 2002 ; however, each outbreak was small, and most involved fewer than six persons. total number of salmonella enterica serovar paratyphi b dt identified in canada (gray bars) and the number of multidrug - resistant s. paratyphi b dt identified over the same period (black bars), by quarter. antimicrobial susceptibility testing was performed on all strains by using the disk - diffusion method as described by the national committee for clinical laboratory standards (8). susceptibilities were determined for ampicillin (a), chloramphenicol (c), ciprofloxacin (cp), streptomycin (s), sulfamethoxazole (su), tetracycline (t), and trimethoprim (tm). of the 237 strains examined for susceptibility, 123 (52%) were susceptible to all antimicrobial agents tested. sixty - seven strains (28%) displayed the pentaresistance phenotype (acssut), and the second most prevalent resistance profile was su (n = 41, 17%). single strains displayed the phenotypes a, t, cssu, asutm, assu, and acsuttm. a significant increase was observed in acssut strains over the time period from 1998 to 2002 (p < 0.001). rates for the years are as follows : 1998, n = 2 (2%) ; 1999, n = 4 (18%) ; 2000, n = 2 (10.5%) ; 2001, n = 23 (39%) ; and 2002, n = 36 (58%). no large outbreaks were reported during this time period. to determine if the pentaresistance phenotype was caused by sgi1, polymerase chain reaction (pcr) was used to detect integrons and the left (thdf - s001) and right (s044-yidy) junctions of sgi1 as previously described (2). of the 67 strains identified with the acssut phenotype, 63 contained 1.0-kb and 1.2-kb integrons and left and right junctions of sgi1, which suggests that these strains contained sgi1 inserted into the same location on the chromosome as was described for dt104 (1). one strain with the acssut phenotype contained 1.0-kb and 1.2-kb integrons and gave a pcr product for the left junction amplification reaction but not the right junction, which suggests that a portion of sgi1 downstream of the integrons was missing. three strains with the acssut phenotype did not give the characteristic size products for integron (0.7 kb, n = 2 ; 2.0 kb, n = 1), and all were negative for the junction pcr, which suggests that these strains most likely harbored other resistance genes giving the acssut phenotype. only four strains with the acssut phenotype were identified from a nonhuman source (one poultry, three environmental). although the three environmental isolates contained sgi1, the acssut strain isolated from poultry did not ; instead, it contained a 2-kb integron. of the human isolates for which source was reported for the acssut strains (n = 53), all were isolated from stool, with the exception of three that were isolated from blood. to ensure the sgi1 was intact, a selected number of isolates were subjected to additional pcr with primers representing all regions of the 44-kb sgi1 element (table). dna from all of these strains gave positive reactions for all primer sets described, which suggests sgi1 was intact in these strains. s. paratyphi b dt recovered from 2000 to 2002 were subtyped by using pulsed - field gel electrophoresis (pfge) after dna extraction and digestion with blni according to the standardized salmonella protocol (9). pfge - generated dna profiles were entered into the bionumerics software program version 3.0 (applied maths, st. cluster analysis was performed by the unweighted pair - group method with arithmetic averages, and dna relatedness was calculated on the basis of the dice coefficient. in addition, all pfge patterns were visually compared and assigned a number or letter identification (10). a dendrogram depicting the s. paratyphi b dt blni macrorestriction patterns of the 139 strains available to subtype (total = 142), visual comparison of the fingerprints revealed a total of 63 unique fingerprint patterns that grouped into 24 clusters. analysis of the dendrogram revealed that all but three strains with the acssut phenotype were grouped into three closely related clusters named clusters 1 to 3 (figure 2). the three strains that did not cluster with the other acssut strains did not harbor sgi1 as described above. dendrogram depicting the dna fingerprints of salmonella enterica serovar paratyphi b dt identified from 2000 through 2002. cluster 2 contained 17 strains that represented 7 subtypes, all of which showed < 7 band differences between strains from cluster 1. cluster 3 contained 15 strains that represented 6 subtypes, all of which showed fewer than seven band differences between subtypes in cluster 2, but had more than seven band differences between the cluster 1 fingerprints. five were other acssut identified in 1998 (n = 1) and 1999 (n = 4), and all were identified in cluster 2. in addition, one s. paratyphi b dt recently shown to harbor sgi1 that was isolated in singapore from a fish was grouped into cluster 3 (4). the canadian isolates in clusters 1 to 3 have been identified from multiple provinces, including british columbia, alberta, manitoba, ontario, and qubec, representing western and central regions of canada. the emergence of multidrug - resistant s. paratyphi b dt was documented recently in the netherlands and scotland (11,12). some isolates had the acssut susceptibility pattern and did not harbor any plasmids, which suggests the resistance is of chromosomal origin (12). however, in neither study was the presence of sgi1 or other resistance genes examined. we demonstrate the rapid emergence of multidrug - resistant s. paratyphi b dt in canada. the increase is due to three clusters, all of which contain the multidrug - resistant genomic island sgi1. that three closely related clonal groups were present suggests sgi1 may have inserted into the genome of s. paratyphi b dt in three separate events, as shown by clusters 1, 2, and 3, or the insertion may have occurred once, with strains diverging over time. we also identified three strains with the acssut phenotype that did not contain sgi1 sequences, which emphasizes the need to monitor genotypic resistance factors and not just phenotypic resistance traits to understand the dissemination of antimicrobial resistance. the emergence of multidrug - resistant enteric pathogens is a concern because of the lack of suitable antimicrobial agents available to treat invasive infections. one organism that emerged in the 1990s is multidrug - resistant s. typhimurium dt104, which harbors sgi1 (3). along with the multidrug - resistant phenotype, however, in vitro studies have not shown any increase in invasiveness or survival in mammalian cells (15,16). whether multidrug - resistant dt104 is more virulent remains to be determined, the underlying question remains : why has this clone of dt104 emerged as a major pathogen ? selective pressure, resulting from the widespread use of antimicrobial drugs in animals for growth promotion or prophylaxis, may have played a role in disseminating this organism. however, this factor may not completely account for its prevalence, because other multidrug - resistant strains of dt104 have emerged but have not disseminated internationally. for example, additional determinants in sgi1 may contribute to the fitness or virulence of salmonella strains harboring it. in the present study, three closely related clusters of s. paratyphi b dt carrying sgi1 have emerged in canada and make up 46% (64 of 139) of all strains identified in 2001 and 2002. furthermore, three additional s. paratyphi b dt strains with the acssut phenotype were identified that do not harbor sgi1 and do not appear to be rapidly increasing in canada. in this hypothesis, sgi1 may predict the next emerging salmonella serotype. other factors, such as processing food products and the structure of the food distribution system, could play a role in disseminating these organisms. we continue to monitor this pathogen and are designing studies to improve understanding of the epidemiology of s. paratyphi b dt in canada. | we document an increase in the number of multidrug - resistant salmonella enterica serovar paratyphi b dt+ identified in canada. most of these strains harbor salmonella genomic island 1 (sgi1). further studies are needed to determine factors contributing to the observed emergence of this multidrug - resistant strain. |
fetal malnutrition (fm) is a significant contributor to perinatal morbidity and mortality.123 it is a state of poor nutrition in - utero resulting from inadequate supply and/or utilization of nutrients1 leading to the fetus failing to acquire adequate amount of fat, subcutaneous tissues, and muscle mass during intrauterine growth.1 fm describes the underweight / wasting aspect of the clinical syndrome seen in malnourished newborns. this clinical state may be present at almost any birth weight.2 globally, the incidence of fm is between 2% and 10% of total births with highest incidence in developing countries.2 a study conducted in ile - ife, nigeria, reported an incidence of 18.8% for fm in term newborns.3 the assessment of the nutritional status of the fetus has been of considerable interest to clinicians because of its potential serious sequelae on multiple organ systems manifesting as perinatal problems and/or long - term central nervous system sequelae.34 recent studies have also demonstrated the evidence that fm may have a far - reaching effect on adult life such as susceptibility to cardiovascular disease and non - insulin dependent diabetes mellitus.567 various anthropometric indices have been used to identify babies that suffered suboptimal fetal growth (weight, length, mid - arm circumference (mac) and head circumference (hc),89 proportionality indices (ponderal index [pi],8910 mac / hc ratio, body mass index [bmi ]), and clinical assessment of nutritional (can) status and its score.91112 can score which contains nine clinical signs, namely, hair, cheeks, neck, arms, chest, abdomen, back, buttocks, and legs, as developed by metcoff11 to differentiate malnourished from appropriately nourished babies4 has also been used widely by researchers to determine fm in the newborn period. there is a dearth of research in the combined use of can score, bmi, and the other anthropometric indices as a means of assessing fm in newborns in the west african subregion. the aims of the present study are to assess the nutritional status of term newborns at birth using can score, bmi, pi, mac / hc, and birth weight and to compare the relative efficiency of can score and the anthropometric indices in the identification of fm in term newborns. the findings from this study will provide renewed insight into the easier and better methods of determining fm in term newborns. the study was carried out on consecutive, singleton, live born babies of 37 completed weeks through 41 weeks of gestation at the inborn unit of luth between may 1, 2010, and november 30, 2010. all the anthropometric measurements, except birth weight, were carried out by the investigator with trained assistance where needed, within 48 h of baby 's birth. all the neonates were weighed completely nude at birth by the delivery room staff using the infant weighing scale (weighmaster model, usa) which records the weight to the nearest 10 g. using the olowe intrauterine growth chart,13 birth weights for gestational age below the 3 percentile and above the 97 percentile on the chart were taken as small for gestational age (sga) and large for gestational age (lga), respectively. the hc was measured using a flexible nonstretchable tape measure with due attention to the appropriate prominences.14 the measurement was to the nearest 0.1 cm. the mac was measured using the same tape at the midpoint between the acromion and the olecranon process with the forearm flexed at 90 at the elbow.14 in the abducted arm with the elbow flexed (in almost all the term babies), a skin crease appears which corresponds approximately with the midpoint of the arm.141516 the readings obtained were recorded to the nearest 0.1 cm. pi was computed from the formula : pi = weight (g)/length (cm) 100 and pi < 2.2 was considered as malnutrition. the mac / hc ratio was calculated for each infant and the value plotted on a standard curve designed for nigerian newborn.161718 bmi was calculated for each baby and the value was plotted on the bmi curve designed by brock. for newborn babies.18 can status was done within 48 h of life on the basis of the superficial readily detectable signs of malnutrition in the newborn as described by metcoff [table 1].4 this consisted of inspection and estimation of loss of subcutaneous tissues and muscles in the designated areas. hair, cheeks, neck and chin, arms, back, buttocks, legs, chest, and abdomen were examined and then scored. a maximum score of 4 was awarded to each parameter with no evidence of malnutrition, and the lowest score of 1 was awarded to parameter with the worst evidence of malnutrition. the total rating of the 9 can score signs was the can score for the subject. fm was defined as can score < 25.41920 the nine signs for clinical assessment of nutritional status in the newborn statistical analysis was performed using the statistical package for social sciences (spss)for windows, version 17.0 (spss inc, chicago released 2008, ibm corp, new york). student 's t - test was used to compare the mean anthropometry between males and females. one hundred and thirty - seven were male while 145 were female, with a male : female ratio of 1:1.07. eight babies (2.8%) were of low birth weight and sga while 15 (5.3%) were lga. the anthropometric characteristics of the study population showed that the mean birth weight was 3260 460 g and mean length of 49.37 2.26 cm while the mac and hc were 34.63 1.19 cm and 10.83 1.01, respectively. about 53.2% of the babies were born to multiparous mothers and 39% of the mothers had various illnesses during pregnancy (high blood pressure [30% ], malaria [15.9% ], and retroviral disease [37.8% ]). the prevalence of fm as detected by can score was 14.5% while bmi detected 13.1% of the study participants as malnourished. pi and mac / hc detected fm in 10.3% and 2.8% of the study population, respectively. the incidence of fm is slightly more in male babies, but it was not statistically significant except with mac / hc ratio which detected no female newborn with fm. relationship between sex and fetal malnutrition two hundred and fifty - nine babies (91.8%) were appropriate for gestational age (aga), 15 (5.3%) were lga, and 8 (2.8%) were classified as sga using olowe 's intrauterine growth chart. can score identified 15.4% of the aga babies as fm and only one of the sga babies as fm. tables 3 and 4 show the comparison of can score with anthropometry in detection of fm. pi, mac / hc, and birth weight showed very low sensitivity of 19.5%, 12.3%, and 2.4%, respectively, in detecting fm ; however, all showed very high specificity (90.9%, 98.6%, and 97.1%, respectively) in detecting fm. comparison between clinical assessment of nutritional score and anthropometric indices in detection of fetal malnutrition in term newborns the sensitivity, specificity and predictive value of anthropometric indices in determining fetal malnutrition about 53.2% of the babies were born to multiparous mothers and 39% of the mothers had various illnesses during pregnancy (high blood pressure [30% ], malaria [15.9% ], and retroviral disease [37.8% ]). the prevalence of fm as detected by can score was 14.5% while bmi detected 13.1% of the study participants as malnourished. pi and mac / hc detected fm in 10.3% and 2.8% of the study population, respectively. the incidence of fm is slightly more in male babies, but it was not statistically significant except with mac / hc ratio which detected no female newborn with fm. relationship between sex and fetal malnutrition two hundred and fifty - nine babies (91.8%) were appropriate for gestational age (aga), 15 (5.3%) were lga, and 8 (2.8%) were classified as sga using olowe 's intrauterine growth chart. can score identified 15.4% of the aga babies as fm and only one of the sga babies as fm. tables 3 and 4 show the comparison of can score with anthropometry in detection of fm. pi, mac / hc, and birth weight showed very low sensitivity of 19.5%, 12.3%, and 2.4%, respectively, in detecting fm ; however, all showed very high specificity (90.9%, 98.6%, and 97.1%, respectively) in detecting fm. comparison between clinical assessment of nutritional score and anthropometric indices in detection of fetal malnutrition in term newborns the sensitivity, specificity and predictive value of anthropometric indices in determining fetal malnutrition fm is prevalent in the developing countries.321 studies have documented that the nutritional status of the newborn is important for identifying perinatal risks42223 as any deviation from the normal is associated with an increased risk of morbidity and mortality.24 in the present study, we evaluated 282 babies for fm. the mean birth weight of the babies was 3260 460 g which was higher than 3067 g recorded in ile - ife5 and 3080 g in jos,24 both in nigeria, and 2600 g reported in india.25 it was however comparable with the 3290 g documented by metcofff4 in the usa. the prevalence of sga in term babies in the present study is low (2.8%) compared to the prevalence reported by adebami.3 in ile - ife (12.1%) and mehta.25 in india (25%) while metcoff4 documented sga prevalence of 11% in term newborns in oklahoma, usa. the low prevalence of sga in the present study may be due to the different intrauterine growth curves used. the olowe intrauterine growth chart13 used in this study classified babies < 2 standard deviations below the mean or below the 3 percentile as sga, whereas the brenner intrauterine growth chart which uses the 10 percentile as the cutoff for sga was used in the study by metcoff4 and adebami.3 a similar study in india by sankhyan.9 had also reported a lower prevalence of sga when local indian intrauterine growth chart was used (9.1%) compared to when international growth charts which had higher cutoff weights at each gestational age was used (45.4%) resulting in higher sensitivity but low specificity. the prevalence of fm in the present study using can score (14.5%) is slightly lower than 18.8% documented in ile - ife by adebami.3 and is 23-fold lower than that documented in india by sankhyan.9 and mehta.25 the reason for the lower prevalence in our study may be related to the demographics and socioeconomic status of the population studied. a well - nourished and healthy mother is more likely to deliver a healthy and well - nourished baby. the prevalence of fm using can score and anthropometry in various gestational age groups of infants had been reported by different researchers. it has been demonstrated that birth weight alone may not reflect the fetal state of nutrition of a newborn adequately. previous researchers had demonstrated that not all sga babies had fm and that some aga babies may have features of fm. in the study by metcoff,4 the prevalence of fm sankhyan.9 documented features of fm in 57.1% of sga and 3.8% of aga newborns. adebami.3 detected fm in 11.5% of term aga babies using can score. in the present study, can score identified fm in 15.4% of term aga babies indicating that these babies suffered from fm in spite of having normal birth weight. among sga babies, only one baby (2.4%) was malnourished using can score ; thus, the term fm and sga are not synonymous. this finding is also similar to that of other researchers such as metcoff that not all sga babies were fm. the anthropometric parameters are important to reflect intrauterine growth and to define a baseline to follow - up the nutritional progress of the infant. many authors have proposed that the assessment of body proportions may be more useful than single measurements for age alone for assessing newborn nutrition.892627 bmi provides a high estimate of body fat mass as it has a positive correlation with skinfold thickness and other methods of estimating the percentage of body fat,15 and it takes advantage of the physiologic principle regarding sparing length at the expense of weight during mild to moderate malnutrition.28 in our study, bmi detected fm in 13.1% of the babies and on comparing with can score, it was noted that the sensitivity of bmi in detecting fm was the highest among anthropometric parameters, suggesting bmi may be a sensitive indicator of fm. soundarya.29 also found bmi to be a sensitive indicator of fm in their study and proposed a combination of bmi and pi in the screening of babies for fm. this is lower than the prevalence recorded by mehta.25 (29.19%) and kayshap.27 (27.8%) in india. it is, however, comparable with the lower prevalence of 8.1% documented by adebami.3 both mehta.25 and kashyap.27 combined the late gestational preterm babies of 35 weeks to the analysis of their results and thus may have contributed to their higher prevalence. some babies with normal pi were found to be malnourished using can score in the present study. can score identified 34 (13.4%) of the babies with normal pi as malnourished. the prevalence of fm in the present study using mac / hc ratio was very low (2.8%). this is lower than the 49.76% recorded in term babies in india by mehta.25 although both studies used different cutoff values in determining fm.252730 it is interesting to note that not all babies classified as malnourished by anthropometry were found malnourished by can score and vice versa. the present study, like others in the literature, had also shown that can score identified more babies with fm compared to anthropometry making it a reliable tool in the detection of fm. we concluded that the newborn baby with fm is a high - risk newborn and his postnatal survival greatly depends on careful observation and documentation of the evidence of his adverse intrauterine life and a proactive management of his anticipated complications. a simple and easy way of identifying fm at birth is ideal and will make for judicious use of scarce resources in developing countries. the different anthropometric indices measure different aspects of the well - being of a newborn while can score measures the visible wasting observed in malnourished newborn. all the parameters, in one - way or the other, reflect the adverse intrauterine nutrition these newborns suffered. therefore, the use of multiple methods of determining fm will increase the likelihood of identification of most babies with fm. the combination of can score and bmi to the routine assessment of newborns at birth may further improve newborn care and buttress the need to look out for these high - risk babies for anticipatory care and follow - up. | background : fetal malnutrition (fm) which describes the underweight / wasting seen in newborns is a significant contributor to perinatal morbidity and mortality and requires proper documentation. the objective of this study was to assess the nutritional status of term newborns at birth using clinical assessment of nutritional (can) status score and four other anthropometric indices and to compare the efficiency of can score and the anthropometric indices in identifying fm in term newborns.materials and methods : the study was conducted on live singleton babies delivered 3742 completed weeks of gestation at the inborn unit of lagos university teaching hospital without major congenital abnormalities or severe perinatal illness. birth weights and lengths were recorded at birth. ponderal index (pi), body mass index (bmi), and mid - arm / head circumference (mac / hc) ratio were calculated and the values were compared with standard curves. the can score consisted of inspection and estimation of loss of subcutaneous tissues and muscles. fm was defined as can score < 25. data were analyzed using the statistical package for social sciences statistics software version 17.0.results:two hundred and eighty - two newborns were analyzed. fm was identified in 14.5%, 10.3%, 13.1%, and 2.8% of newborns using can score, pi, bmi, and mac / hc ratio, respectively. out of the fm babies identified by can score, pi, mac / hc, and bmi identified 19.5%, 12.3%, and 53.7% of them as fm also. bmi was the most sensitive anthropometric index for detecting fm.conclusion:can score is a simple clinical tool for identifying fm and when used in conjunction with bmi will enhance fm detection. |
exogenous ethanol (etoh) causes elevated brain and hepatic homocysteine (hocys) levels, decreased brain and hepatic taurine levels, and increased apoptosis rates within embryonic chick brains and livers [13 ]. exposure to either teratogen causes reduced brain masses, elevated brain lipid hydroperoxide (lpo) levels, elevated brain membrane lipid peroxidation intermediates, and elevated brain caspase-3 activities [48 ]. hocys catabolism uses remethylation pathways and the transsulfuration pathway (figure 1). in remethylation pathways, hocys is remethylated back to methionine by using either betaine homocysteine methyltransferase (ec 2.1.1.15 ; non - folate - dependent remethylation), or the cobalamin - dependent enzyme, methionine synthase (ec 2.1.1.13), which uses 5-methyltetrahydrofolate as the methyl donor. in the transsulfuration pathway, hocys is converted to cystathionine through the use of cystathionine -synthase (ec 4.2.1.22) and cystathionine is ultimately converted into -ketobutyrate, reduced glutathione (gsh), or taurine [9, 10 ] (figure 1). etoh - induced decreased taurine levels within embryonic chick brains [2, 3 ] may be devastating because taurine 's oldest known function is regulating osmotic stress. during mammalian brain swelling, astrocytes release taurine into extracellular spaces creating a hypertonic environment and cause h2o efflux from brain cells [11, 12 ]. hypotonicity causes astrocytes and neurons to incorporate cysteine sulfinic acid and synthesize taurine through the use of cysteine sulfinic acid decarboxylase (csad ; ec 4.1.1.29) [11, 13 ]. ironically, elevated levels of d / l - homocysteine, l - cysteate, l - glutamate, and l - aspartate are all potent inhibitors of na - dependent cysteine sulfanilic acid uptake in chick astrocytes, chick neurons, and rat astrocytes. hence, etoh - induced reductions in brain taurine levels coupled with etoh - induced increased brain hocys levels [13 ] may alter neural osmotic regulation [11, 13 ]. taurine, and especially hypotaurine, has membrane - protective and antioxidant effects in a variety of systems [11, 14, 15 ]. taurine supplementation ameliorated etoh - induced increased lipid peroxidation, alcohol - induced liver damage, and etoh - induced cytochrome p450 - 2e1 (cyp 2e1) activities within sprague - dawley rats. because cats have extremely low csad activities and primates have low brain csad activities, dietary taurine is essential for cats and is conditionally essential for primates [11, 14, 15, 17 ]. taurine deficiencies in cats cause destabilization and fragmentation of retina and tapetum membranes ; increased frequencies of retinopathy and blindness ; increased frequencies of spontaneous abortions and stillbirths ; altered immune systems where the proportions, morphology, and functions of leukocytes are altered ; and myocardial failure (dilated cardiomyopathy) [11, 14, 15, 18 ]. neuroblasts and glioblasts cell division and migration within the cortex and cerebellum of cat brains is normally completed by the third neonatal week. in taurine - deprived kittens, cell division and migration within the cortex and cerebellum is delayed [15, 18 ]. taurine supplementation ameliorated reduced synaptic connections, reduced neuron and glial cell numbers, and increased apoptosis rates within the cortex of fetal rat brains experiencing intrauterine growth restriction. consequently, taurine is important in embryonic and neonatal development. because etoh reduced taurine levels within embryonic chick brains [2, 3 ], the overall objective of this study was to determine if taurine supplementation alleviated etoh - induced oxidative stress within embryonic chick brains. oxidative - stress measures included rates of apoptosis (caspase-3 activities), homocysteine (hocys) levels, reduced glutathione levels (gsh), membrane fatty acid composition, and lipid hydroperoxide (lpos) levels. unlike mammalian embryos, avian embryos are closed systems (cleidoic egg) that offer the ability to solely observe embryonic responses to a teratogen as compared to combined maternal responses, fetal responses, and maternal to fetal transport as seen within eutherian embryos. all experiments used fertile, specific pathogen - free white leghorn chicken eggs (gallus domesticus) purchased from a commercial hatchery. chickens raised at this commercial hatchery ingest a standardized diet causing minimal differences in yolk composition from generation to generation. animal care was in compliance with the guide for the care and use of laboratory animals, 8th edition (national academies press, washington dc, usa, 2011). control eggs were injected with approximately 25 l of h2o or a 1 : 1 (v / v) mixture of 0.01 m taurine and h2o (4 mol taurine / kg egg) into each air sac during the first three days of development (e02). experimental eggs were injected with approximately 25 l of a 1 : 1 (v / v) mixture of 47.5% etoh and h2o, (1.5 mmol etoh / kg egg) ; a 1 : 1 (v / v) mixture of 95% etoh and h2o (3 mmol etoh / kg egg) ; a 1 : 1 (v / v) mixture of 47.5% etoh and 0.01 m taurine (1.5 mmol etoh / kg egg and 4 mol taurine / kg egg) ; or a 1 : 1 (v / v) mixture of 95% etoh and 0.01 m taurine (3 mmol etoh & 4 mol taurine / kg egg) into each air sac during the first three days of development (e02). all embryos were incubated in a forced air incubator at 37.5c and turned every 4 hours with relative humidity ranging from 80 to 90%. at 11 days of incubation [20, theoretical stage 37 ], blood was drawn from chorio - allantoic blood vessels and serum collected by centrifugation (1,000 g). chick embryos were then decapitated, brains excised, and all serum and isolated brains stored at 80c, until subsequent biochemical analyses. because chicks normally hatch at 21 days of development, embryos at 11 days of development [20, theoretical stage 37 ] have completed approximately 52% of total embryogenesis (midembryogenesis) and embryonic exposure to etoh and/or taurine during the first three days of development (e02) represents approximately 14% of embryogenesis. theoretical stage 37 [20, 11 days of development ] was selected because exogenous etoh (3 mmol etoh / kg egg) caused elevated brain hocys levels ; increased brain caspase-3 activities ; increased brain lipid hydroperoxide levels (lpos) ; decreased brain membrane polyunsaturated fatty acids (pufas) levels ; and decreased brain taurine levels at theoretical stage 37 [13, 68 ]. the 4 mol of exogenous taurine / kg egg dosage was recently deemed to be semi - safe. this dosage failed to impact brain masses, brain caspase-3 activities, or brain lipid hydroperoxide (lpo) levels within chick embryos at 18 days of development [20, theoretical stage 44 ] (unpublished data). in order to monitor apoptosis within brain homogenates, embryonic chick brains were homogenized in 400 l of ice - cold 50 mm hepes [4-(2-hydroxyethyl)-piperazineethanesulfonic acid ] buffer (ph 7.4), 1 mm dtt (dithiothreitol), 0.1 mm edta (disodium ethylenediaminetetraacetic acid), and 0.1% chaps (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), and nonsoluble material was removed by centrifugation at 10,000 g (10 min at 4c). aliquots from each supernatant were assayed for total protein and caspase-3 activity [5, 22 ]. as the fluorogenic acetylated - aspartate - glutamate - valine - aspartate-7-amino-4-methylcoumarin (acdevd - amc) substrate was cleaved by activated caspase-3, the liberated amc (7-amino-4-methylcoumarin) was detected by fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. caspase-3 activities are reported as units of caspase-3/mg protein, and one unit of caspase-3 is defined as the release of 1 pmol of amc liberated per minute from the 0.3 mm acdevd - amc substrate at 30c. in order to study the effects of exogenous etoh and/or taurine on brain hocys levels, the isolation and quantification of hocys was achieved by modifying the technique of yi.. embryonic brains were homogenized in 500 l of ice - cold 50 mm hepes buffer (ph 7.4), 1 mm dtt, and 0.1 mm edta, and nonsoluble material was removed by centrifugation at 10,000 g (5 min at 4c). supernatants were deproteinated by the addition of 400 l of 0.4 m perchloric acid (pca). protein - containing precipitates were removed by centrifugation at 10,000 g (5 min at 4c) and the ph was readjusted to approximately 6.5 to 7.5 by adding 15 l of 6 m naoh to each supernatant. hocys and methionine derivatives were prepared as previously described [1, 22, 23 ]. the separation and quantification of hocys derivative was achieved by hplc using a 15 cm 4.6 mm discovery bio - wide pore c 18 hplc column (5 m beads) using a high - pressure liquid chromatography system (hplc) equipped with a fluorescence detector. the mobile phase was 50 mm sodium monophosphate, 1 mm octane sulfonic acid, and 2% acetonitrile and adjusted to ph 2.7 with 85% phosphoric acid. the isocratic flow rate was 1 ml / min (1800 to 2100 psi) and the excitation wavelength was 230 nm and the emission wavelength was 418 nm. using these hplc conditions, hocys concentrations within biological samples were determined from a standard curve prepared from known concentrations of d / l - homocysteine (0 to 10 mol hocys). effects of exogenous etoh and/or taurine on reduced glutathione (gsh) levels within embryonic chick brains utilized the protocols of anderson [24, 25 ]. embryonic brains were homogenized in 500 l of ice - cold 50 mm sodium phosphate buffer (ph 7.4) and 0.1 mm edta, and nonsoluble material was removed by centrifugation at 10,000 g (5 min at 4c). an additional aliquot of 50 l was removed from each supernatant and deproteinated by adding 190 l of 4% 5-sulfosalicylic acid and the protein - containing precipitate removed by centrifugation at 15,000 g (5 min at 4c). an aliquot of 200 l was then removed and 1 ml of ellman 's reagent (0.1 mm 5,5-dithio - bis(2-nitrobenzoic acid) in 0.1 m sodium phosphate buffer, ph 8.0) was added to each aliquot and incubated at 37.5c for 30 min. as reduced gsh was converted into oxidized gssg (oxidized glutathione disulfide), 5,5-dithiobis(2-nitrobenzoic acid) was converted into 2-nitro-5-thiobenzoic acid, and the absorbance was measured at 412 nm. serum aliquots of 50 l were deproteinized by adding 50 l of cold 0.2 m 5-sulfosalicylic acid to each sample, and nonsoluble material removed by centrifugation at 11,500 g (2 min at 4c). columns containing 1 ml of dowex 1 4 resin over 3 ml of dowex-50w-8 resin in 12 ml reservoirs fitted with 1.5 cm frits were prewashed with 12 ml of 1 m hcl. after 40 l of 100 m homoserine was added to each supernatant as an internal standard, supernatants were layered onto a dual - bed column and taurine and homoserine were eluted with 4 ml of h2o. opa (ortho - phthalaldehyde) derivatives were synthesized according to waterfield 's approach and filtered through 0.2 m millex - fg13 ptfe filters. aliquots of 20 l were injected onto a 15 cm 4.6 mm discovery bio - wide pore c 18 hplc column (5 m beads) on a scm 1000 high - pressure liquid chromatography system (hplc) equipped with dual detectors including a fluorescence detector and a uv - vis detector. the uv - vis detector was set at a wavelength of 250 nm and the excitation wavelength of the fluorescence detector was 350 nm with an emission wavelength of 476 nm [2, 3 ]. the mobile phase was 0.05 m nah2po4 (ph 5.4) mixed with a 1 : 1 solution of methanol - water (43 : 57, v / v). the isocratic flow rate was 1 ml / min (1900 to 2100 psi). using this protocol, the retention time of homoserine (internal standard) was 3.683 0.299 min and the retention time of taurine was 4.504 0.287 min. because reactive oxygen species (oxygen radicals (o2) and hydroxyl radicals (ho)) can attack any unsaturated neutral lipid or any unsaturated phospholipid, it became imperative to determine if exogenous etoh and/or taurine altered brain membrane fatty acid composition. embryonic brains were homogenized in 400 l of ice - cold 50 mm hepes buffer (ph 7.4), 1 mm dtt, and 0.1 mm edta, and total lipids were extracted by the folch technique. samples were concentrated by the evaporation of solvent under n2 and stored overnight at 40c. neutral lipids were separated from membrane lipids on silicic acid columns [46, 29 ]. liquid chromatography columns (12 ml reservoirs) were packed with anhydrous 100200 mesh sil - r to a bed height of 3.5 cm. after columns were washed several times with chloroform, total lipids were dissolved in 0.5 ml of 2 : 1 chloroform - methanol (v / v) and applied to a column. neutral lipids were eluted with 4 ml of chloroform, and membrane lipids, including phospholipids and some glycolipids, were eluted with 4 ml of methanol. after fractionated samples were dried under n2, membrane lipid samples were saponified and liberated fatty acids were methylated according to metcalfe.. fatty acid methyl esters (fames) were separated on an omega - wax capillary gas chromatography (gc) column (30 m 0.53 mm 0.25 m film thickness) in a gc. injector temperature was 225c and the flame ionization detector was set at 250c with a he carrier gas rate of 2025 ml / min. after a 5 min delay, the column temperature was increased to 205c at a rate of 2c / min. after methyl pentadecanoate was added as an internal standard (48.75 nmol / sample), individual fames within each biological sample were identified by their retention times, quantified as compared to the internal standard, and data expressed as % mol. lipid hydroperoxides (lpos) are peroxidation intermediates and were extracted by homogenizing brains in 500 l of ice - cold 50 mm hepes buffer (ph 7.4), 1 mm dtt, and 0.1 mm edta. samples were deproteinated by adding 500 l of meta - phosphoric acid (20 mg / ml in methanol) to each sample, and protein - containing precipitates were removed by centrifugation (5,000 g for 5 min). nonpolar lipid hydroperoxides were extracted from each supernatant by adding 3 ml of chloroform, followed by centrifugation for 5 min at 2,000 g. an aliquot of 1 ml was removed from each organic layer and spectrophotometrically assayed for lpos by using lipid hydroperoxide assay kits. lpos are highly unstable and readily promote the conversion of fe to fe and were detected by using thiocyanate ions as the chromogen. membrane fatty acid composition is reported as % mol. because percentages may form binomial distributions rather than normal distributions, all % mol data were subjected to arcsine transformations prior to analysis of variance (anova) followed by post hoc tukey tests (honestly significant difference tests). other data sets were not subjected to arcsine transformation before anova analyses were followed by post hoc tukey tests (honestly significant difference tests). the significance level in post hoc tukey tests was p 0.05. early embryonic exposure to etoh (3 mmol / kg egg) and taurine (4 mol / kg egg) caused approximately a 1.8-fold and a 1.6-fold decrease in brain masses at 11 days of development, respectively (table 1). while the combination of exogenous etoh and taurine caused reduced brain masses, the reductions were insignificant. brain caspase-3 activities increased by approximately 1.8-fold when exposed to either 1.5 mmol etoh / kg egg or 3 mmol etoh / kg egg as compared to controls. meanwhile, exposure to 4 mol of exogenous taurine / kg egg caused approximately a 1.7-fold increase in brain caspase-3 activities as compared to controls. the combined treatments of etoh and taurine (1.5 mmol etoh and 4 mol taurine / kg egg or 3 mmol etoh and 4 mol taurine / kg egg) also caused increased brain caspase-3 activities as compared to controls (table 1). hence, etoh and/or taurine caused increased brain apoptosis rates. early embryonic etoh (3 mmol / kg egg) exposure (e02) caused increased brain and hepatic caspase-3 activities and reduced brain masses at 11 days of development (midembryogenesis) [13, 68, 32 ]. however, the observations that exogenous taurine also caused reduced brain masses and promoted increased brain caspase-3 activities are novel and indicate that exogenous etoh and/or taurine are embryopathic. in a preliminary study in this laboratory, a 50 mg of exogenous taurine / kg egg dosage (400 mol taurine / kg egg) during early embryogenesis (e02) caused reduced percentage of living chick embryos at 11 days of development (theoretical stage 37) to 31.9 10.2% as compared to 86.7 3.1% within controls (t = 10.80 ; df = 6 ; p 0.0001) (unpublished data). in this study, living embryos were defined as possessing a beating heart, and there were 4 rounds of injections (n = 4) with 18 to 21 eggs within each injection group. in this study, a correlation coefficient (r) of 0.52 (f = (1, 22) 8.20 ; p = 0.009) was observed when serum taurine levels were correlated to brain caspase-3 activities across all groups. this last observation indicates that exogenous taurine is more toxic in chick embryos at 11 days of development as compared to chick embryos at 18 days of development. the exogenous dosage of 4 mol taurine / kg egg during the first three days of development previously failed to impact brain caspase-3 activities within 18-day chick embryos (late embryogenesis) and was, therefore, thought to be semi - safe (unpublished data). in this study, an exogenous dosage of 4 mol taurine / kg egg during the first three days of development caused increased brain caspase-3 activities within 11 day chick embryos (midembryogenesis) (table 1). taurine - induced toxicity within chick embryos was first reported by van gelder and belanger who reported that a single injection of 100 mol taurine/50 g egg (2 mmol taurine / kg egg) caused increased incorporation of taurine into developing hearts and brains at 15 days of development and was associated with ataxia, reduced muscle strength, a loss of motor coordination, and reduced embryo viability. consequently, we have still not found a safe exogenous taurine dosage for chick embryos at 11 days of development (tables 1, 2, and 3). exogenous etoh, taurine, and the combination of etoh and taurine all caused increased brain hocys levels (table 1). exposure to 3 mmol etoh / kg caused a 1.9-fold increase in brain hocys levels and exposure to 4 mol exogenous taurine / kg egg caused a 2.3-fold increase in brain hocys levels as compared to controls. the combined treatments of etoh and taurine (1.5 mmol etoh and 4 mol taurine / kg egg and 3 mmol etoh and 4 mol taurine / kg egg) both caused elevated brain hocys levels (table 1). when brain caspase-3 activities were correlated to brain hocys levels across all groups, pearson 's product moment (r) was 0.35 (f = (1, 47) 6.48 ; p = 0.0004). thus, etoh- and taurine - induced increased brain caspase-3 activities correlated to etoh- and taurine - induced increased brain hocys levels (table 1). etoh - induced and taurine - induced increases in brain hocys levels can promote the synthesis of reactive oxygen species (ros). two hocys molecules can undergo autooxidation and form a dimer (homocysteine : oxidized disulfide) by liberating two hydrogen ions and two electrons. in doing so, hydrogen peroxide and hydroxyl radicals can be generated and thus produce reactive oxygen species, that is, hydroxyl radicals (ho) and oxygen radicals (o2). early embryonic exposure to etoh (1.5 mmol etoh / kg egg and 3 mmol / kg egg) and taurine (4 mol / kg egg) caused approximately a 1.2-fold to 1.3-fold decrease in brain gsh levels at 11 days of development, respectively (table 1). the combined treatments of etoh and taurine (1.5 mmol etoh and 4 mol taurine / kg egg or 3 mmol etoh and 4 mol taurine / kg egg) both caused approximately a 1.2-fold to 1.0-fold decrease in brain gsh levels as compared to controls. total brain thiol (gsh and gssg) levels were largely unaffected (table 1). when brain caspase-3 activities were correlated to brain gsh levels within all groups, pearson 's product moment (r) was 0.29 (f = (1, 48) 4.40 ; p = 0.004). thus, as apoptosis rates increased (brain caspase-3 activities), brain gsh levels decreased. recently, we demonstrated that etoh (3 mmol / kg egg) exposure during the first three days of development (e02) caused decreased brain selenium - dependent glutathione peroxidase (gpx ; ec. 1.11.1.9) activities and non - selenium - dependent gpx activities at 11 days of development (midembryogenesis) (unpublished data) (p < 0.05). glutathione peroxidases (gpx), both selenium - dependent and non - selenium - dependent, are well - known first - line defense antioxidant enzymes because they catalyze the reduction and ultimate removal of lipid hydroperoxides (lpos), and other peroxides, as follows [35, 36 ] : consequently, etoh - induced reduction in brain gsh - dependent gpx activities could certainly contribute to oxidative stress and slow the catabolism of etoh - induced increased lpo levels (table 1). increased endogenous serum taurine levels were caused by exogenous etoh (1.5 mmol etoh / kg egg and 3 mmol / kg egg), taurine (4 mol / kg egg), and etoh and taurine (1.5 mmol etoh and 4 mol taurine / kg egg and 3 mmol etoh and 4 mol taurine / kg egg) (table 1). early embryonic exposure to 1.5 mmol exogenous etoh / kg egg and 3 mmol exogenous etoh / kg egg caused approximately a 2.2-fold increase and a 3.1-fold increase in serum taurine levels, respectively. meanwhile, exposure to 4 mol of exogenous taurine / kg egg caused approximately a 2.4-fold increase in serum taurine levels. the combined treatments of etoh and taurine (1.5 mmol etoh and 4 mol taurine / kg egg and 3 mmol etoh and 4 mol taurine / kg egg) both caused elevated serum taurine levels as compared to controls (table 1). when serum taurine levels were correlated to brain caspase-3 activities within all groups, pearson 's product moment (r) was 0.61 (f = (1, 24) 11.54 ; p = 0.003) and when serum taurine levels were correlated to brain hocys levels within all groups, pearson 's product moment (r) was 0.43 (f = (1, 24) 6.05 ; p = 0.003). therefore, etoh - induced and taurine - induced increases in serum taurine levels correlated to increased apoptosis rates and increased hocys levels within embryonic chick brains. presumably, exogenous taurine caused increased endogenous serum taurine levels (table 1) by diffusing into the yolk and then entering the embryo during vascularization. previously, it was demonstrated that exogenous taurine exposure during the first three days of embryonic development (e02) caused increased endogenous chick serum taurine levels during late embryogenesis (18th day of development) (unpublished data). this last observation has now been extended to midembryogenesis (11 days of incubation ; table 1). while it was not surprising that exogenous taurine caused increased serum taurine levels (table 1), it was surprising to find that early exogenous etoh exposure caused increased levels of serum taurine (table 1). one hypothesis in explaining this phenomenon is etoh - induced increases de novo taurine synthesis rates. 4.1.1.29) then catalyzes the decarboxylation of cysteine sulfinic acid to hypotaurine and taurine (figure 1). chick embryos can synthesize taurine from cysteine, and very high cysteine lyase (ec. 4.4.1.10) activities are found in the area vasculosa within 10-somite (approximately 33 hr) chick embryos. cysteine lyase catalyzes the synthesis of cysteine sulfinic acid through the following reaction : chick neurons and glia readily incorporate cysteine sulfinic acid, and incorporated cysteine sulfinic acid is quickly converted into cysteic acid and is decarboxylated, via cysteine sulfinic acid decarboxylase (ec. besides synthesizing taurine from cysteine, adult rats, adult chickens, and chick embryos also have the ability to synthesize taurine from serine [3942 ]. in this very old evolutionary pathway, inorganic sulfate is added to atp and converted to 3-phosphoadenosine-5-phosphosulfate (paps) followed by the transfer of the sulfate from paps to a hydroxylated organic compound, such as serine, to form taurine and is catalyzed by the pyridoxine - dependent enzyme, paps transferase (ec. this pathway is illustrated as follows : (3)atp+so42 atp - sulfurylaseadenosine-5-phosphosulfate (aps)(4)aps+atp aps kinaseadp + 3-phosphoadenosine-5-phosphate (paps)(5)paps+serinepaps transferasetaurine we suspect that the hypothesis concerning exogenous etoh - induced increased de novo taurine synthesis rates within chick embryos is false. elevated d / l - hocys levels are a potent inhibitor of na - dependent cysteine sulfanilic acid uptake into chick astrocytes and neurons and should slow de novo brain taurine synthesis [11, 13 ]. etoh - induced and taurine - induced increases in brain hocys levels have been observed in this study (table 1), and previous reports have demonstrated that exogenous etoh (3 mmol / kg egg) during the first three days of development (e02) caused reduced brain and hepatic taurine levels at 11 days of development [2, 3 ]. thus, an alternative hypothesis predicts etoh - induced increases in membrane fluidity and permeability may stimulate an efflux of taurine, and possibly k, from neural cells into the circulatory system. etoh - induced increases in membrane fluidity are documented [7, 27 ] and etoh - induced and hocys - induced decreased chick brain membrane phosphatidylcholine (pc) levels [5, 44 ], increased chick brain membrane phosphatidylethanolamine (pe) levels [5, 44 ], and decreased chick brain membrane pufa levels [4, 68, 22, 27, 44 ] have been reported. etoh - induced increases in taurine and pe levels within chick allantoic fluid have been reported and allantoic taurine and pe levels exceeded serum levels. as cells lose cylindrically shaped phospholipids, such as pc, and gain triangularly shaped phospholipids, such as pe, membrane fluidity and permeability increase [7, 27, 46 ]. an etoh- or hocys - induced taurine efflux from brain and hepatic cells into the circulatory system could create hypertonic environments and promote cell shrinkage that is typically observed during apoptosis [11, 17 ]. in biopsies of adult human gliomas, histology revealed significant correlations between apoptotic cell density and taurine efflux in nonnecrotic (r = 0.73, p < 0.003) and necrotic (r = 0.63, p 0.0005) biopsies and, thus, the authors argued that taurine efflux is a potential marker of apoptosis. increased extracellular taurine levels have been implicated as the mechanism in apoptosis - related cell shrinkage in several cell types [4850 ] and p38 mitogen - activated protein kinase activation before taurine and k efflux have been associated with caspase-3 activation. early embryonic etoh exposure altered brain membrane fatty acid composition during midembryogenesis (table 2). etoh (3 mmol / kg egg) caused a 2.2-fold reduction in brain membrane docosahexaenoic acid (22 : 6, n-3) levels ; a 1.7-fold reduction in docosapentaenoic acid (22 : 5, n-6) levels ; a 1.5-fold reduction in arachidonic acid (20 : 4, n-6) levels ; a 2.2-fold reduction in linolenic acid (18 : 3, n-6) levels ; and a 2.3-fold reduction in palmitolenic acid (16 : 3, n-3) levels as compared to controls (table 2). meanwhile, etoh caused a 1.5-fold increase in brain membrane palmitic acid (c16 : 0) levels and a 3.5-fold increase in myristic acid (14 : 0) levels when compared to controls (table 2). early embryonic taurine (4 mol / kg egg) exposure also altered brain membrane fatty acid composition during midembryogenesis (table 2). taurine caused a 1.4-fold reduction in brain membrane docosahexaenoic acid (22 : 6, n-3) levels and a 1.7-fold reduction in adrenic acid (22 : 4, n-6) levels as compared to controls. meanwhile, taurine - induced increases in brain membrane linoleic acid (18 : 2, n-6) levels were observed. the combination of etoh and taurine (3 mmol etoh and 4 mol taurine / kg egg) caused a 3.0-fold reduction in brain membrane docosahexaenoic acid (22 : 6, n-3) levels ; a 1.6-fold reduction in docosapentaenoic acid (22 : 5, n-6) levels ; a 2.2-fold reduction in adrenic acid (22 : 4, n-6) levels ; a 1.4-fold reduction in arachidonic acid (20 : 4, n-6) levels ; and a 1.2-fold decrease in palmitoleic acid (16 : 1, n-7) levels as compared to controls. meanwhile, a 5.6-fold increase in brain membrane linoleic acid (18 : 2, n-6) levels was observed in etoh- and- taurine - treated embryos as compared to controls (table 2). in order to look at etoh - induced and taurine - induced changes in brain membrane fatty acid composition in a simplified and holistic manner, membrane fatty acid levels (table 2) were converted into two ratios (table 3). the first ratio was the sum of unsaturated / saturated membrane fatty acids (unsaturated/ saturated fatty acids) and the second ratio was the sum of long - chain polyunsaturated fatty acids / short - chain membrane fatty acids (long - chain pufas/ short - chain membrane fatty acids) (table 3). in this study, long - chain pufas are defined as having hydrocarbon lengths 20 carbons, and short - chain fatty acids had hydrocarbon chain length 20 carbons. when unsaturated/ saturated fatty acids ratios were correlated to long - chain pufas/ short - chain membrane fatty acids in all groups, a pearson 's product moment (r) value of 0.37 (f = (1, 31) 4.99 ; this correlation coefficient indicates that long - chain pufas are relatively more unsaturated as compared to short - chain fatty acids. etoh - treated embryos had a 1.5-fold decrease in unsaturated/ saturated membrane fatty acid levels and a 1.9-fold decrease in long - chain pufas/ short - chain membrane fatty acid levels as compared to controls (table 3). taurine - treated embryos and etoh- and- taurine - treated embryos demonstrated insignificant differences in unsaturated/ saturated membrane fatty acid levels as compared to controls. however, taurine - treated embryos had a 1.3-fold decrease in long - chain pufas/ short - chain membrane fatty acid levels and etoh- and- taurine - treated embryos had a 2.0-fold decrease in long - chain pufas/ short - chain membrane fatty acid levels as compared to controls. thus, etoh - treated, taurine - treated-, and etoh- and- taurine - treated embryos all demonstrated decreased levels of membrane long - chain pufas as compared to controls (table 3). because reduced pufa levels are observed during lipid peroxidation [7, 10, 27 ], brain lipid hydroperoxides (lpos) levels lpos are lipid peroxidation intermediates and etoh - treated embryos had a 5.0-fold increase in lpo levels, taurine - treated embryos had a 3.6-fold increase in lpo levels, and etoh- and- taurine - treated embryos had a 4.8-fold increase in lpo levels as compared to controls (p < 0.05). when long - chain pufas/ short - chain membrane fatty acid values were correlated to brain lpo levels within all groups, a pearson 's product moment (r) of 0.78 (f = (1, 28) 42.86 ; p < 0.0001) was observed. taurine, and especially hypotaurine, has membrane - protective and antioxidant properties in a variety of systems undergoing oxidative - stress [11, 14, 15 ]. taurine supplementation ameliorated etoh - ameliorated lipid peroxidation, alcohol - induced liver damage, and etoh - induced increased cytochrome p450 - 2e1 (cyp 2e1) activities within rats. consequently, etoh - induced and/or hocys - induced taurine efflux from the brain into the circulatory system [2, 3 ] may remove a powerful antioxidant from the neurons and/or astrocytes that are undergoing etoh - induced oxidative stress (tables 2 and 3). etoh - induced formation of reactive oxygen species via the oxidation of fe, increased xanthine oxidase activities, and increased cytochrome p450 - 2e1 (cyp 2e1 ; ec 1.14.13n7) activities have been discussed [7, 27 ]. etoh- and taurine - induced increased reactive oxygen species levels can attack long - chain pufas and momentarily create lpo intermediates which cleave long - chain pufas into less saturated membrane fatty acids and produce a number of cytotoxic reactive aldehydes (tables 2 and 3) [27, 31 ]. while the sources of reactive oxygen species during membrane lipid peroxidation in taurine - treated chick embryos are unclear (tables 2 and 3), the commonality between etoh - induced membrane lipid peroxidation and taurine - induced lipid peroxidation (tables 2 and 3) elevates brain hocys levels as seen in both exogenous treatments (table 1). this paper has failed to demonstrate that an exogenous dosage of 4 mol taurine / kg egg can ameliorate etoh - induced lipid peroxidation, oxidative - stress, and apoptosis within embryonic chick brains during midembryogenesis. both exogenous etoh and exogenous taurine treatments during early embryogenesis (e02) caused increased apoptosis rates (increased caspase-3 activities), increased oxidative - stress (decreased gsh levels), and increased membrane lipid peroxidation (reduced long - chain pufas and increased lpo levels) within embryonic chick brains (tables 1, 2, 3). etoh - induced and taurine - induced increases in brain hocys levels were associated with decreased brain gsh levels and brain taurine levels (tables 1 and 2) and may indicate etoh - induced and taurine - induced decreased metabolism via the transsulfuration pathway (figure 1) that may contribute to elevated hocys levels. because increased brain hocys levels can be a source of reactive oxygen species, elevated hocys levels appear to be associated with increased rates of apoptosis, oxidative - stress, and lipid peroxidation within embryonic chick brains and possibly cause a taurine efflux from brain and hepatic cells into the blood and may contribute to apoptosis - related osmotic stress and cell shrinkage (table 1) [2, 3 ]. while the exogenous dosage of 4 mol / kg egg failed to impact brain masses, brain caspase-3 activities, or brain lpo levels within chick embryos at 18 days of development (late embryogenesis), this same dosage clearly promotes increased rates of membrane lipid peroxidation and apoptosis within embryonic chick brains at 11 days of development (midembryogenesis) (tables 1, 2, 3). increased exogenous taurine - induced toxicity, as seen in embryonic chick brains at midembryogenesis as compared to late - embryogenesis, may not be surprising because chick brain and hepatic glutathione - dependent glutathione peroxidases (gpx ; ec. 1.11.1.9) activities increase by 1.4-fold to 2.8-fold when comparing embryos at midembryogenesis (11 days of development) to late - embryogenesis (18 days of development). that is, the protective capabilities of antioxidant enzymes are greater during later stages of development as compared to midembryogenesis. while a lower exogenous taurine dosage may have antioxidant capabilities within chick brains during midembryogenesis as compared to the prooxidant capabilities of the 4 mol / kg egg dosage used in this study, this paper calls for caution and more study. energy drinks has become popular and a concern within european and north american communities [52, 53 ]. taurine levels in energy drinks can range from 10 mg (79.9 mol) to 1000 mg (7990 mol). thus, a pregnant woman, weighing from 140 pounds (63.5 kg) to 180 pounds (81.6 kg), who consumes a single drink of alcohol mixed with an energy drink that contains 1000 mg taurine (7990 mol), consumes etoh mixed with an exogenous taurine dosage ranging from 97.9 mol / kg to 125.8 mol / kg. these dosages are from 25.5-fold to 31.5-fold higher than the 4 mol exogenous taurine / kg egg dosage used in this study which caused apoptosis and membrane lipid peroxidation within chick brains at midembryogenesis. this is a concern and warrants caution because a truly safe exogenous taurine dosage within a human fetus is currently unknown. | because taurine alleviates ethanol- (etoh-) induced lipid peroxidation and liver damage in rats, we asked whether exogenous taurine could alleviate etoh - induced oxidative stress in chick embryos. exogenous etoh (1.5 mmol / kg egg or 3 mmol / kg egg), taurine (4 mol / kg egg), or etoh and taurine (1.5 mmol etoh and 4 mol taurine / kg egg or 3 mmol etoh and 4 mol taurine / kg egg) were injected into fertile chicken eggs during the first three days of embryonic development (e02). at 11 days of development (midembryogenesis), serum taurine levels and brain caspase-3 activities, homocysteine (hocys) levels, reduced glutathione (gsh) levels, membrane fatty acid composition, and lipid hydroperoxide (lpo) levels were measured. early embryonic etoh exposure caused increased brain apoptosis rates (caspase-3 activities) ; increased brain hocys levels ; increased oxidative - stress, as measured by decreased brain gsh levels ; decreased brain long - chain polyunsaturated levels ; and increased brain lpo levels. although taurine is reported to be an antioxidant, exogenous taurine was embryopathic and caused increased apoptosis rates (caspase-3 activities) ; increased brain hocys levels ; increased oxidative - stress (decreased brain gsh levels) ; decreased brain long - chain polyunsaturated levels ; and increased brain lpo levels. combined etoh and taurine treatments also caused increased apoptosis rates and oxidative stress. |
epithelial ovarian carcinoma is no longer felt to be a uniform disease, both in the pathology and oncology literature. it is now divided into two categories in pathology articles, tumors arising from a precursor lesion with a better prognosis versus tumors arising de novo with a worse prognosis. the most common type of epithelial ovarian carcinoma, high grade serous carcinoma, is in the second category. these two categories are based on differences in genetic mutations with clinical implications for targeted chemotherapy [1, 3 ]. cytotoxic and antiangiogenesis medications targeting various cellular receptors and pathways have the potential to improve response rates in patients. angiogenesis or tumor vascularity can be indirectly assessed on imaging by the degree of enhancement of the mass. although ovarian carcinomas are now viewed as two distinct groups in the pathology literature, little is known about whether there are imaging differences between the groups. thus far, most imaging studies of ovarian tumors, including those using dynamic contrast enhanced (dce) mri, have not evaluated the enhancement of the subtypes of ovarian cancer and only a minority of patients in these reports have had serous carcinoma [410 ]. for ovarian masses, the focus in imaging has been on differentiating benign from malignant masses [410 ]. in contrast, on imaging studies for tumors such as renal cell carcinoma, it is recognized that the histologic subtypes have different enhancement characteristics, for example, papillary versus clear cell, and this difference is evident on postcontrast scans obtained in the clinical setting [1113 ]. however, it is not known if similar observations can be made for the subtypes of ovarian cancer. therefore, we performed a pilot study to determine if there is a significant difference in enhancement to distinguish high grade serous carcinoma from other ovarian cancers in routine clinical practice. the institutional review board issued a waiver of informed consent for our retrospective study, which was conducted in compliance with hipaa guidelines. the pathology database was searched to identify patients who had ovarian cancer at oophorectomy between december 2004 and january 2011, followed by a search for preoperative mri studies in this group. inclusion criterion was the availability of mr scans with pre- and postcontrast gradient echo mr imaging of the pelvis with fat saturation, and 37 scans in 37 patients met this criterion and formed the study group. the median time interval between mri and oophorectomy was 21 days (mean 25 days, range 665 days). the mri was done prior to chemotherapy or any other treatment in all patients with primary ovarian cancer in our study. pathology reports were reviewed following analysis of the mri scans. for data analysis, patients were divided into 4 categories based on histology of the malignant ovary : high grade serous carcinoma, other epithelial ovarian carcinoma, other nonepithelial ovarian cancer, and borderline tumor. all mri scans were performed with fat saturated gradient echo (gre) sequences before and after intravenous gadolinium contrast administration ; 3d - gre technique was used in 28 patients and 2d - gre technique was used in 9 patients. the scans were performed between 2004 and january 2011 at multiple institutions on magnets from different vendors and were retrospectively reviewed at our institution on a single picture archiving and communication system (pacs) (centricity, ge medical systems, milwaukee, wi, usa). 3d - gre scan parameters were as follows : median tr 4.4 ms (range 39.1 ms), median te 1.9 ms (range 0.852.9 ms), median slice thickness 2.5 mm (range 1.59 mm), median nex 0.75 (range 0.371), median field of view 26 24 cm (range 20 38 cm), median frequency matrix 256 (range 156320), and median phase matrix 160 (range 98256). 2d - gre scan parameters were as follows : median tr 250 ms (range 172520 ms), median te 3.7 ms (range 1.25.6 ms), median slice thickness 5.5 mm (range 4.5 - 8.5 mm), nex 1, median field of view 28 24 cm (range 20 34 cm), median frequency matrix 256 (range 232320), and median phase matrix 166 (range 128224). four postcontrast phases were performed in 12 patients, 3 phases in 17 patients, 2 phases in 3 patients, and 1 phase in 5 patients. the first postcontrast scan was obtained in the axial plane in 32 patients, in the sagittal plane in 3 patients, and in the coronal plane in 2 patients. the mri scans were independently reviewed by two readers, each with several years of experience in gynecologic imaging. the highest enhancing solid component was selected for signal intensity (si) measurement with the largest possible circular region of interest (roi). this roi was placed on the same region of enhancing tissue on all postcontrast phases, and cross - referencing was performed when necessary. mean roi size was 43.1 mm (range 1.8177.5 mm) for reader 1 and 56.8 mm (range 7291 mm) for reader 2. percent enhancement of the lesion was calculated as [(si lesion post si lesion pre)/si lesion pre ] 100 where si post = lesion signal intensity on post contrast scan and si pre = lesion signal intensity on pre contrast scan. in addition, to compare the si of other pelvic structures between the pre and postcontrast scans, the si of the buttock fat immediately superficial to the gluteus muscle was obtained for all phases. the ratio of the lesion si to fat si was calculated for the same phase in each patient and percentage of change in the lesion / fat si ratio from pre- to post contrast. since the delay between contrast injection and scan acquisition was not available from retrospective review, si of the right external iliac artery and vein was also obtained for each phase at the level of the ovarian mass to calculate an arterial / venous ratio. all patients had a precontrast enhancement measurement and between 1 and 4 post - contrast enhancement measurements. the percentage change in enhancement between the pre - contrast measurement and each post - contrast measurement was calculated, and the maximum of these values was taken to be the highest percent enhancement (hpe). percentage change in enhancement between consecutive pairs of phase the change in lesion / fat ratio between the pre - contrast measurement and each post - contrast measurement was also calculated and the maximum was found. the arterial to venous (a / v) ratio was calculated at each phase and the maximum was found. we examined the association between maximum a / v ratio and the highest percent enhancement using scatterplots. finally, comparisons of hpe and maximum change in lesion / fat ratio were calculated using generalized estimating equations (gee) models with an exchangeable correlation structure. gee models employ a sandwich variance estimate to account for the correlation between multiple ovarian enhancement measures from a single patient. lesion and lesion / fat hpe were transformed to the log scale for modeling purposes. results of all analyses are presented separately for the two independent readers and p values < 0.05 were considered statistically significant. all analyses were performed using sas software, version 9.2 for windows (sas institute, cary, nc, usa) and r version 2.11.0 (r development core team ; 2010). pathology revealed a total of 55 malignant ovaries in the study population of 37 patients, and 18 patients had bilateral malignancy and 19 patients had unilateral malignancy. high grade serous carcinoma occurred in 25 ovaries (46%), other epithelial carcinomas (well or moderately differentiated endometrioid carcinoma, clear cell carcinoma, mucinous carcinoma, and carcinosarcoma) occurred in 12 ovaries (22%), other non - epithelial cancers (carcinoid, dysgerminoma, immature teratoma, struma ovarii, granulosa cell tumor, and metastatic breast cancer) occurred in 8 ovaries (14%), and borderline tumors occurred in 10 ovaries (18%). in the 18 patients with bilateral malignant ovaries, the same histology was seen bilaterally in 12 patients with high grade serous carcinoma, 2 patients with borderline tumor, 1 patient with carcinosarcoma, and in 1 patient with metastatic breast cancer. two patients had an endometrioid carcinoma in one ovary and a borderline tumor in the other ovary. sixteen patients had stage i disease (43%), 1 patient had stage ii disease (3%), 15 patients had stage iii disease (41%), and 2 patients had stage iv disease (5%). precise staging was not available for the remaining patients (n = 3, 8%), 1 of whom was clinically classified as having advanced disease. thirty of the 37 patients (81%) had optimal surgical debulking defined as residual disease < 1 cm. the average size of the largest ovarian mass in each patient was 9.3 5.1 cm as measured by reader 1. tables 1 and 2 summarize the enhancement of the ovarian masses by histologic type and reader. table 1 gives the percentage of lesion enhancement while table 2 gives the percentage of (lesion / fat) enhancement which accounts for possible changes in technical parameters between pre- and postcontrast scans and therefore has lower enhancement values compared with table 1. on the first postcontrast phase, the mean and median percent enhancement of ovarian malignancies was less than 100% in most cases indicating that the signal intensity of the tumor group as a whole did not double. including all post contrast phases, the highest mean and median percent enhancement seen was between 100% and 200% for the invasive tumors and approaching 100% for borderline tumors. the percent enhancement of high grade serous carcinoma was not significantly different from other invasive ovarian malignancies (reader 1 p = 0.865 ; reader 2 p = 0.353). the percent enhancement of invasive ovarian malignancies was more than borderline tumors and approached statistical significance (reader 1 p = 0.102 ; reader 2 p = 0.072). illustrative examples of the enhancement of high grade serous carcinoma, endometrioid carcinoma, and borderline tumor are shown in figures 1, 2, 3, and 4. there was variability in the enhancement of all tumor groups as seen in the wide range of enhancement values in tables 1 and 2 and figures 1 and 2. the spread of enhancement values versus the calculated arterial / venous ratio is plotted in figure 5. the mean peak arterial / venous si ratio for the 3d mri scans (n = 28 scans) was 2.2 (median 1.5, range 0.87.7) and for the 2d mri scans (n = 9 scans) was 1.3 (median 1, range 0.73.0). the concordance correlation coefficient (95% ci) for agreement between the two readers on peak percent enhancement of the lesion was 0.80 (95% ci : 0.70, 0.90). in our study of clinically performed mri scans, there was no significant difference in the enhancement of high grade serous carcinoma compared to other invasive ovarian cancers. these results suggest that in clinical practice enhancement can not be used to distinguish high grade serous carcinoma from other ovarian malignancies. in addition, there was also a wide range in the degree of enhancement of all the tumor groups including high grade serous carcinoma as shown in table 1 and in figures 1 and 2. although some of this variability is possibly due to technical factors such as contrast injection and scan delay, a plot of enhancement versus arterial / venous ratio in figure 5 did not show a pattern for higher enhancement with higher arterial / venous ratios suggesting that other factors may also play a role. enhancement of ovarian masses depends on the delivery and retention of contrast in the lesion. the vascular supply, capillary network, and leakage of contrast into the extravascular interstitial space contribute to the accumulation of contrast within the mass and greater enhancement. angiogenesis is known to occur in cancers and supports tumor growth [14, 15 ]. however, microvascular density alone may not correlate with the degree of enhancement and other factors are also likely responsible. the pericyte coverage index which is a measure of vascular maturity has been reported to correlate negatively with enhancement amplitude. vascular endothelial growth factor receptor (vegfr-2) which affects angiogenesis and vascular permeability has also been shown to be expressed by more cells in invasive tumors compared to benign masses. however, variable genetic aberrations may result in variable expression of vegfr-2 as well as other factors. an analysis of genetic mutations in high grade serous carcinoma found tp53 mutations in the majority of cases (96%) but a lower prevalence of mutations in other genes. using the renal cell carcinoma model, a recent study on clear cell renal cell carcinoma showed that tumors with different chromosomal aberrations showed different levels of enhancement on ct. this suggests that the morphologic appearance can differ even within the same histologic subtype of tumor depending on the genetic makeup. in addition, these genetic variations could account for overlap in the morphologic appearance of the different histologic subtypes of ovarian cancer. more detailed analysis correlating genetic analysis with imaging would shed light on the enhancement of ovarian malignancies. which focused on distinguishing malignant from benign ovarian lesions, there was lower mean percent enhancement of the ovarian malignancies in our study. this is expected given the probable higher contrast injection rate, smaller scan delay, and higher temporal resolution with dce - mri. in their study, a preselected solid portion of the tumor was imaged at 5-second intervals following injection of contrast at 2 cc / second. the study analyzed ovarian masses from 41 patients, 16 of whom had invasive epithelial ovarian carcinomas, and the median percent enhancement of the invasive masses was 176.6% (range 129.1225.5%). the majority of tumors in this study by thomassin - naggara. were endometrioid carcinomas compared to high grade serous carcinoma in our study. the publications by dilks. have imaged the entire ovarian mass at 30-second intervals after contrast and selected the highest enhancing component to measure signal intensity similar to the image review method used in our study [9, 10 ]. an initial publication by this group demonstrated higher percentage enhancement and wash - in rate in ovarian malignancies compared with benign masses. however, the malignant group had only one case of high grade serous carcinoma and included borderline tumors limiting conclusions about the different histologic subtypes of ovarian cancer. a more recent publication by the same group included 8 cases of serous cystadenocarcinoma as well as borderline tumors in a cohort of 36 malignant lesions. the patients were imaged at 30-second intervals for 2 minutes and malignant tumors showed a percentage enhancement of 81 33.5%. this is similar to the degree of enhancement seen in our study on the first post contrast phase. when the authors separated borderline from invasive tumors, malignant tumors had a percentage enhancement of 89.5 28.8% compared with 38.8 22.1% for borderline tumors and this difference was statistically significant (p = 0.001). in the study by thomassin - naggara., the median percent enhancement of invasive tumors was also higher than borderline tumors measuring 176.6% (range 129.1225.5%) versus 79.3% (range 55.5155.3%). similar to both these prior dce mri studies, invasive ovarian tumors in our clinical study showed higher enhancement compared with borderline ones [7, 10 ]. a limitation of our study is that clinically performed studies were reviewed retrospectively and it is possible that the post contrast phases were delayed resulting in an underestimation of the degree of enhancement of the ovarian mass due to washout. overall, the highest percentage of enhancement achieved (table 1) was greater than the percentage of enhancement in the first post contrast phase suggesting continued enhancement of the mass on subsequent phases. in addition, previous mri studies with contrast kinetics time curves suggest that rapid washout of contrast is not typical in the initial imaging time period [5, 7, 8, 19 ]. one mri study with various histologic subtypes of ovarian cancer showed overall progressive enhancement of malignant ovarian masses from 60 seconds to 120 seconds. other limitations of our study include technical differences between the scans, such as the use of both 2d and 3d technique and the lack of a standardized imaging protocol. on clinically performed contrast enhanced mri studies, enhancement of high grade serous ovarian carcinoma is not significantly different from other ovarian malignancies. invasive tumors enhanced greater than borderline tumors. unlike the model of renal cell carcinoma where differing enhancement of papillary and clear cell types is seen both qualitatively and quantitatively in clinical practice without strict adherence to scan timing, such a difference was not evident in our study on the subtypes of ovarian cancer. the role of wash - in rate of contrast may be helpful in learning more about the enhancement of high grade serous carcinoma and is an added value of dce - mri scans over clinically performed mri. high temporal resolution scanning and quantification of contrast kinetics in ovarian cancers may have a potential role in the imaging evaluation of patients [19, 20 ]. | purpose. to assess if there is a significant difference in enhancement of high grade serous carcinoma of the ovary compared with other ovarian malignancies on clinically performed contrast enhanced mri studies. methods. in this institutional - review board - approved study, two radiologists reviewed contrast enhanced mri scans in 37 patients with ovarian cancer. readers measured the signal intensity (si) of ovarian mass and gluteal fat pre- and postcontrast administration. percentage enhancement (pe) was calculated as [(post - pre)/precontrast si ] 100. results. pathology revealed 19 patients with unilateral and 18 patients with bilateral malignancies for a total of 55 malignant ovaries - high grade serous carcinoma in 25/55 ovaries (45%), other epithelial carcinomas in 12 ovaries (22%), nonepithelial cancers in 8 ovaries (14%), and borderline tumors in 10 ovaries (18%). enhancement of high grade serous carcinoma was not significantly different from other invasive ovarian malignancies (reader 1 p = 0.865 ; reader 2 p = 0.353). enhancement of invasive ovarian malignancies was more than borderline tumors but did not reach statistical significance (reader 1p = 0.102 ; reader 2 p = 0.072). conclusion. on clinically performed contrast enhanced mri studies, enhancement of high grade serous ovarian carcinoma is not significantly different from other ovarian malignancies. |
an intracranial or spinal subarachnoid hemorrhage (sah) can result from the rupture of an aneurysm in the anterior, posterior, radicular, or radiculomedullary cervical spinal arteries. hemodynamic stress caused by arteriovenous malformation and coarctation of the aorta are well - known etiologic factors in formation and rupture of a spinal artery aneurysm.3)4)6)7)12)16) however, occurrence of an sah due to the rupture of an isolated aneurysm in the cervical anterior spinal artery is very rare. we report on a case of an sah that resulted from the rupture of an aneurysm in the branch of the anterior spinal artery in a patient with end - stage common bile duct cancer. a 47-year - old male was transferred to our emergency room in a semicomatose state. initially, he presented with posterior neck pain that gradually became more aggravated during the transfer. brain computerized tomography scans showed subarachnoid and intraventricular hemorrhages that were denser in the cisterns around the pons and medulla than in the basal and anterior interhemispheric cisterns (fig. we found an aneurysm in the anterior communicating artery and performed embolization of the aneurysm using detachable coils (fig. we did not notice a small aneurysm in the branch of the anterior spinal artery until the following day during a review of the angiograms (fig. we expected spontaneous occlusion of the aneurysm ; therefore, we decided to follow this aneurysm with cerebral angiography. we took this course of action because the family did not want the aneurysm to be clipped. in addition, we believed that endovascular treatment carried the risks of both parent artery occlusion and additional hemorrhaging. on the sixth day of hospitalization, follow - up cerebral angiography showed that the aneurysm was still present in the anterior spinal artery branch with a small filling defect in the aneurysm sac (fig. therefore, we decided to continue to follow the aneurysm with cerebral angiography. due to progression of the patient 's end - stage common bile duct cancer, his condition slowly worsened. the angiograms showed no significant interval change in the aneurysm from previous examinations (fig. he returned to his hometown where he died of the cancer on the 103rd day after the sah. both arteriovenous malformations of the spinal arteries and coarctation of the aorta are known to cause hemodynamic stress on the arteries and formation and rupture of aneurysms.3)4)6)7)12)16) however, rupture of an isolated aneurysm in the anterior spinal artery has rarely been reported. to the best of our knowledge, only eight cases of sahs resulting from the rupture of an isolated aneurysm in the cervical anterior spinal artery have been reported.8)9)14)15)17)18 - 20) in our case, the aneurysm was located in the branch of the cervical anterior spinal artery in a patient with common bile duct cancer. to the best of our knowledge, this is the first case in which an sah resulted from the rupture of an aneurysm that was located in the branch of the anterior spinal artery in a patient with common bile duct cancer. the strategy for treatment of spinal artery aneurysms remains controversial. among the eight reported cases of ruptured anterior spinal artery aneurysms, two cases were treated conservatively,8)20) and one of these two cases resulted in death.8) a review of the literature identified three cases of aneurysms located in the cervical radicular,5)11)21) radiculomedullary, or posterior spinal artery, which resulted in death among five conservatively treated cases.1)5)10)11)21) in contrast, all patients who were treated with embolization, surgical resection, or aneurysm clipping recovered completely or with some sequelae. thus, we agree with the opinion that surgical or endovascular intervention is the ideal treatment of choice for a ruptured cervical anterior spinal artery aneurysm.8) however, conservative medical treatment has been successful in some cases. in the case of a patient with a small, ruptured aneurysm measuring approximately 2.5 1.3 mm in size and located in the anterior spinal artery at the first cervical vertebra level, the aneurysm disappeared from the angiograms after 108 days of conservative management. the authors chose conservative treatment because they thought that obliteration of the aneurysm would be difficult without disrupting the blood flow of the parent artery. due to its spontaneous disappearance, the authors assumed that the aneurysm was a dissecting aneurysm.8) in a second case, a ruptured aneurysm measuring approximately 3 2 mm in size and located at the c5 - 6 radiculomedullary artery was found in a patient with behet 's disease ; a follow - up angiography one month later showed no aneurysm following high - dose methyl - prednisolone therapy, which was initiated on the day of admission.1) another case report described a ruptured aneurysm in a patient with sjgren 's syndrome. the small saccular aneurysm, which was located in a radiculomedullary branch of the right vertebral artery at the c3 level, had a diameter of approximately 2 mm. the authors presumed that the aneurysm had formed due to the immunological vasculitis that is associated with sjgren 's syndrome and treated the patient with glucocorticoids, cyclophosphamide and azathioprine. the aneurysm was not detected during digital subtraction angiography one year after the sah.10) in addition, longatti. considered the mass effect of aneurysms or blood clots as the only indication for surgical treatment.13) therefore, conservative medical treatment can be another management strategy when the aneurysm is associated with dissection or immunological vasculitis, as in behet 's disease and sjgren 's syndrome. this may be particularly true if the parent artery can not be preserved, the patient 's condition is not suitable for surgical treatment, or endovascular treatment poses more hazards than benefits. spontaneous disappearance of an aneurysm between one month and one year after the sah has been reported. thus, waiting at least one year for spontaneous occlusion of the aneurysm to occur is possible.1)2) we could not confirm the disappearance of the anterior spinal artery aneurysm, however, the aneurysm did not re - rupture during the 103 days after the sah. in addition, the caliber of the parent artery of the aneurysm can be considered too small to make a true saccular aneurysm. thus, the author believes that the aneurysm in this case may have been a dissecting aneurysm. an aneurysm rupture at the cervical spinal level results in development of intracranial hemorrhages as well as spinal sahs. recurrent hemorrhage has been the cause of death in all patients who have received conservative treatment, and rebleeding occurred from the day of admission to the 17th hospital day.11)21) thus, an operative intervention should be performed immediately, if indicated, as in cases of intracranial aneurysm rupture. an patient with end - stage cancer with an sah due to the rupture of an aneurysm in the branch of the anterior spinal artery received conservative management. this expectative strategy was chosen because his family did not want open surgical management and endovascular treatment might carry the risk of parent artery occlusion and injury. the patient survived for 103 days after the sah without a re - rupture of the aneurysm. a conservative management strategy is a viable option for patients with conditions that are unsuitable for surgical or endovascular management. | incidence of aneurysm of the anterior spinal artery is known to be very low and the standard treatment strategy has not yet been established. the author experienced a case of subarachnoid hemorrhage (sah) caused by the rupture of an aneurysm in the branch of the cervical anterior spinal artery, which was managed conservatively. the patient had end - stage common bile duct cancer and survived for 103 days after onset of the sah without a re - rupture of the aneurysm. |
the potential to respond to environmental stimuli through dynamic rearrangements of synapto - dendritic networks, as well as by regulating the generation of new neuronal and glial cells, renders the brain highly mutable. these phenomena, collectively known as neuroplasticity, are critical to promote neuronal adaptations ; its failure is now increasingly considered to be a major component in many neuropsychiatric conditions. among these, depressive spectrum disorders are a paradigmatic example of the importance of neuroplastic alterations in the adult brain. recent studies provide a comprehensive picture of the effects of stress, a major trigger factor in depression, in the (de)regulation of neuroplasticity ; the latter is, in turn, related to the emergence of physiological and behavioral alterations comprised in the symptomatic profile of depressive disorders. although these molecular and physiological mechanisms regulating neuroplastic processes are relevant for the onset of depressive symptoms, they have also been implicated in the action of antidepressants (ads). so far, and although there is still much to be elucidated, it is becoming evident that the triad stress - neuroplasticity - depression constitutes fertile ground for new findings. although different forms of neuroplasticity are affected in depression, a debate endures concerning the exact neurobiological significance of postnatal hippocampal cell genesis, both for the development of depressive pathology and for the therapeutic action of ads. from the bulk of evidence gathered so far, it is increasingly appreciated that alterations in cell genesis are involved in the pathology and treatment of depression ; however, there are several conflicting reports regarding its relevance. first, there is a necessary ' difficulty to approach this question in humans suffering from depression ; postmortem studies in humans and animal models of depression have, nevertheless, provided important insights. second, it seems to exist a major prevalence of studies focusing on the functional implications of neurogenesis, in disregard of gliogenesis, a parallel cell - genesis process likely to be of relevance in this context. lastly, because these events are highly dynamic, the adoption of different experimental models and time frames when analyzing the participation of cell genesis in the pathology and treatment of depression is critical to have a complete perspective of the topic. on account of these experimental dissimilarities, an integrative, and careful, interpretation of data published in the last years suppression of hippocampal cell proliferation in naive animals through irradiation, pharmacological approaches or through the use of transgenic models of cytogenesis ablation has been shown to be associated with the development of deficits in different behavioral dimensions commonly affected in depression. strikingly, most of the studies in which analyses were performed shortly after cytogenesis ablation did not reveal significant deficits in most behavioral domains normally assessed in the characterization of animal models of depression (figure 1). however, recent reports in which abrogation of cytogenesis is maintained for long periods (over 4 weeks) or in which the behavioral analysis was conducted only 4 weeks after the cessation of cytogenesis suppression, reported multidimensional behavioral deficits that emerged only weeks after the antiproliferative insult. importantly, the specific late manifestation of depressive - like behavior and cognitive disabilities in animals in which cytogenesis had been suppressed illustrates how manipulating lengthy neuroplastic phenomena is associated with the non - immediate development of behavioral impairments, which are only fully manifested once newborn cells are expected to be incorporated in local neuroglial circuits. this view has been recently supported by the demonstration that the specific inhibition of 4-week old new hippocampal neurons causes deficits in memory retrieval in mice ; remarkably, inhibiting the activity of either younger or less - plastic older neurons does not produce effects in this cognitive domain. an exception must be made in respect to anxiety behavior, because disruption of hippocampal cytogenesis is associated to the immediate development of heightened anxiety, which is commonly comorbid in depressed patients. in fact, it has been demonstrated that immature newborn neurons display a major role in anxiety behavior control. in contrast with the slow reconfiguration of neuroglial networks promoted by the addition of new cells in the adult brain, are the rapid synaptic and dendritic morphological changes. these underlie the short - term impacts on distinct emotional and cognitive processes observed in the onset of depressive symptoms. indeed, defects on neuronal cytoarchitecture have been documented in postmortem analysis of brain tissue from depressive patients, which seem to be ameliorated by chronic ad treatment. these structural defects include cortico - limbic dendritic atrophy in depressed subjects, accompanied by abnormalities in glial cell structure. importantly, in animal models of depression, these changes are associated with hallmarks of depressive behavior, such as anhedonic behavior, behavioral despair and cognitive disabilities. besides this common participation of slow cytogenesis processes and rapid dendritic rearrangements in the pathology of depressive spectrum disorders, these two neuroplastic phenomena, with very different temporal dynamics, constitute the substrate by which ads (partially) exert their therapeutic actions. thus, the reestablishment of normal neuroglial networks seems to be achieved in a biphasic manner (figure 1) : in a short - term context, ad actions rely on rapid modulatory effects upon genes involved in the restructuring of the synaptic network ; later on, the generation of new fully matured neuronal and glial cells will have an impact on the long - term remission from emotional and cognitive disabilities manifested during a depressive episode. in fact, despite triggering an immediate pro - proliferative response, this early effect corresponds only to the onset of a slow neuroadaptation whose neurobiological importance can only be fully appreciated later on, once new cells attain complete maturation and functionality, and are integrated in the local neurocircuitry. taken together, and because mammalian neurogenesis is described to take 46 weeks, the overall outcome of ad 's therapeutic action upon neuroplasticity may only be entirely manifested after this period. remarkably, this period correlates with the time latency that typically prescribed ads take to fully manifest their action in depressive patients. although the neuroplastic alterations occurring during the onset, treatment and remission from depression are being increasingly characterized, comprehension of the processes (namely genetic and epigenetic programs) that orchestrate these alterations is still limited. future research focusing on these processes should also be extended to the still underexplored glioplastic component of this disorder. furthermore, local neuroplastic adaptations are likely to occur in articulation with systemic neuroendocrine and immunological alterations, which are still to be integrated in the complex puzzle of mechanisms implicated in depression. | brain neuroplasticity is increasingly considered to be an important component of both the pathology and treatment of depressive spectrum disorders. recent studies shed light on the relevance of hippocampal cell genesis and cortico - limbic dendritic plasticity for the development and remission from depressive - like behavior. however, the neurobiological significance of neuroplastic phenomena in this context is still controversial. here we summarize recent developments in this topic and propose an integrative interpretation of data gathered so far. |
measurement of muscle strength in rehabilitation is important for the evaluation of treatment outcomes. simple manual muscle testing can be performed, or a measuring device can be used for quantitative measurement of muscle strength. in particular, a hand - held dynamometer (hhd) is a relatively inexpensive and very portable instrument, and has been used in a variety of situations for measuring muscle strength. previous reports have demonstrated the high reliability of measuring muscle strength with an hhd1, 2. however, it has also been reported that the results of measuring muscle strength with an hhd are influenced by the position of the testee s lower extremity, fixation of the device, and the skill of the examiner3,4,5,6,7. for improving reliability, a strap or a fixing frame has been used to make fixation holding the measuring device by hand8, and both methods provide high reliability. also, using an hhd attached with a belt has been reported to be high reliable, overcoming the problem of examiners skills9,10,11,12. in terms of measurement modes, it has been reported that the break test requires greater strength of the examiner to resist the strength of the examinee than the make test13,14,15. furthermore, the reliability of the results of the break test has been reported to be low16. when measuring muscle strength with an hhd attached by a belt, muscle strength is measured by pushing a part of the body against the sensor pad. in measurement of isometric knee extension strength, a sensor pad is attached to the front of the distal lower extremity to measure strength. some patients complain of pain and discomfort at the distal lower extremity when this method is used, and the pain becomes a limiting factor, preventing the exertion of full muscle strength, particularly in cases of high muscle strength values. when the thickness and shape of the cushion of the sensor pad are changed for the measurement of isometric knee extension strength using an hhd attached by a belt, values of muscle strength are reproducible, but muscle strength values are strongly influenced by the pain experienced at the time of measurement. therefore, to improve the appropriateness of measured values of muscle strength, it may be necessary to use a pad of sufficient thickness with an appropriate curve to eliminate pain. in rehabilitation, the muscle strength of elderly individuals and subjects with reduced muscle mass is often measured. compared with healthy adults, soft tissue at the front of the distal lower extremity is often thin, and, accordingly, the measurement procedure can be painful. therefore, in measurement of isometric knee extension strength using an hhd with attachment of a fixation belt, pain at the front of the distal lower extremity remains a problem. to overcome this problem, mobie, a new device for measuring muscle strength using a pull sensor, rather than pushing a limb against the sensor, was developed. the intra - class correlation coefficient (icc) is used as a statistical analysis method of reliability. however, in cases of data with large individual differences and measurements with marked variability, inter - examiner differences and errors become relatively small and the coefficient becomes large. recently, the reliability of an hhd was examined using standard error of measurement (sem) and minimal detectable change (mdc) in addition to icc17,18,19,20,21,22. there are random errors and systematic errors, and the former can be reduced by increasing the number of subjects and repetition of measurements, whereas the latter is an error in which measurements deviate with a specific bias, and it can not be minimized by increasing the number of subjects or measurements. therefore, it is necessary to extract systematic errors. systematic errors are proportional and constant. proportional errors increase or decrease with the true value with a specifi bias, whereas constant errors have aspecific bias irrespective of the true value. systematic errors can be identified using bland - altman analysis23, and using the degree of variability between two measurements, it is possible to determine the reliability of the measurement and its appropriateness for clinical application. in this study intra - examiner reliability and inter - examiner reliability of an hhd with a pull sensor the subjects were 54 healthy adults (35 males and 19 females ; mean age, 23.26.8 years ; height, 166.38.4 cm ; weight, 60.012.6 kg) (table 1table 1.the characteristics of the subjects (n=54)age (yrs) 23.2 6.8height (cm) 166.3 8.4 weight (kg) 60.0 12.6bmi (kg / m) 21.6 4.1 data are means sd). the 35 subjects (21 males and 14 females) in their 20s were examined the most. the aims and contents of the study permission to conduct this study was provided by the human research ethics committee of tokyo college of allied medicine physical therapy. a manual muscle strength monitor (mobie ; sakai medical co., ltd.) mobie is an hhd with a thin sensor. unlike conventional hhds that measure force by pushing the body against the sensor, this device senses muscle strength by pulling a distortion gauge, and muscle strength can be measured with a fixation belt attached. to examine inter - examiner differences in the mobie hhd measurements, measurements were performed by two examiners : one female physical therapist (pt) (examiner a) with 21 years of clinical experience and skill in handling hhds, and one male third - grade student on a four - year physical therapy course (examiner b). he practiced the method of the measurement that had defended notes the day before the measurements began. on the first day, examiner a and examiner b measured one leg of each subject, and the next day they measured the other leg. for all measurements, isometric knee extension was performed at maximum effort for approximately 3 seconds. for the measurements, the subjects sat on the edge of a bed with their feet not touching the floor, and their arms crossed in front of the body. subjects were asked ; to maintain the trunk in the upright position ; to keep the buttocks on the bed ; and to exert muscle power to extend the knee joint without a swing movement. prior to measurement, an appropriate posture for measurement was explored. since the muscle to be measured is the femoral quadriceps, the occurrence of compensatory movement due to exertion of muscle power in isometric knee extension was examined in two postures : supporting both arms at the back of the bed with the pelvic tilted backward and the trunk in extension, similar to the posture for manual muscle testing ; and with both arms crossed in front of the trunk. elevation of the pelvis was more likely to appear in the posture similar to that for manual muscle testing than in the posture with both arms crossed in front of the trunk, and it was necessary for the examiner to fix both sides of the pelvis manually in addition to controlling the device to block this compensaory movement. therefore, considering rapid and simple measurement with blocking of compensaory movement in clinical practice, the posture with both arms crossed in front of the trunk was selected for this study. the pad of the measuring device was attached around the distal lower extremity and controlled with a belt fastened to the examination pole so that the lower extremity was perpendicular to the floor. the sensor was set parallel to the floor with the fixation belt, and positioned midway between the lower extremity and the examination bed. since this measuring device senses tension, a hand of the examiner supported to the sensor and to prevent gravity - derived downward tension during calibration and measurement. in addition, to avoid popliteal pain by compression measurement, a bath towel was spread over the site. icc was calculated based on the muscle strengths measured by examiners a and b, and intra - examiner reliability and inter - examiner reliability were examined using bland - altman analysis. when no systematic error was recognized, the mdc of the 95% confidence interval (mdc95) was calculated. mdc95 and sem were calculated according to the following formula.mdc95 = sem 1.96.sem = standard deviation of difference between two measurements (s) /. inter - examiner reliability was examined with icc (1,1) and (1,3) over the three measurements. the first and second measured values were examined bilaterally using bland - altman analysis. for inter - examiner reliability, icc (2,1) and icc (3,1) were calculated to examine both the random effect and the fixation effect, respectively. in both intra - examiner reliability and inter - examiner reliability, systematic errors and the limit of agreement (loa), the permissible range of errors between two measurements, were calculated. loa was calculated according to the following formula.[(d 1.96s) + tse loa ] ~ [(d + 1.96s) tse loa]d : average of difference between two measurements se loa= t : t value for the degree of freedom n 1 for data analysis, ibm spss statistics 21 was used. table 2table 2.the average values of the measured strengtha examiner (kgf) b examiner (kgf) right (average 3 times) 39.4 10.6 39.5 11.2 left (average 3 times) 39.4 10.6 39.2 9.9 right 1 test39.0 10.739.1 11.62 test 39.5 10.4 39.3 11.13 test 39.9 10.8 40.2 11.1 left 1 test38.5 10.639.3 9.62 test 39.5 10.539.1 10.33 test 40.2 10.739.3 10.1data are means sd shows the averages of muscle strength as measured by examiner a and examiner b. the average right muscle strength was 45.6 9.0 kgf in males and 28.2 3.8 kgf in females by examiner a, and 45.9 8.4 kgf in males and 27.9 4.9 kgf in females by examiner b. with regard to intra - examiner reliability, both icc (1,1) and icc (1,3) were 0.9 or higher (table 3table 3.the intra - examiner reliabilities of the measured strengthicc(1,1) icc(1,3) loa bland - altman analysisadding errorproportional error95% confidence intervalpresence or absence the slope of the regression linepresence or absence examiner0.960.993.97 1.05absence 0.14absence a right to 3.17 to 0.26examiner0.940.986.50 2.01absence 0.06absence a left to 4.5 to 0.01examiner0.950.985.951.34absence 0.16absence a right to 5.35 to 0.74examiner0.940.984.861.13absence 0.21absence a left to 5.06 to 0.75). bland - altman analysis indicated there were no constant or proportional errors by examiner a or examiner b (table 3). the loa of the right extremity was 3.97 to 3.17 by examiner a and 5.95 to 5.35 by examiner b, while the loa of the left extremity was 6.50 to 4.50 by examiner a and 4.86 to 5.06 by examiner b. since no systematic error was found, mdc95 of the right and left extremities were 4.7 kgf and 7.3 kgf, respectively, by examiner a, and 7.5 kgf and 6.7 kgf, respectively, by examiner b. these results indicate that when muscle strength was measured twice in the same subject, mdc95 within 4.7 kgf for the right extremity by examiner a was considered a measurement error and mdc95 more than 4.7 kgf was judged as true change caused by intervention for the subject. in terms of inter - examiner reliability, both icc (2,1) and icc (3,1) in the first through the third measurements were 0.9 or higher for the right extremity and 0.8 or higher for the left extremity (table 4table 4.the intra - examiner reliabilities of the measured strengthicc(2,1)icc(3,1)loa bland - altman analysisadding errorproportional error95% confidence intervalpresenceor absence the slope of the regression linepresenceor absence a right 0.920.929.02 1.74absence 0.17absence 1 testto 14.50to 1.522 test0.920.928.53 1.48absence 0.13absence to 14.21to 1.683 test0.90 0.90 10.262.13absence 0.04absence to 16.0to 1.53a left 0.890.90 10.172.52absence 0.17absence 1 testto 14.53to 0.922 test0.90 0.90 8.971.13absence 0.02absence to 15.73to 0.753 test0.880.889.311.06absence 0.1absence to 17.34to 2.66). in the first through the third measurements, neither constant nor proportional errors were identified by bland - altman analysis (table 4). no subject complained of pain due to the pad attached around the lower extremity while muscle power was being exerted. to investigate intra - examiner and inter - examiner reliability of isometric muscle strength as measured using the mobie hhd with a pull sensor, measurement was repeated three times by a pt with clinical experience and a pt student without clinical experience. previously, we reported that for subjects in their 20s24, which accounted for the largest proportion of subjects, isometric knee extension strength of males and females was 60.4 8.1 kgf and 37.1 8.9 kgf, respectively. compared with these results, the muscle strength of the subjects in this study was slightly weaker. therefore, compensaory movement was unlikely to have occurrde and both intra - examiner variability and inter - examiner variability in measurements were smaller, and systematic errors were unlikely to have occurred. with regard to intra - examiner reliability, icc (1,1) was 0.94 to 0.96, and the reliability was judged as no systematic error was found, indicating that there is no problem with intra - examiner reliability. when icc (1,1) and icc (1,3) were compared, icc (1,3) was slightly higher showing that reliability was higher, as expected, when the same examiner carried out the same measurement more than once. with regard to inter - examiner reliability, both icc (2,1) and icc (3,1) were 0.88 to 0.92, and the reliability was considered icc (2,1) and icc (3,1) were slightly reduced as measurement was repeated from the first through the third time, and the variability was attributed to fatigue. however, since icc ranged between 0.88 and 0.92 and no constant or proportional errors were found, there was no problem inter - examiner reliability. from the study of examiner a with clinical experience and skill in handling hhds, and those by examiner b, a pt student with no experience of measuring muscle strength with hhd, the examiner can carry out measurement even if he / she has no experience on the operation of the device as long as he / she gives the subjects the notification at measurement and measures according to the predetermined procedure. although pain remains a problem in muscle strength measurement with an hhd and a fixation belt, the device used in this study caused no pain and had no influence on the muscle power exerted. therefore, when knee extension strength is measured using an hhd with a pull sensor, the measurement is accurate and, since the subjects experienced no pain while exerting muscle power, measurement is possible without inducing pain in cases of thin soft tissue at the front of distal lower extremities in frail elderly people. moreover, quantitative muscle strength measurement is expected to be possible in a variety of subjects. in this study, the subjects were healthy adults, but compensatory movement is likely during the exertion of muscle power by subjects with high muscle power levels such as athletes. however, compensatory movement is unlikely to occur in the elderly. whether knee joint extension without a swing movement is possible or not influences the measurement errors of the measuring device. it will be necessary to further investigate the utility of measurements with a pull - type hhd using subjects with different muscle power levels, elderly people, and patients. | [purpose ] the purpose of this study was to examine the intra - examiner and inter - examiner reliabilities of measurements of knee extensor muscle strength using a pull - type hand - held dynamometer (hhd). [subjects ] fifty - four healthy adults (35 males ; average age, 23 years) participated in this study. [methods ] knee extensor muscle strength of each leg was measured three times using the hhd. to examine the intra- and inter - examiner reliabilities, measurements were performed by two examiners, a physical therapist and a physical therapy student. [results ] the intra - examiner reliabilities, icc (1, 1) and icc (1, 3) ranged from 0.940.99. the inter - examiner reliabilities, icc (2, 1) and icc (3, 1) ranged from 0.900.92 for the right leg, and 0.880.90 for the left leg. neither constant nor proportional errors were found by bland - altman analysis. [conclusion ] intra - examiner and inter - examiner reliabilities were acceptable, indicating that muscle strength can be measured with the pull - type hhd without dependence on skill of measurement. pain was not caused by measurements with the pull - type hhd. |
during xenopus gastrulation, mesoderm migrates to the inside of the embryo and moves along the blastocoel roof to establish the three germ layer structure. this process involves several morphogenetic cell movements including mesendoderm extension and convergent extension. during mesendoderm extension, cells migrate along the blastocoel roof in contact with fibronectin (fn) fibrils (winklbauer, 1990 ; davidson., 2002). in convergent extension, cells are polarized and elongated mediolaterally, then the cells are intercalated. this movement forms the dorsal mesodermal structure and extends the anteroposterior body axis (shih and keller, 1992 ; wallingford., 2002). the noncanonical wnt pathway has been implicated in the regulation of convergent extension (kuhl, 2002 ; tada., one of the intracellular signaling components, xenopus dishevelled (xdsh), plays a pivotal role in this process. when the function of xdsh is inhibited, the polarity of the mesodermal cells is not established normally (wallingford., 2000). because these cell movements are accompanied by dynamic changes in cell polarity, morphology, and motility, it is very likely that cytoskeletal dynamics are carefully regulated. we decided to focus on myristoylated alanine - rich c kinase substrate (marcks). mammalian marcks has been shown to interact with actin (arbuzova., 2002). it has been reported that xenopus marcks is expressed maternally and throughout embryogenesis (ali., 1997 ; 1997), but its role in development was not well understood. here, we report that the loss of marcks function severely impaired gastrulation movements. marcks regulates the cortical actin formation, cell adhesion, protrusive activity, and cell polarity control during gastrulation. we further show that marcks is necessary for the protrusive activity regulated by the noncanonical wnt pathway. these findings show that marcks regulates the cortical actin formation that is requisite for dynamic morphogenetic movements. to investigate the function of marcks in xenopus development, we conducted loss of function experiments using antisense morpholino oligonucleotides (mo). first, we examined the specificity of marcks mo (fig. the mo specifically and effectively inhibited epitope - tagged marcks protein synthesis, leading us to expect that it could inhibit the endogenous marcks protein synthesis. using marcks mo, we analyzed marcks function in development. when it was injected into the dorsal marginal zone (dmz) of four - cell embryos, the phenotype of marcks mo was partially rescued by coinjection of marcks mrna (fig. as discussed below, however, cell biological effects of marcks mo were efficiently rescued by marcks mrna. over- and under - expression of marcks may have opposite effects at a cellular level, but both of these effects may negatively influence gastrulation movements. (a) both 500 pg of marcks mrna and 5 pmol of marcks mo impaired gastrulation movements, when either was injected into the dorsal marginal region. (b) statistical data of the gastrulation - defective phenotype caused by marcks mrna and mo. (c) expression of chordin at the gastrula stage, detected by in situ hybridization. (d) somites (left) and notochord (right) were immunostained with 12/101 and mz15 antibodies, respectively. (e) 5 pmol of marcks mo was injected into the two dorsal blastomeres at the four - cell stage ; the dmz explants were isolated, and the expression of mesodermal markers was detected by rt - pcr. (f) 2 pmol of marcks mo inhibited the activin mrna - induced elongation of animal caps. it has been reported that marcks - like protein (xmlp) is also expressed in xenopus embryo (zhao., 2001). although xmlp is similar to marcks (23% amino acid identity), xmlp - mo injected embryos showed malformations of the anterior axis and eye defect, but the gastrulation defect was not reported (zhao., 2001). they seem to play distinct roles in xenopus development. to determine whether this gastrulation defect was caused by a defect in mesodermal differentiation, we examined the expression of the dorsal mesodermal markers. at the gastrula stage, marcks mo - injected embryos expressed chordin at the same level as control embryos (fig. the notochord and somites were formed in the marcks mo - injected embryos, but the extension of these tissues was severely inhibited (fig. we also tested the expression of the mesodermal markers in dmz explants by rt - pcr (fig. these results indicated that the phenotype was caused, not by a defect in mesoderm differentiation, but by a defect in morphogenetic movements. next, we tested whether the loss of marcks function affects the animal cap elongation, which mimics convergent extension movements during gastrulation (fig. 1 f). marcks mo blocked the elongation by activin, and it was rescued by coinjecting marcks mrna without the utr, suggesting that marcks is required for convergent extension. during mesodermal convergent extension, the cells become polarized, align mediolaterally, and are then intercalated. to test how marcks is involved in this process, the convergent extension in dmz explants was observed microscopically. marcks mo, rhodamine dextran, and mrna encoding membrane - binding venus (mb - venus) were coinjected into one of the two dorsal blastomeres (fig. mb - venus mrna alone was injected into the other dorsal blastomere. at the gastrula stage, these explants adhered to the fn, and convergent extension movements occurred subsequently in the mesoderm (kinoshita. in the absence of marcks mo, red and nonred cells were polarized and intercalated. in the marcks mo - injected explants, the nonred cells, which were assumed to lack the mo, were polarized and showed convergent extension. in contrast, the red cells (mo - injected cells) were not polarized and did not participate in the intercalation. (a) 5 pmol of marcks mo, rhodamine dextran, and the mrna for 100 pg of mb - venus were coinjected into one of the two dorsal blastomeres at the four - cell stage. dmz explants were cultured on a cover glass coated with fn, and convergent extension movements were observed. (b) control or 5 pmol of marcks mo was coinjected with 100 pg of venus mrna into two blastomeres of four - cell embryos. dmz explants were cultured on an fn - coated dish until sibling embryos reached the late neurula stage. marcks mo, control mo, venus mrnas (green), and rhodamine dextran (red) were coinjected dorsally as indicated. cells from the control- and marcks - mo injected explants were mixed, plated on fn - coated dishes, incubated for 6 h, and fixed in formaldehyde. cells that did not adhere to the dish were removed by washing five times with pbs. (d) 3 pmol of marcks mo inhibited the protrusive activity of cells in dmz explants. dmz explants were cultured on an fn - coated dish until sibling embryos reached the early neurula stage. in addition to convergent extension, an important mechanism regulating gastrulation movements is mesendoderm extension (davidson., 2002). to test whether marcks is required for this process, dmz explants were cultured on fn - coated dishes according to the method developed by davidson. 2 b). when venus mrna and the control mo were coinjected, the venus - expressing cells dispersed broadly, and some cells migrated to the front. we examined 15 explants and confirmed that none of the marcks mo - injected cells reached the leading edge of the migrating mesendoderm. we examined whether marcks mo affects the adhesion to fn of cells dissociated from the dmz explants. marcks mo and venus mrna (green) or rhodamine dextran (red) was injected dorsally (fig. 2 c). dissociated cells were cultured on fn - coated dishes, and cells that adhered to the dish were counted (fig. 2 c). when marcks mo was coinjected with venus, the adherence of venus - expressing cells was extremely reduced. in contrast, when marcks mo was coinjected with rhodamine dextran, these red cells rarely adhered to the dish. a few cells containing marcks mo were found on the dish, but these cells were rounded up and did not spread out on the dish. the effect of marcks mo on the cell adhesion was rescued by coinjection with marcks mrna (unpublished data). mesodermal cells had many filopodia - like protrusions when the dmz explants adhered to a fn - coated dish (fig. the effect of marcks mo on the protrusive activity was rescued by coinjection with marcks mrna. thus, marcks is required for the protrusive activity, which may directly correlate with the control of cell adhesion and motility. the inhibition of cell adhesion and migration on the fn fibrils that cover the blastocoel roof may contribute to the gastrulation defect caused by marcks mo. the actin - binding domain of mammalian marcks binds to actin filaments and cross - links them in vitro (hartwig., 1992). cells expressing marcks - venus were dissociated from dmz explants and cultured on an fn - coated dish. the cells were then fixed and f - actin was stained with phalloidin. as shown in fig. (a) cells expressing 200 pg of marcks - venus mrna were dissociated from dmz explants and plated on a fn - coated cover glass. (b) wild - type, ga, and sd mutants were expressed in the dmz explants and observed. (c) the actin - binding domain of xenopus moesin was fused with venus (venus - actin bd) and expressed in cho cells. (d) mo was injected into one blastomere of two - cell embryo with mb - rfp mrna as a tracer. (e) western blot with an antiactin antibody (left) and coomassie blue (cbb) staining (right). (f) marcks mo and mb - rfp were coinjected and animal cap cells were immunostained with antiactin antibody. ga is an unmyristoylated mutant in which the second glycine residue is replaced with alanine. sd is a pseudophosphorylation mutant whose potential phosphorylation sites were replaced with aspartic acid, which is expected not to bind to actin filaments (hartwig., 1992). to detect f - actin, we used the f - actin binding domain of moesin fused to red fluorescent protein (rfp ; campbell., 2002), designated rma (rfp - moesin actin binding domain). it has been shown biochemically that this domain binds to f - actin (turunen., 1994 ;, the corresponding domain of moesin fused with gfp was successfully used to analyze actin dynamics (dutta., 2002). we confirmed that our construct (venus - moesin actin - binding domain) colocalized with stress fibers and cortical actin stained with phalloidin in cho cells (fig. embryonic cells, the rma was localized to the cell cortices, and cytochalasin b treatment, which disrupts actin filaments, dispersed the rma to the cytoplasm (fig. when these marcks - venus genes were expressed, the wild - type and ga forms were associated with the plasma membrane and colocalized with rma, but sd was in the cytoplasm. when the cells were treated with cytochalasin b, ga dispersed to the cytosol with rma, whereas wild - type remained on the membrane. this result suggested that the association of marcks with the membrane was regulated by two mechanisms, myristoylation and binding to the cortical actin. we also found that ga and sd mutants did not inhibit gastrulation movement when they were overexpressed (fig. these results suggest that both myristoylation and actin biding are required for its function. to test whether marcks regulates cortical actin formation, marcks mo animal caps were isolated at the late blastula stage, fixed, and stained with phalloidin. 3 d, marcks mo significantly reduced the amount of cortical actin stained by phalloidin. the amount of actin protein was not affected, however, judging from western blotting and immunocytochemistry using an antipan actin antibody (fig. it has been demonstrated that xdsh, an essential cytoplasmic component in this pathway, regulates cell polarity and protrusive activity in dmz cells (wallingford., 2000). the wnt pathway activates rhoa and rac (habas., 2001, 2003), which have been shown to regulate the protrusive activity (tahinci and symes, 2003). thus, the pathway may directly regulate actin cytoskeletal dynamics. to investigate the relationship between the wnt pathway and cortical actin, we examined the localization of xdsh. cells were dissociated from the dmz explants expressing xdsh - venus, cultured on an fn - coated dish, and stained with phalloidin. as shown in fig. 4 a, xdsh was colocalized with cortical actin, even in the lamellipodial and filopodial protrusions. marcks is required for the cortical actin dynamics regulated by the noncanonical wnt signaling pathway. (a) 250 pg of xdsh - venus mrna was expressed in dmz explants. (b) 100 pg of xdshdix - venus mrna was expressed in dmz explants. (c) xwnt11 and xfz7 mrnas (200 pg each) were coexpressed in animal cap explants with mb - rfp. the effect of marcks mo was rescued by coinjection of 200 pg of marcks mrna. (d) mb - rfp was injected with or without mrna encoding dominant - negative xwnt-11 (2 ng). when rma was expressed during convergent extension, it was located at the tips of elongated mesodermal cells (fig. 4 b). we showed previously that xdsh - venus was also accumulated in the same region (kinoshita., 2003). mammalian dishevelled interacts with actin filament through the nh2-terminal dix domain (capelluto., whether the tip localization of xdsh was due to the interaction between the dix domain and f - actin, we tested the localization of xdsh lacking the dix - domain (xdshdix). 4 b, the xdshdix was located at the tip, indicating that this localization is not due to interaction between f - actin and the dix domain. this result is consistent with the finding that dishevelleddix can mediate the noncanonical wnt signaling in xenopus and zebrafish (heisenberg., 2000 ; the actin depolymerizing reagent, latrunculin a, dispersed both rma and xdshdix to the cytosol. essentially, the same result was also obtained using cytochalasin b (unpublished data). these results strongly suggest that xdsh interacts with f - actin either directly or indirectly and mediates the wnt signaling to the actin cytoskeleton. to examine whether the wnt pathway regulates the protrusive activity, we coexpressed xwnt11 and xfz7 (xenopus frizzled-7) in animal cap explants with the membrane - binding rfp. 4 c, the coexpression of xwnt11 and xfz7 dramatically promoted lamellipodia- and filopodia - like protrusions and it was inhibited by xdd1, a dominant negative xdsh mutant (sokol, 1996 ; wallingford., 2000). when marcks mo was coinjected, this activity was severely inhibited (fig. 4 c). in addition, dorsal mesodermal cells expressing dominant - negative xwnt-11 (tada and smith, 2000) significantly reduced the number of protrusions (fig. 4 d), these results strongly suggest that the wnt signaling pathway regulates cortical actin dynamics and that marcks is required for this process. here, we have shown that marcks plays an essential role in regulating cortical actin dynamics in xenopus development. marcks mo inhibited cell movements, cell shape change, cell adhesion, and interaction with fn probably through the defect it caused in the cortical actin dynamics. marcks is required not only for gastrulation but also for the neural tube formation. when marcks mo was injected into the dorso - anterior blastomeres of eight - cell embryos to target the neuroectoderm, neural tube closure was severely impaired (fig. it has been shown that marcks - deficient mouse shows neural tube closure defect (stumpo. the regulation of the cortical actin cytoskeleton by marcks may be important for a proper cellular response to signals such as wnt and the fn / integrin pathways. pkc has been involved in the noncanonical wnt pathway (sheldahl., 1999) and the integrin pathway (vuori and ruoslahti, 1993). it would be interesting to determine how the activity of marcks is regulated during development. procedures for the plasmid construction, rna synthesis and sequences of morpholino oligos were described in the online supplemental material. the rfp plasmid is a gift from r. tsien (university of california, san diego, ca). in situ hybridization in xenopus was performed as described by harland (1991). for rt - pcr analyses, the procedure for whole - mount immunostaining was performed as described in kurata. the antibodies were mz15 for notochord (a gift from f. watt, imperial cancer research fund, london, uk) and 12/101 for somites western blotting was performed using a mouse monoclonal antipan - actin antibody was purchased from neomarkers (ms-1295-p0). for the animal cap explants, marcks mrna or mo was coinjected with 0.5 pg activin mrna into the animal pole of two - cell embryos. the animal cap was dissected from stage-9 embryos. for dmz explants, mrna or a mo these explants were cultured in 1 steinberg 's solution until sibling embryos reached stage 17. to dissociate cells from the explants, the explants were incubated in the ca - mg free medium for 2 h. for the cytological observation, explants and dissociated cells were cultured in 1 steinberg 's solution on an fn - coated dish (4000030 ; iwakil), or on a cover glass coated with fn (1 g / cm, f1141 ; sigma - aldrich). to stain f - actin, cells were fixed in 4% pfa and stained with pbs 0.5% triton x-100 containing a 40-fold dilution of bodipy 581/589 phalloidin (b-3416 ; molecular probes) or alexa fluor 488 phalloidin (a-12379 ; molecular probes). for confocal microscopy, images were captured using 510 software (carl zeiss microimaging, inc.). procedures for the plasmid construction, rna synthesis and sequences of morpholino oligos were described in the online supplemental material. the rfp plasmid is a gift from r. tsien (university of california, san diego, ca). in situ hybridization in xenopus was performed as described by harland (1991). for rt - pcr analyses, the procedure for whole - mount immunostaining was performed as described in kurata. the antibodies were mz15 for notochord (a gift from f. watt, imperial cancer research fund, london, uk) and 12/101 for somites western blotting was performed using a mouse monoclonal antipan - actin antibody was purchased from neomarkers (ms-1295-p0). for the animal cap explants, marcks mrna or mo was coinjected with 0.5 pg activin mrna into the animal pole of two - cell embryos. the animal cap was dissected from stage-9 embryos. for dmz explants, mrna or a mo these explants were cultured in 1 steinberg 's solution until sibling embryos reached stage 17. to dissociate cells from the explants, the explants were incubated in the ca - mg free medium for 2 h. for the cytological observation, explants and dissociated cells were cultured in 1 steinberg 's solution on an fn - coated dish (4000030 ; iwakil), or on a cover glass coated with fn (1 g / cm, f1141 ; sigma - aldrich). to stain f - actin, cells were fixed in 4% pfa and stained with pbs 0.5% triton x-100 containing a 40-fold dilution of bodipy 581/589 phalloidin (b-3416 ; molecular probes) or alexa fluor 488 phalloidin (a-12379 ; molecular probes). for confocal microscopy, images were captured using 510 software (carl zeiss microimaging, inc.). | myristoylated alanine - rich c kinase substrate (marcks) is an actin - binding, membrane - associated protein expressed during xenopus embryogenesis. we analyzed its function in cytoskeletal regulation during gastrulation. here, we show that blockade of its function impaired morphogenetic movements, including convergent extension. marcks was required for control of cell morphology, motility, adhesion, protrusive activity, and cortical actin formation in embryonic cells. we also demonstrate that the noncanonical wnt pathway promotes the formation of lamellipodia- and filopodia - like protrusions and that marcks is necessary for this activity. these findings show that marcks regulates the cortical actin formation that is requisite for dynamic morphogenetic movements. |
perianal mucinous adenocarcinoma is a rare disease often associated with a long - standing anal fistula, representing approximately 23% of large bowel cancers. the occurrence of a carcinoma in association with a fistula is probably due to chronic inflammation, although the rarity of the condition precludes any definite assumption in regard to the etiologic relationship of the fistula and carcinoma. in most patients, early diagnosis of this disease detection is usually late as the symptoms often initially mimic benign inflammatory conditions of the anorectal region and biopsies fail to reveal the infiltrating carcinoma. therefore a tumor is often found in progress. due to the rarity of this tumor and the lack of sufficient patients for controlled trials, abdominoperineal resection (apr) is advocated in the vast majority of patients to definitively remove the entire lesion and lessen the risk of local recurrence. local resection is sometimes performed, but only in cases where the tumor margins can be confidently excised without damage to the anal sphincters. in order to prevent local recurrence, the apr has to be performed with wide local excision of both perianal soft tissue and the overlying skin. recently locally advanced rectal cancer has been considered an indication for preoperative chemoradiotherapy (crt). preoperative crt for rectal cancer has been shown to increase the probability of tumor resectability and to decrease the rate of local recurrence. radiotherapy alone or combined with chemotherapy can be a curative treatment for squamous cell carcinoma of the anal canal. on the other hand, the role of radiotherapy has not been established for perianal mucinous carcinoma. the aim of this article is to report a rare case of large perianal mucinous adenocarcinoma, arising from an anorectal fistula, successfully resected after preoperative radiotherapy. a 60-year - old man with a 45-year history of perianal fistula who had recently been suffering from mucinous discharge, pain and perianal induration was referred to hospital for treatment. two years before, he had undergone surgery at another institution for incision and drainage of recurrent perianal abscess. physical examination revealed an indurated, ulcerative lesion 7 cm in diameter with an external anal fistula opening (fig. the internal opening of the fistula was indicated at the right side of dentate line. magnetic resonance imaging (mri) demonstrated a large demarcated tumor at the level of the anorectal junction with extension to the right side of the ischiorectal fossa and presacral space (fig. no enlarged pelvic lymph node was observed, nor was there evidence of distant metastasis. neither colonoscopy nor barium enema showed any evidence of mucosal lesion in the colon or rectum. laboratory data showed renal dysfunction caused by pyelonephritis, and the carcinoembryonic antigen serum level was extremely elevated to 40.8 ng / ml (normal < 5 ng / ml). to increase resectability and decrease the rate of local recurrence, preoperative radiotherapy was proposed without chemotherapy because of the renal dysfunction. preoperative radiotherapy was given with 40 gy in 25 fractions for 5 weeks through posterior and bilateral portals. 1c, d) and carcinoembryonic antigen serum level gradually decreased to 13.0 ng / ml. the resected specimen revealed that the tumor had developed in the ischiorectal fossa, but there was no evidence of cancer extension to the mucosal surface of the anal canal and rectum (fig. histological examination of the excised specimen showed foci of mucinous adenocarcinoma involving the lower internal and external anal sphincter muscles, and the resected margins did not show any residual tumor (fig. 3). according to the uicc tnm staging criteria, the treatment outcome with preoperative radiotherapy revealed minimal response (grade 2). perianal mucinous adenocarcinomas arising from anorectal fistulae are rare tumors resulting in severe problems concerning diagnosis and treatment. it is suggested that adenocarcinoma developing in the perianal region is often associated with a long - standing anal fistula. early diagnosis of a perianal tumor is difficult because of the absence of a tumor within the lumen of the bowel and the slow growth of a lesion hidden within the ischioanal fossa. as local excision is inadequate in most cases, apr with wide excision of both ischiorectal fossae and the overlying skin is recommended. on the other hand, radiotherapy alone or combined with chemotherapy can be a curative treatment for squamous cell carcinoma of the anal canal. in the present case, total removal of the tumor without any residual tumor was quite difficult because of the size of the tumor and the fact that it had infiltrated the surrounding tissue. recently, preoperative crt has become common practice for stage ii and iii rectal cancers. it has been well documented that neoadjuvant crt induces tumor regression and downstaging, and therefore increases tumor resectability and r0 resection rate. therefore we performed preoperative radiotherapy to improve the resection rate and decrease the local recurrence rate. after treatment the tumor was markedly shrunk and could be completely resected without skin grafting. this suggests that preoperative radiotherapy with or without chemotherapy is appropriate for treatment of perianal mucinous carcinoma arising from an anorectal fistula if adequate surgical margins can not be ensured. the role of crt in the treatment of perianal mucinous adenocarcinoma has not yet been established. unfortunately there are an insufficient number of reported cases undergoing this therapy to determine the prognosis for individual patients [7, 8 ]. further tests are required to standardize the treatment of preoperative crt for locally advanced perianal mucinous adenocarcinoma arising from an anorectal fistula. in conclusion, if surgical treatment for anorectal fistula is unsuccessful in the long term, mucinous adenocarcinoma should be suspected. the result of this case suggests that preoperative radiotherapy appears to be a valuable alternative treatment for patients with perianal mucinous adenocarcinoma arising from an anorectal fistula. the advantage of this approach can result in downstaging and r0 resection, which prevents local recurrence. | a case of mucinous adenocarcinoma arising on a long - standing anorectal fistula is described. a 60-year - old man with a long history of mucinous discharge, pain and perianal induration underwent a biopsy of the external opening of the fistula that showed a mucinous infiltrating adenocarcinoma. due to the large size of the tumor and the fact that it had extended into the surrounding tissue, preoperative radiotherapy was performed. radiotherapy was given with 40 gy in 25 fractions for 5 weeks through posterior and bilateral portals. after radiotherapy the tumor was markedly shrunk and the serum level of carcinoembryonic antigen was also improved. abdominoperineal resection was performed 8 weeks after the termination of radiotherapy. histological examination of the resected specimen revealed that the invasion of the tumor remained within the sphincter muscle and that no cancer cells were present on the surgical margin. the histological effect of radiotherapy was judged as grade 1b. this treatment can result in downstaging and r0 resection, which also has a possibility to prevent local recurrence. this case suggests that preoperative radiotherapy may play an important role in the definitive treatment of locally advanced perianal mucinous adenocarcinoma. |
however, nearly 10% of abnormal thyroid function was observed according to thyroid function survey using general health checkup system for the adult in japan. this implicates that pregnant women in japan also have higher prevalence of thyroid dysfunction. up to now, there are no large population studies regarding the prevalence of thyroid dysfunction in pregnant women in japan. a well - known complication is low intelligence quotient in the infants born from mothers with overt hypothyroidism. thyroid dysfunctions are also associated with several obstetrical complications such as preeclampsia and placental abruption [710 ]. consequently, there is a question whether the management of thyroid dysfunction could reduce these complications on both mother and child during pregnancy. currently, no consensus has been reached on the universal screening for thyroid function for pregnant women. acog recommended thyroid function screening during pregnancy should be limited to women with symptoms of thyroid disease and those with a history of thyroid disease or other medical conditions associated with it. however, this conventional screening may be insufficient because most of pregnant women that have thyroid dysfunction are asymptomatic and without a history of thyroid disease. therefore, consideration should be given to extend the screening objects including women with possible thyroid disease except for women with symptoms of thyroid disease and those with a history of thyroid disease. here, we conducted a study to see the incidence of thyroid dysfunction in pregnant women having obstetrical or medical complications. if we obtain a high incidence of thyroid dysfunction in some high - risk groups, a more reasonable approach for screening would be provided. this study was undertaken retrospectively and there was no need to be approved by a suitably constituted ethics committee of our institution. we retrospectively reviewed the medical charts of women with obstetrical or medical complications that were admitted to the perinatal center of miyazaki medical association hospital from january 2001 to april 2011. our hospital mainly deals with referral cases in the central part of miyazaki province, japan. the total number of deliveries was 4381 in the study period. in miyazaki province, when antepartum high - risk factors are diagnosed, women are advised to visit high - level perinatal centers where they finally deliver their babies. high - risk factors include prenatal medical complications such as diabetes, obstetric complications such as hypertensive disorders, and fetal complications such as growth restriction. additionally, some emergencies may occur and they are transferred to the 8 high - level centers. as a consequence, 80% of total deliveries (pregnant women without risk) were carried out in private clinics. the rest deliveries with high - risk factors were carried out in 8 high - level centers. pregnant women with the following complications were selected : women with symptoms of thyroid disease and those with a personal history of thyroid disease (thyroid disease), intrauterine growth restriction (iugr), diabetes mellitus (diabetes), hypertension, intrauterine fetal death (iufd), and placental abruption (abruption). the signs and symptoms of hyperthyroidism included tremors, nervousness, insomnia, excessive sweating, heat intolerance, tachycardia, hypertension, and goiter. the signs and symptoms of hypothyroidism included fatigue, muscle cramps, constipation, cold intolerance, and hair loss. the history of thyroid disease was either known hyperthyroidism such as graves ' disease or hypothyroidism such as hashimoto thyroiditis, subacute thyroiditis, and iodine deficiency. iugr was defined as sex - specific birth weight less than the 10th percentile for gestational age according to the japanese standard growth curve for singletons. diabetes was defined as either preexisting diabetes mellitus or gestational diabetes mellitus based on 75 g glucose tolerance test. gestational diabetes mellitus is diagnosed, if one or more of these readings are elevated in this study ; at fasting 100 mg / dl, at 1-hour 180 mg / dl and at 2-hour 150 mg / dl. preeclampsia was diagnosed when hypertension (systolic 140 mmhg or 90 mmhg diastolic) and proteinuria (dipstick 2 +) developed after 20 weeks of gestation. gestational hypertension was diagnosed when hypertension (systolic 140 mmhg or 90 mmhg diastolic) without proteinuria developed after 20 weeks of gestation. chronic hypertension was defined as hypertension diagnosed prior to conception or within the first 20 weeks of pregnancy. placental abruption was determined by the presence of retroplacental hematoma and clinical presentations (any one or combination of genital bleeding, abdominal pain, pregnancy - induced hypertension, premature labor, premature rupture of membrane, iufd, or nonreassuring fetal status). in case of placental abruption with pregnancy - induced hypertension or iufd, a total of 838 pregnancies displaying obstetrical or medical complications were found in the study period. from the 838 pregnancies, we selected the 392 cases that had examined thyroid function test from 1st to 3rd trimesters. they were iugr (n = 115), diabetes (n = 115), hypertension (n = 63), iufd (n = 52), abruption (n = 15), and thyroid disease (n = 32). the test of thyroid function was done in our hospital. serum concentrations of tsh and free t4 (ft4) were measured by electro chemiluminescence immunoassay or chemiluminescent enzyme immunoassay (abbott japan, architect tsh, ft4). mu / l in 3rd trimester) and normal - to - high ft4 (> 0.7 ng / dl). mu / l in 1st trimester, > 3.0 in 2nd and 3rd trimesters) and normal - to - low ft4 (1.8 ng / dl) with normal - to - high tsh was categorized as hyperthyroidism. mu / l) in conjunction with low ft4 (< 0.7 ng / dl) or high tsh (10.0 mu / l) in conjunction with high ft4 (1.8 ng / dl). the alterations in thyroid function during pregnancy can pose challenges to the interpretation of laboratory thyroid tests. therefore, we used trimester - specific reference intervals for thyroid function, following the guidelines of the american thyroid association. the following clinical characteristics were also collected : maternal age, parity, gestational age at examination of thyroid function (weeks), gestational age at delivery (weeks), birth weight (g), and cesarean delivery. perinatal outcomes were investigated and included evaluations of umbilical artery ph (ua ph) and neonatal death (nd). the incidence of thyroid dysfunctions including hyperthyroidism or hypothyroidism was examined and compared among the groups of iugr, diabetes, hypertension, iufd, abruption, and thyroid disease. data are expressed as number, incidence (%), or mean sd. probability values < 0.05 were considered significant. in the 392 women, the mean gestational age at delivery was 34.3 6.3 weeks (table 1). the incidence of thyroid dysfunction of each group is as follows : 31% in thyroid disease, 25% in iugr, 30% in diabetes, 27% in hypertension, 12% in iufd, and 7% in abruption (table 2). the incidence of thyroid dysfunction was statistically insignificant in iugr, diabetes, ht, and abruption. the group of thyroid disease accounted for only 10% of all thyroid dysfunctions (figure 2). hypothyroidism was more prevalent than hyperthyroidism in the study groups except for the thyroid disease group. 26 cases of subclinical hyperthyroidism and 55 cases of subclinical hypothyroidism were found in the study groups (table 2). the incidences of thyroid dysfunction in the diabetes (30%) and thyroid disease (31%) were slightly higher than those of other groups as previously reported [17, 18 ]. on the other hand, the incidences of thyroid dysfunction in iugr (25%) and hypertension (27%) were also high. besides, this study showed that the screening for women with thyroid disease (symptoms of thyroid disease and personal history of thyroid disease) could pick up only 10% of affected women. in other words, it would miss 90% of affected women with thyroid dysfunction (figure 1). therefore, current screening strategy for thyroid dysfunction during pregnancy is not sufficient. in our study, hypothyroidism was more prevalent than hyperthyroidism and most of cases ware categorized as subclinical disease (table 2). in contrast to overt diseases, subclinical hypothyroidism and subclinical hyperthyroidism are not associated with poor pregnancy outcomes [19, 20 ]. however, apart from preterm delivery or miscarriage, it was also reported that major obstetrical complications such as hypertension, iufd, or abruption were closely associated with subclinical hypothyroidism [2123 ]. we also found that the incidence of thyroid dysfunction in the dm group was similar to the groups of thyroid disease, iugr, and hypertension. high frequency of antithyroid antibodies in pregnant women with gestational diabetes mellitus was also reported. acog and the endocrine society recommended that thyroid testing should be limited to women with symptoms of thyroid disease and those with a history of thyroid disease or other medical conditions associated with it, such as type 1 diabetes or autoimmune disorders [11, 25 ]. according to our results, we recommended to screen thyroid function in women having the above - mentioned complications. furthermore, three cases of overt hypothyroidism were noticed in the groups of dm and ht. we were not able to detect three cases of overt hypothyroidism, if thyroid testing was limited to women with thyroid disease. one of the reasons was the trimester - specific range for tsh in this study. mu / l. the normal range of tsh during pregnancy is narrow when compared to that of general population. our perinatal center is a secondary hospital and mainly deals with referral cases due to thyroid disease, iugr, diabetes mellitus, hypertension, iufd, and placental abruption. high hcg level at first trimester resulted in hcg - induced hyperthyroidism. in our study, first, the current study was done in one secondary hospital dealing with referral cases only. as a consequence, there was a higher proportion of high - risk deliveries and a higher incidence of cesarean deliveries in our hospital. a regional population - based study should be needed to verify the prevalence of thyroid dysfunction and the relevance of perinatal prognosis in low - risk population. second, we did not investigate thyroid function of the child who was born from a mother with thyroid dysfunction. the outcome of infant should be obtained to verify the effect of thyroid dysfunction during pregnancy. the presence of antibodies influences clinical course of pregnancy independently from thyroid dysfunction. in conclusion, we demonstrated that the prevalence of thyroid dysfunction is increased in pregnant women with obstetrical or medical complications. under the currently recommended screening method, the majority of thyroid dysfunctions may be missing. with a full awareness of high incidence of thyroid dysfunction in pregnant women with obstetrical or medical complications | objective. we conducted the study to see the incidence of thyroid dysfunction in women with obstetrical high - risk factors. methods. we retrospectively reviewed medical charts of high - risk pregnant women who had examination for thyroid function during pregnancy. women were divided according to clinical presentation, symptoms of thyroid disease and those with a personal history of thyroid disease (thyroid disease, n = 32), intrauterine growth restriction (iugr, n = 115), diabetes mellitus (diabetes, n = 115), hypertension (n = 63), intrauterine fetal death (iufd, n = 52), and placental abruption (abruption, n = 15). the incidence of thyroid dysfunctions including hyperthyroidism or hypothyroidism was compared. results. the overall prevalence of thyroid dysfunction was 24.7%. the incidence of thyroid dysfunction in each group was as follows : 31% in thyroid disease, 25% in iugr, 30% in diabetes, 27% in hypertension, 12% in iufd, and 7% in abruption. except iufd, the incidence was not statistically significant from the group of thyroid disease (thyroid disease versus iufd, p = 0.03 by 2 test). thyroid disease represented for only 10% of all thyroid dysfunctions. conclusion. testing of women with a personal history or current symptoms of thyroid disease during pregnancy may be insufficient to detect women with thyroid dysfunction, who will become at high - risk pregnancy. |
keeping this in view, during the mid-1980s the government of india decided to assess the extent of yaws problem in the country8. based on the data so obtained, the government took the initiative to renew its efforts to eradicate yaws based on the premise that yaws eradication can not only alleviate the suffering of the tribal populations but also assist in bringing them out of economic deprivation9. moreover, since women and children are at particularly high risk, the disease has a negative impact on reproductive and general health and on nutritional status as well. several other factors helped india decide to launch the eradication programme, including - (i) epidemiological : there is no extra - human reservoir of infection and the disease is endemic only in limited albeit difficult and hard to reach areas ; (ii) technological : simple diagnostic tests and effective low cost treatments are available ; the use of a single intra - muscular injection of long - acting benzathine penicillin to cases and their contacts can cure the disease and interrupt transmission ; (iii) historical : the disease was almost eradicated during the 1950s ; and (iv) perhaps most importantly, the government of india was convinced that eradication of yaws was an attainable goal and was committed to achieve the goal10. india launched an anti - yaws campaign in 1952 with assistance from who and unicef. from 1952 to 1964, the campaign detected and treated about 200 thousand cases in four states, namely orissa (now odisha), andhra pradesh, maharashtra and madhya pradesh5. the prevalence of yaws declined from 14.4 to 1.1 per cent in odisha, from 5.6 to 0.3 per cent in andhra pradesh, and 5.5 to 1.4 per cent in the bastar district of madhya pradesh. the active yaws eradication efforts were gradually abandoned in the country and the strategy changed from active case finding and treatment to passive surveillance as part of general health services. it is ironic that yaws control was left for general health services to take care of, in the very areas where there were virtually no health care services existing in the first place. unsurprisingly, as a result the disease began to re - emerge in the late 1970s11. in 1986, the national institute of communicable diseases (nicd), encouraged by the success of smallpox eradication during the 1970s and progress being made in eradication of guinea worm disease in the country during the early 1980s, organized a meeting to develop a strategy for yaws eradication9. the strategy agreed to in the consultation consisted of (i) active search for and treatment of infectious cases, and (ii) health education and social mobilization in the community. cases were to be treated with long - acting benzathine penicillin, thereby rendering them non - infectious ; simultaneously, the family contacts given penicillin shots as prophylaxis. in cases sensitive to penicillin, tetracycline or erythromycin was recommended for a period of 15 days. more recently, studies have shown the efficacy of single - dose azithromycin for treatment of yaws in children12. the strategy was first piloted in koraput district in odisha during 1996 - 1997 to ascertain the feasibility of its implementation as a part of the programme. subsequently, based on positive feedback from the pilot studies, the national programme was extended to four more states, namely andhra pradesh, gujarat, madhya pradesh and maharashtra. by 1999, the programme was expanded to all 51 districts in 10 states where yaws cases were reported in the past (fig. active case search operations were carried out by multipurpose workers and community level functionaries, who visited house - to - house carrying a coloured disease recognition card and other health education materials looking for yaws cases. the treatment consisted of penicillin injections (1.2 million units for those above 10 years and 0.6 million units for children below 10 yr), preceded by testing for sensitivity to penicillin. the active search and treatment operations were carried out at 6-monthly intervals before and after monsoon seasons. in addition to active search, cases which were routinely diagnosed at health centres were also treated and their contacts given prophylaxis. through active case search and routine reporting, the programme detected and treated about 7000 infectious cases and their contacts. the strategy was highly effective, as the number of yaws cases detected in the country started declining rapidly ; from 3571 cases in 1996 to 664 in 2000 (a reduction of more than 80%) and only 46 three years later6. the last case was diagnosed and treated in mayurbhanj district, odisha in 2003613 (fig. 3), thereby achieving the interim goal of yaws elimination set by the programme (no yaws case status) in 200414. at the recommendation of the task force, the government of india on september 19, 2006 formally declared having achieved yaws elimination6. to move from yaws elimination to eradication, defined by the programme as absence of new cases for a continuous period of three years, supported by data indicating absence of transmission through serological surveys among children, a road map was developed based on consensus building among yaws experts in the country10. the road map consisted of three new activities : (i) serological surveys among children between one and five years of age to assess cessation of community transmission of yaws, (ii) rumour reporting and verification, and (iii) institution of cash reward for anyone reporting a yaws case, in addition to the information and communication activities and active search for cases which were continued on yearly basis. many rumours of possible yaws cases were verified as non - yaws over the next five years. and, no one came forward to receive the cash award. during the 2009 - 2011 period, serological surveys were carried out among randomly selected children using rapid plasma reagin (rpr) test with treponema pallidum haemagglutination assay (tpha) for confirmation. of the 18,217 children surveyed in the erstwhile yaws villages, no serological evidence of yaws infection was found (jain sk, personal communication). simulteneously, for comparison purposes, 39319 children in non - yaws villages were tested and none were found positive, collectively indicating cessation of yaws transmission in the community and in the country as a whole (jain sk, personal communication). while the overall programme was planned, implemented and coordinated by nicd (now called national centre for disease control or ncdc), the programme performance at the field level was evaluated by an independent set of experts through appraisal missions. at the highest level, the programme was monitored by a task force under the chairmanship of the director general of health services, government of india. since 2000, six such independent appraisal missions were fielded ; the last and sixth mission consisting of 16 teams was undertaken during may and june 2014 and visited 16 districts in 10 states (jain sk, personal communication). the single key factor responsible for the success was the sustained effort of health care workers on the frontline, supported by programme staff, working under trying field conditions, in the affected areas. the most important among those were as follows : first, yaws is immensely amenable to eradication due to a number of favourable factors - there is no animal reservoir ; only a few localized foci of infection remain ; a single intra - muscular injection of long - acting penicillin completely cures the disease ; and the diagnosis of yaws can be done clinically with minimum training of staff and through community education. however, efforts must be continued until the last case is diagnosed, treated, and cured. second, to achieve the goal of yaws eradication, not only is a high level of political commitment needed but must be sustained over time so that the programme achieves its ultimate goal of eradication. for the field operations, human resources and logistic support were provided by the state governments while the government of india, besides policy support, ensured funding support for supplies, training, search operations, and for monitoring missions and feedback. third, partnership among various sectors and stakeholders engaged in the programme was essential for success. besides the health directorate, other sectors namely department of tribal welfare and other institutions such as panchayati raj, education, forest, etc. they visited the field at regular intervals for verification of the progress being made to ensure that yaws cases are no longer existing and that community transmission is interrupted, even at the risk of their personal safety and security. fourth, the programme was implemented through the existing health care delivery system of the state health directorates. successful programme implementation was facilitated by a target oriented campaign, provision of supportive supervision during field activities, improved communication from the field to ncdc, and regular review meetings of state programme officers to review progress and share lessons learnt. finally and most importantly, the success of the programme could be attributed to a technically sound strategy which proved effective in trying field conditions. the programme was primarily funded by the government out of its own resources and as a part of its national 5-year plan, with additional technical and financial support from the who. the achievement of yaws eradication in india is an excellent example of a triumph of public health over a disease which still afflicts many populations in remote areas in africa and asia. the indian experience clearly shows that with commitment and renewed efforts, the disease can be eradicated from all those countries of the world where yaws is still prevalent. programmes such as these can go beyond health and play a role in enhancing the socio - economic conditions of people long neglected from policy point of view15. as stated above, the eradication campaign in india was conceived with the idea of not only alleviating the suffering of the poor, predominantly tribal population, but also with a view that the programme could contribute to the economic empowerment of remote areas and work as an entry point for primary health care for people living in these areas. the anecdotal information is available (jain sk, personal communication) to suggest that yaws eradication has brought about considerable improvements in the lives of tribal people, and has been instrumental in stimulating productivity and economic growth in these otherwise impoverished and so - called backward areas of the country. with improvements in living conditions, sanitation, and health and nutritional levels, the eradication programme is contributing towards poverty reduction among neglected populations living in hard to reach areas. | yaws, a non - venereal treponematosis, affecting primarily the tribal populations, has been considered historically as one of the most neglected tropical diseases in the world. in 1996, india piloted an initiative to eradicate yaws based on a strategy consisting of active case finding through house - to - house search and treatment of cases and their contacts with long acting penicillin. thereafter, the campaign implemented in all 51 endemic districts in 10 states of the country led to the achievement of a yaws - free status in 2004. in the post - elimination phase, surveillance activities accompanied by serological surveys were continued in the erstwhile endemic districts. these surveys carried out among children between the age of 1 - 5 yr, further confirmed the absence of community transmission in the country. the experience of india demonstrates that yaws can be eradicated in all endemic countries of africa and asia, provided that political commitment can be mobilized and community level activities sustained until the goal is achieved. |
extramedullary plasmacytomas (emps) are uncommon and occur almost exclusively in the head, neck, and upper respiratory tract ; emps in gastrointestinal organs are rare.1 the next most frequent site of mass lesion occurrence is the stomach ; however, this is also extremely rare, accounting for less than 5% of all emps.2 emps of the head and neck are more sensitive to radiotherapy than to surgery.2,3 endoscopic submucosal dissection (esd) is a less invasive therapeutic approach for early gastric cancer and has been accepted as a standard therapeutic approach for early gastric cancers limited to the mucosa.4 here, we report the successful treatment via esd of a patient with a solitary gastric emp confined to the mucosa. a 70-year - old man was admitted to our hospital with the complaint of dyspepsia. we performed gastroscopy and observed a flat lesion with focal erythematous changes in the anterior wall of the antrum (fig. a biopsy revealed poorly differentiated neoplastic cells and atypical lymphocytes, consistent with metastatic carcinoma. a giemsa stain indicated helicobacter pylori negativity. to rule out cancer and systemic diseases such as lymphoma we also performed abdominal and pelvic computed tomography (ct) and positron emission tomography / ct scans ; these yielded unremarkable results, particularly with regard to lymph node and bone lesions. regarding the gastric focal lesion, we performed an endoscopic procedure to confirm the previous endoscopic biopsy result as well as for therapeutic reasons using an insulation - tipped knife (kd-610l ; olympus, tokyo, japan) (fig., the acquired specimen revealed plasma cell infiltration into the lamina propria ; however, these cells did not extend deeply into the submucosal layer. because emps are systemic, the patient was treated with additional oral thalidomide and dexamethasone. later, we performed three endoscopic follow - up exams during a 2-year period and confirmed nonspecific esd scarring. plasma cell neoplasms are classified into four types : solitary myeloma (bone plasmacytoma), multiple myeloma (bone marrow and other systemic involvements), extramedullary (soft tissue) plasmacytoma, and plasmablastic sarcoma.5 emp is defined as an immunoproliferative, monoclonal, plasma cell tumor that develops in an extramedullary organ ; gastric plasmacytomas are a very rare form of emp. gastric tumors account for 2% to 5% of all emps and tend to be identified at a late stage if an endoscopic examination is not performed.6 gastric plasmacytomas can be classified into infiltrative, nodular, ulcerative, and polypoid types, of which the nodular type is most common. most gastric plasmacytomas are large, deeply infiltrating tumors with ulceration ; however, tumor cells are limited to the mucosal and submucosal layers in the early stage.7 almost all patients with emps are treated with radiation therapy, surgery, or combination therapy (surgery and/or chemotherapy or irradiation). however, no general treatment guidelines have been established for gastric emps. additionally, the invasiveness of these initial therapeutic approaches precludes their recommendation for emps of the head and neck.2,3 we also considered the possibility of mucosa - associated lymphoid tissue (malt) lymphoma in the present case, although the endoscopic findings suggested a reduced likelihood of malt lymphoma. extranodal lymphomas mainly occur in the stomach, whereas malt lymphomas mainly occur in the digestive tract. further, h. pylori infection correlates with malt lymphoma, and malt lymphomas express b - cell lineage markers.8 however, in the present case cd138 immunostaining was positive and a giemsa stain was negative. some studies have reported the complete regression of emps after treatment for h. pylori.6,9,10 however, there is insufficient data to demonstrate a relationship between gastric plasmacytoma and h. pylori.11 endoscopic mucosal resection has been established as a reliable therapeutic approach for early gastric cancers limited to the mucosa, but esd has limited efficacy for infiltrative diseases. in this case, esd has the advantages of being less invasive and less expensive and also has a lower incidence of side effects than surgery or radiotherapy.4 in addition, confinement of an emp to the mucosa can be confirmed using endoscopic ultrasonography.10 in this case, plasma cells did not extend deeply into the submucosal layer. however, we administered additional thalidomide and dexamethasone to this patient because emps are systemic and a combination therapy with thalidomide and dexamethasone is known to be highly effective.12,13 although other similar case reports have been published,10,13 further study is needed to confirm the results of solitary gastric emp treatment via esd. nevertheless, our case report suggests that esd can be an alternative therapeutic option for solitary gastric emps confined to the mucosa. | we report a rare case of a gastric plasmacytoma treated with endoscopic resection and oral thalidomide therapy. a 70-year - old man was admitted to our hospital with indigestion. he had no specific medical history and unremarkable laboratory results. gastroendoscopic findings revealed a focal, erythematous, flat elevated lesion in the anterior wall of the stomach antrum. a biopsy revealed atypical lymphocytes. endoscopic submucosal dissection (esd) with an insulation - tipped knife was performed 45 days after diagnosis. radiological and hematological evaluations, including a bone marrow biopsy, were performed and showed no involvement of other organs. the patient was diagnosed with extramedullary gastric plasmacytoma. follow - up gastroendoscopy was performed three times during a 2-year period and showed nonspecific esd scarring. the patient 's condition was found to be stable. |
working memory is one of memory types, and it allows transitory information to be actively held for several seconds on line. several brain regions, such as medial prefrontal cortex, and hippocampus, are known to play critical roles in holding transitory memory. testing working memory in the mouse has typically used the delayed non - matched to position (dnmtp) paradigm. for example, during a working memory test in a t - maze, intervention by the experimenter is inevitable because the maze needs to be cleaned and the animal must be relocated to the start box after sampling. therefore, there is a need to test working memory in operant - conditioning - based paradigm in which all experimental procedures will be scheduled automatically. this touch screen paradigm has several advantages over traditional behavioral testing paradigms (e.g., morris water maze [mwm ], t - maze, and radial - arm maze). first, testing can be scheduled almost automatically, permitting more experimental sessions to be conducted with high accuracy and flexibility. second, the method greatly reduces the need for experimenter intervention during memory test trials, because the experimental procedure is fully automated. third, it can provide the animal with multiple choice locations on the screen, which enables the capability of assessing the animal 's ability to discriminate between choice locations, such as pattern separation. fourth, various visual stimuli (e.g., from simple white rectangles to complex figures) can be presented on the screen. this enables testing of complex cognition, which was not able to test in the traditional behavioral tests. these advantages would advance research on working memory, if touch - screen - based technology were to be applied. unfortunately, a touch - screen system for the working memory test paradigm for mice is not available yet. therefore, in the present study, we developed a delayed matched to position behavioral assessment paradigm using a touch - screen testing system for evaluating working memory in mice. animals were housed in groups (four mice), maintained on a 12-h light / dark cycle, and food and water were provided ad libitum. all animal procedures were conducted in accordance with the guidelines of the institutional animal care and use committee of seoul national university. we used campden instruments bussey - saksida touchscreen chamber (campden instruments ltd, uk) for touch - screen testing. food restriction started at age of 10 weeks, and their weights were maintained to about 80% of initial weight through all behavioral tasks. before mice learned the rules of the cue - response paradigm and dmtp, they were trained to respond to visual stimuli on an lcd monitor to earn a reward. all experiments were conducted as described in the manufacturer 's guide and previous studies. briefly, on day 1, mice were habituated to a touch - screen chamber for 10 min. on day 2, mice were given liquid sweetened milk as a reward. liquid sweetened milk was made by diluting condensed milk (seoulmilk, korea) with the same volume of tap water. in this phase, the animal 's nose poking to the reward magazine located behind the chamber resulted in delivery of reward. on day 3, in initial touch phase, both nose poking to reward magazine and response to visual stimulus on the lcd monitor delivered the reward (fig. next, on days 4 through 6, in the must touch phase, only when mice responded to the visual stimulus reward was delivered (fig. after the must touch phase, mice were trained to avoid responding to a blank window ; incorrect punishment (fig. when the mice responded to a blank window, the room light was illuminated as a punishment signal. sixty trials were conducted within 60 min until mice reached the criterion (70% correct response in consecutive 2 days). after incorrect punishment, mice were trained to associate central cue and correct location choice for a reward. after central cue diminished, white squares were presented both in left and right windows. guided by the central cues, mice could choose either the left or right location to receive a reward. when the animal made an incorrect choice, the room light was illuminated, and additional correction trials were given until the animal made a correct choice. in cue - response training, 60 trials within 90 min were given for 5 sessions. three touches to the left- or right - positioned sample / cue stimulus on the screen by the mouse resulted in presentation of a visual stimulus in a central window. when the animal responded to the central visual stimulus, visual test stimuli were presented both on the left and right side of the screen. as in cue - response training, an incorrect choice resulted in room light illumination and additional correction trials. after successful dmtp rule learning, delay time between final response to the sample location cue stimulus and presentation of the central visual test stimulus was increased to either 3 s or 9 s. animals were housed in groups (four mice), maintained on a 12-h light / dark cycle, and food and water were provided ad libitum. all animal procedures were conducted in accordance with the guidelines of the institutional animal care and use committee of seoul national university. we used campden instruments bussey - saksida touchscreen chamber (campden instruments ltd, uk) for touch - screen testing. food restriction started at age of 10 weeks, and their weights were maintained to about 80% of initial weight through all behavioral tasks. before mice learned the rules of the cue - response paradigm and dmtp, they were trained to respond to visual stimuli on an lcd monitor to earn a reward. briefly, on day 1, mice were habituated to a touch - screen chamber for 10 min. on day 2, mice were given liquid sweetened milk as a reward. liquid sweetened milk was made by diluting condensed milk (seoulmilk, korea) with the same volume of tap water. in this phase, the animal 's nose poking to the reward magazine located behind the chamber resulted in delivery of reward. on day 3, in initial touch phase, both nose poking to reward magazine and response to visual stimulus on the lcd monitor delivered the reward (fig. next, on days 4 through 6, in the must touch phase, only when mice responded to the visual stimulus reward was delivered (fig. after the must touch phase, mice were trained to avoid responding to a blank window ; incorrect punishment (fig. to train this avoidance, when the mice responded to a blank window, the room light was illuminated as a punishment signal. sixty trials were conducted within 60 min until mice reached the criterion (70% correct response in consecutive 2 days). after incorrect punishment, mice were trained to associate central cue and correct location choice for a reward. after central cue diminished, white squares were presented both in left and right windows. guided by the central cues, mice could choose either the left or right location to receive a reward. when the animal made an incorrect choice, the room light was illuminated, and additional correction trials were given until the animal made a correct choice. in cue - response training, 60 trials within 90 min were given for 5 sessions. three touches to the left- or right - positioned sample / cue stimulus on the screen by the mouse resulted in presentation of a visual stimulus in a central window. when the animal responded to the central visual stimulus, visual test stimuli were presented both on the left and right side of the screen. as in cue - response training, an incorrect choice resulted in room light illumination and additional correction trials. after successful dmtp rule learning, delay time between final response to the sample location cue stimulus and presentation of the central visual test stimulus was increased to either 3 s or 9 s. first, in the " cue - delay - response " approach, the experimental animal makes a correct response guided by various cues presented at the start of the test trial. after the cue is diminished, the animal should hold a memory of the cue during the delay and up until the animal 's choice moment is coming. to use this system, it is necessary to create a reference memory by which the cues can guide the animal to make a correct choice. second, in the " delayed matched to position (dmtp) " or " non - matched to position (dnmtp) " paradigms, the animal should first learn the basic rule of the delayed matched or non - matched position to make a correct choice response. after the sampling location, a temporal delay is instituted before the animal is allowed to choose the matching (or non - matching) location. to test working memory in a touch - screen system, we first tested whether mice can learn a reference memory in which they choose between locations on the left or right, following presentation of a visual cue presented in the center (fig. this task also has been used to test rodent 's visuo - motor response. on a given trial, to receive a reward, the mouse must touch a visual cue presented in the center location on the screen, and then choose either the left or right location of subsequent visual choice stimuli, in accordance with the visual cues (fig. 2a) that have different colors and shapes. when choice moments were given 1 second after touching the center cue, however, mice performed poorly this task (fig. their failure to learn the reference rule indicates that using the cue - delay - response method in the touch - screen paradigm is not an appropriate way to test working memory in mice. we next employed a dmtp paradigm in which working memory of mice can be assessed in a lever - pressing operant chamber, to determine whether mice can learn a dmtp rule (fig. 3). to do sampling, the mouse was required to touch the visual stimulus presented in either the left or right position of the screen three times. one second later, the mouse was allowed to make a final sample touch of the visual cue presented in the center of the screen. after the mice made this final touch of the center position, they were then presented with two alternative visual stimuli that were positioned in both the left and right position. when mice chose a position that matched the cue 's position, a sweet milk reward was delivered (fig. when mice made an incorrect choice, correction trials were given until they made a correct choice. as was true in the lever - pressing operant chamber, mice successfully learned the matched to position rule (fig. 3). after four training sessions, mice scored high in accuracy compared to their first session, and the number of correction trials also diminished (fig. 3c). after mice learned the matched - to - position rule, to test working memory, we increased delay time between final sample touch and central visual stimulus presentation (fig. when delay time was increased to 3 s, there was a slight, but significant, decrease in the rate of correct choice to matched position (fig. 4a), compared to the last session of the rule learning. when we further trained mice in a 3-s delay paradigm by twice, animals made correct choices comparable to those of the last session of rule learning (fig. finally, a 9-s delay resulted in a correct matched - to - position learning rate was substantially reduced compared to that in the last session of 3-s delay paradigm (fig. prolonged delay time between final sample touch and central visual stimulus presentation reduced the rate of animal 's correct matched - to - sample responses. for example, increasing delay time from 1 s to 3 s slightly reduced correct choice rate (fig. as more training sessions were given, however, mice made comparable correct choices to those in the 1-s delay condition. this may indicate, in our experimental condition, that a 3-s delay is not long enough to test the working memory of mice. when delay time was increased to 9 s, mice showed substantially reduced correct choice rate, which indicates that their rate of correct choice of matched position is affected by delay time between sampling and choice moment. as clearly mentioned in the previous report, trial - unique, delayed nonmatching - to - location (tunl), the only known working memory testing paradigm using touch screen, failed to test working memory in mice. there are several differences between tunl task and our current paradigm, which successfully assessed working memory of mice. firstly, we employed dmtp paradigm, whereas tunl task used dnmtp paradigm even though it is not clear how this difference affected cognitive performance. secondly, we increased the number of sampling touch to 3 times as in lever pressing dmtp paradigm whereas animals were allowed only one sampling touch in tunl task. we suppose that this multiple sampling may increase retention of transitory memory during delay period. thirdly, in our paradigm, mice were not required to do any specific task to initiate choice phase at the end of delay period, whereas in tunl task, animals are required to poke reward magazine at the end of delay paradigm to initiate choice phase. this may also make our paradigm easier for mice to learn the rule of dmtp. our present approach to assess working memory in mice using touch - screen test could be used to characterize several features of working memory. first, how long mice can retain the memory of a sample position could be tested. although not well investigated, 10 s is commonly used as the time limit in current t - maze - based experiments assessing working memory in mice. using a similar testing protocol in our present study, mice could make correct matched - to - sample choices with as long as a 9-s delay. therefore, with our highly flexible and accurate system, the duration of transitory memory during dmtp tasks could be determined for mice. second, the effect of pattern separation on working memory could be studied in mice. the hippocampal ca1 region is required to make non - matched choices between highly separated choices but not between close choices in the radial - arm maze task. there is no study that assesses whether mice also have hippocampal ca1-dependent temporal separation memory. using our present paradigm, it could be examined whether mice have temporal separation memory, and, if they do, which brain regions are involved in temporal separation memory. | assessing the working memory of the rodent by using a touch - screen system has several advantages (e.g., allowing highly accurate data collection and flexibility in memory task design). however, there is currently no available testing paradigm utilizing touch - screen systems that can assess working memory in the mouse. in this study, we developed a touch - screen testing paradigm in which mice were trained to choose a location that is matched to a sample location after a time delay. consistent with previous studies, this study showed that mice could not only learn the rule in the delayed matched to position (dmtp), but also could retain a transitory memory of the sample position during delay. this indicates that a touch - screen system can provide a dmtp testing platform to assess working memory in the mouse. |
mastitis is the most significant and ubiquitous infectious disease of dairy cattle throughout the world. streptococcus uberis has been repeatedly identified as the most commonly isolated pathogen from clinical and subclinical samples in several countries, including australia, the united kingdom, new zealand, and belgium (14). multilocus sequence typing (mlst) is a highly discriminatory and reproducible means of investigating the molecular epidemiology of s. uberis (510). this has been undertaken primarily on isolates from clinical and subclinical mastitis, which have provided insights into the population diversity (5, 9) and enabled identification of distinct geographic variation in strain prevalence (11). control measures for managing the risk of new s. uberis cases have traditionally focused on reducing the environmental infection pressure by separating the cow from sources of infection, such as pastures and certain bedding materials (12). however, several studies have hypothesized a role for contagious transmission of s. uberis based on mlst or pulsed - field gel electrophoresis (pgfe) strain patterns (9, 10, 13, 14). previous studies have also suggested contagious transmission of s. uberis based on modeling of the temporal patterns of infection (15, 16) and response to mastitis management practices (17). however, most of these studies have been limited to small numbers of isolates and few herds ; no study has estimated the incidence of potentially contagious transmission in herds within the context of the overall incidence of s. uberis mastitis across many herds. transmission of infection by different routes is likely to be driven by a combination of multiple factors, including the infectious pressure and the biology of the pathogen and its host. an accurate understanding of the likely transmission route is fundamental when designing risk - based mastitis control strategies. the aim of this study was to evaluate the heterogeneity of s. uberis strain types between and within herds using mlst and assess the extent of possible within - herd contagious transmission of s. uberis. this study utilized isolates of s. uberis from milk samples collected during a previous study (2). farm selection was through the database administered by national milk records (nmr), chippenham, united kingdom, to identify 250 herds with a record of > 35 cases of clinical mastitis per 100 cows during the previous 12 months (2003 to 2004). a recruitment letter was sent to the farmers inviting them to participate in the study, and 68 responded positively. monthly recordings of somatic cell counts (sccs) from these herds were assessed ; 26 herds with an annual arithmetic mean scc of > 200,000 cells / ml (high group) and 26 with an annual arithmetic mean scc of 50% of the clinical cases illustrated in fig. 2 (listed in descending order of s. uberis clinical case frequency ; one outlier farm [farm 27 ] with very high mastitis prevalence, shown at far left in fig. the mean number of pte mastitis cases following an index case was 1.6 (range, 1 to 15 clinical cases). there were no significant correlations between the proportion of isolates classified as index, pte, or persistent and herd size, total herd mastitis prevalence, herd scc (high / low category pairs), housing versus grazing, bedding, reported milking parlor routine, or within - herd prevalence of s. uberis mastitis. number of s. uberis clinical mastitis cases grouped by case classification for all mlst sequence types across all herds index case (i) : more than one clinical case of mastitis in different cows in the same herd during the study period. these pairs or multiple cases potentially occur through contagious (cow - to - cow) transmission rather than from an environmental reservoir. the first occurrence of one of these sts in a herd was classified as the index case of that st in that herd. potential transmission event (pte) : following an index case in a herd each subsequent clinical cases caused by that specific st in another cow in that herd was classified as a potential transmission event (pte). persistent (p) : when the same st was identified on two or more occasions from the same mammary gland quarter, all but the first case caused by that st were classified as persistent infections. unclassified (u) : clinical cases where the identified st was isolated once in a particular herd but was also identified (as persistent, index, or pte) in another herd. solitary (s) : sts occurred in only one clinical case throughout the study period. all 35 s. uberis mlst sequence types from clinical mastitis isolates associated with potential transmission events (ptes) cases listed in descending order of their overall prevalence as distribution between classification groups. mlst profiles are listed in the following locus order ; arcc, ddl, gki, recp, tdk, tpi, yqil. mastitis clinical cases by classification groups for each farm where s. uberis mastitis cases were identified, with complete mlst profiles in descending number of total identified isolates. out of all 195 sts, 9 were significantly overrepresented (ovr), causing 10 clinical cases across all herds (p = 0.045, fisher 's exact chi - square test). eight of the 9 sts were also identified in persistent infections (table 3). the pte sts were significantly more likely to be classified as persistent cases, accounting for 85% (23 of 27) of the persistent cases (table 3). within the 35 pte sts, the ovr subgroup mastitis cases were significantly more likely to be classified as persistent, accounting for 63% (17 of 27) of the identified persistent infections in the study. a small number of sts of common lineage were identified that caused 40% of the clinical mastitis cases attributable to s. uberis (table 3). phylogenetic analysis illustrates the relative similarity of eight of the nine ovr sts (fig. 3) : st-5, st-6, st-22, st-24, st-35, st-233, st-361, st-512, and st-20 (separate lineage). with the exception of st-20, the ovr sts form part of the global clonal complex previously defined as gcc5 (9). sts varied by > 3 alleles both from the main group and from each other, leading to the majority remaining ungrouped. in this study, st6 is defined as the founder, four of the ovr sts are single - locus variants, and two of the ovr sts are double - locus variants (table 3). st20 is the fifth most commonly isolated st and the only ovr st found to be in a separate lineage. the node size proportionate to the number of clinical mastitis isolates attributed to that st. s. uberis has been classified as an opportunistic environmental pathogen since the early 1970s, following its identification in straw bedding and as a commensal organism on skin and in feces (23), as well as in infected bovine mammary glands. this is in contrast to the more prevalent, obligate pathogens of that era, principally streptococcus agalactiae, which appeared to reside principally or exclusively in the mammary gland, which in turn were classified as contagious pathogens. this broad classification is likely to be an oversimplification of the epidemiology (13, 14, 17). several studies investigating s. uberis mastitis identified temporal patterns of clinical mastitis that were suggestive of contagious transmission (15, 16), occurring as outbreaks in individual herds. there are several published studies using mlst on relatively small numbers of samples collected from a small number of herds that have demonstrated a wide variety of sts, indicating that infections were probably acquired from diverse environmental populations (5, 9, 24). studies have also shown that one pathogenic st can predominate in a herd (5, 9, 13). the same sts (st-5, -6, and -20) previously identified as predominant in different herds were identified as ovr sts in this study. the prevalence of sts in mastitis samples compared to environmental samples has been shown to be significantly different (10), indicating that a variable fitness to colonize between sts in the environment exists or some degree of cow - to - cow transmission occurs. given the st diversity catalogued in the s. uberis mlst database (635 separate sts), the heterogeneity of environmental populations (25), and the limited number of clinical cases per herd during the study period, it seems implausible that multiple cows in the same herd would become infected with the same st if there were no additional fitness or contagious phenotypic attributes specific to these highly prevalent strains. the potential for contagious transmission was described in several studies that demonstrated temporal patterns (15) and herd - level mastitis treatment and control protocols (16, 17) that support the hypothesis of contagious transmission. analysis of the relative prevalence of sts across all herds identified nine strains that were significantly more prevalent (ovr sts) than would be expected if opportunistic infection from the environment were the main transmission route. the nine ovr sts accounted for only 4.7% of the sts identified in the study. however, these sts were diagnosed in 38% of sequenced s. uberis clinical mastitis cases and 63% of persistent mastitis infections. while we recognize the possibility that the distribution of mastitis prevalence reflects an underlying distribution within the s. uberis population in the farm environment, this would seem unlikely, given that the mastitis and environment populations were previously shown to be significantly different (10). of the 52 herds studied, 63% had at least one pair of mastitis cases caused by the same st in different cows. while not conclusive, this result suggests that either contagious transmission or enhanced infectivity among the environmental population may be more common and more important than previously thought. pathogens that acquire virulence genes for intramammary infection may reside in the environment and cause new infections as environmental opportunists. however, it is logical to assume that environmental pathogens with enhanced infectivity will present an increased risk of contagion and pose the ability to maintain bacterial numbers at a high enough level for long enough to contaminate milking equipment with the requisite infectious dose. this may occur especially if their virulence mechanisms involve host immune evasion and the establishment of persistent infections, as suggested in previous studies (26). persistent infections, by definition, are present during more milking periods than transient infections and therefore present an increased number of transmission opportunities for an infectious pathogen (22). in this study, the overwhelming dominance of pte sts and particularly the ovr types in the persistent mastitis classification suggests that these sts would have more opportunities to be transmitted from cow to cow during milking. previous studies identified genetic sequences associated with important infectious processes, such as colonization (20, 27). genomic analysis using isolates from clinical and subclinical mastitis cases show that complex multigene arrangement, rather than the simple presence or absence of virulence determinants, influences bacterial pathogenicity and the outcome of the infection (28). the results of this study confirm the predominant clonal complex in the united kingdom to be cc5, in contrast to those found in australia and new zealand, where cc86 and cc143 are more common (5, 10, 11). cc143 isolates were also identified in china (29), whereas only singleton sts were identified in india (7). while some of these studies involved only a limited number of isolates, their results do indicate geographic variation in the s. uberis population. the more extensive management systems practiced in these countries and different pre- and postmilking hygiene protocols may predispose to different patterns of s. uberis mastitis in these countries, and it is plausible that contagious transmission may not be as likely under those conditions. this study provides the first estimate of the prevalence of these potentially contagious patterns between herds in the united kingdom and the distribution of incidence rates of potentially contagious transmission events within herds. the finding that potential contagious transmission contributed > 50% of the total clinical mastitis in 33% of the herds studied suggests that contagious transmission may be a major barrier to conventional control protocols designed to limit new s. uberis infections that focus on environmental factors alone. previous studies have discussed contagious transmission as a means of explaining dramatic clinical mastitis outbreaks in individual herds (15, 16). in this study, 1 of 52 herds (farm 27) displayed a temporal pattern of clinical cases that would have fallen within a previously proposed definition of an outbreak (15) ; in this single herd, 71% of the clinical cases were classified as ptes (fig. 2, farm 27), with high ratios of index to pte cases for several ovr sts (e.g., 1:15 for st-6 and 1 to 6 for st-20). however, this study highlights the potential role of insidious, low - level cow - to - cow transmission at index - to - pte ratios of 1:1 or 1:2 as a major component of the ongoing mastitis incidence in the majority (63%) of herds. this insidious manifestation of potentially contagious mastitis may arguably be more important and more challenging than the rare, dramatic mastitis outbreaks. it is only with the aid of molecular epidemiology that estimates of the relative importance of these two modes of transmission can be attempted. the selection criteria used in the recruitment of herds provided a reasonable cross section of herds and management practices at the time of data collection. however, herds with a substantially higher incidence of s. uberis clinical mastitis or significantly different management practices may exhibit higher rates of ptes than those identified in this study. this study has identified several specific sts, which warrant further investigation due to their higher prevalence and wider distribution. further work is required to elucidate the underlying molecular mechanisms, host - pathogen interactions, and influence of management interventions in the colonization of mammary glands by s. uberis. this may allow us to understand how and why different patterns of potentially contagious or environmental mastitis appear to predominate in different herds. the variability observed between herds in this study may provide some explanation of the apparently intractable difficulties faced by the dairy industry in reducing s. uberis mastitis prevalence in spite of the adoption of control measures for environmental pathogens, as suggested previously (17). a more tailored risk - based approach using a combination of pathogen classification, analysis of mastitis patterns, and individual herd management practices may be more successful. | multilocus sequence typing was successfully completed on 494 isolates of streptococcus uberis from clinical mastitis cases in a study of 52 commercial dairy herds over a 12-month period. in total, 195 sequence types (sts) were identified. s. uberis mastitis cases that occurred in different cows within the same herd and were attributed to a common st were classified as potential transmission events (ptes). clinical cases attributed to 35 of the 195 sts identified in this study were classified pte. ptes were identified in 63% of the herds. pte - associated cases, which include the first recorded occurrence of that st in that herd (index case) and all persistent infections with that pte st, represented 40% of all the clinical mastitis cases and occurred in 63% of the herds. pte - associated cases accounted for > 50% of all s. uberis clinical mastitis cases in 33% of the herds. nine sts (st-5, -6, -20, -22, -24, -35, -233, -361, and -512), eight of which were grouped within a clonal complex (sharing at least four alleles), were statistically overrepresented (ovr sts). the findings indicate that 38% of all clinical mastitis cases and 63% of the ptes attributed to s. uberis in dairy herds may be caused by the nine most prevalent strains. the findings suggest that a small subset of sts is disproportionally important in the epidemiology of s. uberis mastitis in the united kingdom, with cow - to - cow transmission of s. uberis potentially occurring in the majority of herds in the united kingdom, and may be the most important route of infection in many herds. |
uterine myomas, also referred to as fibroids, are benign tumors of the smooth muscle of the uterus and are the most commonly encountered type of tumor in women, occurring in nearly 50%70% of women of childbearing age. the status of fibroids during pregnancy is especially complex, because fibroids can affect the pregnancy and pregnancy can affect the fibroids. degeneration has been estimated to occur in 10% of fibroids during pregnancy, and is characterized by abdominal pain, leukocytosis, and mild fever. enterouterine fistulas can occur following : (1) carcinoma of either the intestine or uterus with subsequent invasion of the other structure and communication of the lumina, (2) rupture of the uterus (either spontaneous or produced during labor), or (3) inflammatory disease (such as pathological puerperium and appendicular abscess) causing peritonitis, diverticulitis with abscess formation and crohn disease. a 25-year - old patient presented with an 87-cm posterior wall fibroid diagnosed for the first time during pregnancy at 12 weeks. the course of the pregnancy was smooth, with mild enlargement of the fibroid to the size of 109 cm. the patient delivered by caesarean section at a private clinic due to malpresentation (breech presentation). in the first week postpartum, the patient experienced malodorous vaginal discharge. the patient sought medical advice and antibiotics were given for 14 days, but the discharge did not resolve and the patient felt a mass inside the vagina. the patient presented to tanta university hospitals where she was admitted, and under general anesthesia, an examination revealed a solid mass extending from the cervix into the vagina. a biopsy was taken from the mass for histopathological examination, and it was identified as a degenerated cellular fibroid polyp. antibiotics also were given for 14 days after a culture and sensitivity test, but the discharge still did not resolve. at this point, the patient became frustrated and sought medical advice from more senior practitioners, who advised her to receive gonadotropin - releasing hormone analogues for 2 months. nonetheless, the discharge was not cured ; it became more malodorous as well as greenish and stool - like in character. the patient developed abdominal pain, distension, vomiting, and absolute constipation 1 week later and was readmitted to tanta emergency hospitals where thorough investigations were performed, including a pelvic ultrasound and a computed tomography scan with gastrointestinal tract contrast. an examination revealed cachexia and toxicity facies, the patient 's vital signs were a blood pressure of 100/60 mm hg, a pulse of 110 beats per minute and a temperature of 38.1. an abdominal examination revealed distension, tenderness with guard and rigidity, and resonance over all quadrants of the abdomen. a pelvic examination demonstrated the presence of tender fornices and a stool - like offensive discharge coming through the vagina, and examination using a speculum found a stool - like discharge coming through the cervix. the following laboratory studies were performed : a complete blood count, urinalysis, a liver function panel, a kidney function panel, and serum electrolytes. ultrasound revealed the absence of peristalsis in the small bowels and a large posterior wall fibroid measuring 107 cm with degeneration. computed tomography with gastrointestinal tract contrast revealed fistulous communication between the ileum and uterus. written consent was obtained from the patient and her relatives for a range of treatment options, including hysterectomy. a midline infraumbilical incision was made by the surgical team, and we found the abnormal invasion of the fibroid to the ileum approximately 30 cm from the ileocecal junction (figure 1). dissection of the intestine from the uterus showed the site of the fistula very clearly at the posterior wall of the uterus through the fibroid (figure 2). the gynecologists managed the case by myomectomy and repair of the endometrial cavity with placement of an intrauterine catheter for antiseptic washing and to prevent asherman syndrome (figure 3). two intraperitoneal drains were inserted, and the catheter was removed 7 days after the operation. written consent was obtained from the patient and her relatives for a range of treatment options, including hysterectomy. a midline infraumbilical incision was made by the surgical team, and we found the abnormal invasion of the fibroid to the ileum approximately 30 cm from the ileocecal junction (figure 1). dissection of the intestine from the uterus showed the site of the fistula very clearly at the posterior wall of the uterus through the fibroid (figure 2). the gynecologists managed the case by myomectomy and repair of the endometrial cavity with placement of an intrauterine catheter for antiseptic washing and to prevent asherman syndrome (figure 3). two intraperitoneal drains were inserted, and the catheter was removed 7 days after the operation. a fistula is defined as an abnormal connection between two epithelium - lined organs that do not connect. in the uterus published perhaps the largest review of enterouterine fistulas in 1956, describing 80 cases, 42 of which followed an obstetric injury, 17 resulted from inflammatory processes, 12 followed curettage, and nine were related to carcinoma. they may also occur as complications of dilatation and curettage, with a range of post - abortive times of presentation. an enterovaginal fistula was described by meier - boni. in 1990. in that case, an enterovaginal fistula was found 1 year after vaginal hysterectomy ; radiological imaging revealed a fistula between the terminal ileum and the vagina in an 81-year - old patient. fadel described an ileo - uterine fistula after myomectomy that was asymptomatic and discovered accidentally during hysterosalpingography for infertility. in our case, we found a fistulous tract between a degenerated posterior wall fibroid and the ileum, and administered a questionnaire to patient and her relatives in an attempt to identify the causes of that complication. several factors may have contributed to the formation of the fistula, with possible explanations including post - caesarean infection, operative trauma to the bowel during the caesarean section leading to an infection - caused adhesion between the fibroid and the injured bowel facilitating fistula formation, and potential uterine perforation during uterine sounding - a preliminary step when excising the uterine polyp - which could have led to an infection at that site that may have facilitated fistula formation. the surgical team excised the fistula and re - anastomosed the gut, following a procedure similar to the operative steps carried out by meier - boni.. myomectomy and repair of the uterine cavity was performed by a gynecology team, with insertion of an intra - uterine foley catheter for seven days to prevent asherman syndrome. our management strategy was in agreement with mcfarlane. and duttaroy and madhok, who preserved the uterus, whereas dewdney. and gutierrez. preferred hysterectomy, because the patients in their cases were 50 and 44 years of age, respectively. ileo - uterine fistula is a rare complication of uterine fibroids that occurred in our patient following degenerative changes in a myoma during pregnancy with presentation following delivery by caesarean section. it was treated by myomectomy and conservative treatment of the uterus after division of the bowel loops and resection and anastomosis of the bowel ends. | uterine fibroids are benign tumors of the myometrium with a diverse range of manifestations. fibroids can dramatically increase in size during pregnancy due to the increase in estrogen levels. after delivery, the fibroids usually shrink back to their pre - pregnancy size. uterine myomas may have many complications, including abnormal uterine bleeding, infertility, pressure on nearby organs, degeneration, and malignant transformation. no previous reports have indicated that a fistula may develop between a uterine fibroid and the bowel loops, although previous studies have documented the occurrence of fistulas from the uterus to the bowel following myomectomy or uterine artery embolization performed to treat a myoma. in our case report, we document the rare complication of a fistula occurring between a degenerated myoma in the posterior wall and the ileum 1 week postoperatively in a patient who underwent a caesarean section but did not have a history of uterine artery embolization. |
accordingly, humans are continually exposed to multiple genera of fungi via various routes, but particularly by the ingestion of food, allowing for the colonization of the gastrointestinal tract. depending on the interaction between the host s mucosal defense mechanisms, fungal virulence factors, and antifungal utilization, colonization may be transient or persistent and local disease may ensue. of the numerous pathogenic fungi, candida is the dominant genus responsible for fungal diseases in humans.1 candida albicans is the species with the highest prevalence among human yeast isolates and is the main opportunistic yeast pathogen in most warm - blooded animals.1 symptomatic mucosal candidiasis (mc) arises in subjects colonized with candida who are predisposed by illness, debility, or a local reduction in host resistance to an overgrowth of their own indigenous flora. candida species are frequently isolated from the oral cavity and are detected in 31%60% of healthy individuals.1,2 colonization rates generally increase with the severity of illness and duration of hospitalization.3 in a recent study, the frequency of oral yeast carriage in the competent host varied as a function of age.2 the colonization rates increase from 24% in persons aged 57 years, to 59% in persons above the age of 60. in the hospitalized non - hiv infected individual, c. albicans accounted for 70%80% of oral isolates and c. glabrata and c. tropicalis each represented approximately 5%8%, while the other non - albicans candida species were recovered only rarely.4,5 in the past decade, there has been a significant increase in the frequency of non - albicans candida species isolated from hiv - infected individuals with mc.6,7 in the 1980 s, non - albicans candida species accounted for 3.4% of oral isolates from hiv - infected patients, while in the 1990 s, 16.8% of isolates recovered from hiv patients were non - albicans candida species. 6,7 the more commonly recovered non - albicans candida species, include c. glabrata, c. parapsilosis, c. tropicalis, and c. dubliniensis. in 5%10% of circumstances the more common combinations include c. albicans with c. glabrata, c. krusei, c. dubliniensis, or c. tropicalis. several local and systemic host and exogenous factors increase the prevalence of gastrointestinal (gi) tract candida carriage and population levels.8 the acuteness and extent of candidal infections increase with the number and severity of predisposing factors. the role of cd4 t cells is to be the normal gastrointestinal mucosal defense mechanism against candida species and this relation is highlighted by the frequent occurrence of oropharyngeal candidiasis (opc) and esophageal candidiasis (ec) in patients with low cd4 t cells and acquired immunodeficiency syndrome (aids).8,9 in hiv infection, oral carriage of yeast and risk of mucosal invasion increase with a progressive reduction in cd4 t cells.8,10 the anti - candida protective mechanism of cd4 t cells at a mucosal level is still incompletely understood. recently, investigators have shown that cytokines, especially gamma interferon, can inhibit the transformation of candida blastoconidia to the more invasive hyphal phase.8,11 in addition, several investigators have shown that a decrease in e - cadherin levels and a loss of cd4 t cells in the mucosa are associated with episodes of acute opc.12 the most commonly reported cause of higher gi yeast carriage rates and symptomatic oral candidiasis is the use of antibiotics.1,8 elimination of bacterial competition is almost certainly the important mechanism by which antibiotics affect candida numbers in vivo. it is important to note that the introduction of highly active antiretroviral therapy (haart), including protease inhibitors, has significantly reduced the prevalence of oropharyngeal and esophageal candidiasis in hiv - infected patients. in the first 1224 months after the introduction of haart, the prevalence of opc decreased from 50%80% down to ~10%.13 in addition, a decrease of 25%50% in the occurrence of ec was also documented. unfortunately, there have been no further studies describing the epidemiology, incidence, or significance of either opc or ec since 2004. based upon epidemiological studies, it is apparent that humans are exposed repeatedly to candida in food and other sources. the natural history of this commensal oral candidiasis has been known since the era of hippocrates, although hippocrates used the term aphthae to describe this infection.1 several clinical forms of oral candidiasis exist ; thrush is the most commonly and widely recognized and is also called acute pseudomembranous candidiasis (figure 1). oropharyngeal candidiasis remains the most frequent opportunistic fungal infection among hiv - positive patients and is frequently the initial manifestation of hiv infection. currently, it is estimated that approximately 80%90% of hiv - infected patients develop opc at some time during the progression of their disease from hiv infection to aids.1,13,14,15 c. albicans is the species responsible for the majority of cases of opc.1,15 the ability of c. albicans to adhere to buccal epithelial cells is critical in establishing oral colonization. after colonization, the organisms may persist for months or years in low numbers in the absence of inflammation. these low numbers are the result of effective host defense mechanisms in the oral cavity. genotyping of candida strains obtained from hiv - infected patients with either opc or ec indicate a genotype distribution frequency similar to that seen in non - hiv - infected subjects, suggesting that hiv - associated mc is not caused by a unique or particularly virulent strain of candida, but likely results from defects in host defense mechanisms.16 symptoms of opc can be extremely variable and range from asymptomatic oral lesions, to a sore, painful mouth, a burning tongue, and associated dysphagia. clinical signs include diffuse erythema and white patches that appear as discrete lesions on the surfaces of the buccal mucosa, throat, tongue, and gums.1,14 severe opc may ultimately impair quality of life and result in a reduction of fluid or food intake. the most serious complication of untreated opc is extension of the infection into the esophagus resulting in decreased nutritional intake. candida is the most common cause of infectious esophagitis and, after the oropharynx, the esophagus is the second most common site of gastrointestinal candidiasis. the prevalence of candida esophagitis (ce) has increased mainly because of its association with hiv - infected individuals. approximately 10%15% of patients with aids will eventually suffer from this entity during their lifetime.1,15,1719 the same organisms that are recovered from the esophageal surface are generally the same organisms identified in oral secretions. c. albicans remains the most common organism identified in ce. in contrast to oral candidiasis, little is known about host and yeast factors operative in the pathogenesis of esophageal candidiasis and experimental models have not been established. however, it is likely that the usual yeast virulence factors and defects in host defense mechanisms are responsible. the high prevalence of esophagitis in connection to aids indicates the critical role of cell - mediated immunity in normally protecting the esophagus from candida invasion. candida esophagitis tends to occur later in the natural history of hiv infection and almost invariably at lower cd4 t cell counts (range 10105, mean 79, median 30 cells).1,1719 it is not uncommon for patients with advanced aids, and near the end of life, suffering from severe esophageal candidiasis manifested by the inability to have any form of oral intake. although ce may arise as an extension of opc, in approximately 10% of cases the esophagus maybe the only site involved affecting the distal two - thirds, rather than the proximal one - third, which is the area more commonly affected (figure 2). an occasional feature of ce is the complete lack of clinical symptoms despite extensive objective esophageal involvement.1,1719 a reliable diagnosis can only be made by direct visualization of the esophagus along with histological evidence of tissue invasion in biopsy material.22,21 however, clinical criteria may be accepted as a basis for initiating antifungal therapy in high risk patients. the differential diagnosis of ec must include gastroesophageal reflux disease (gerd), idiopathic hiv - ulcers, and viral esophagitis due to either cytomegalovirus or herpes simplex virus.1,20,21 numerous antifungal agents are available for the treatment of opc and ec in the hiv - positive patient (tables 1 and 2). in addition, guidelines for the management of mucosal candidiasis in hiv - infected patients have been published by the centers for disease control, national institutes of health, and the hiv medicine association of the infectious diseases society of america.22 however, several factors should be considered when selecting antifungal agents for patients with hiv infection. in hiv - positive patients, antifungals similarly, the time to clinical response also tends to be delayed in this population.17,22,23 moreover, the relapse rate is higher in patients with hiv than in any other patient population. 1,17,22,23 for unknown reasons, a subgroup of hiv - positive patients experience recurrent episodes of opc and thus receive numerous courses of antifungals during their lifetime. as their hiv infection progresses they tend to experience shorter disease - free intervals between episodes of mucosal candidiasis and thus have greater antifungal exposure, which may ultimately lead to the development of clinical and in vitro antifungal resistance and its associated morbidity and mortality. it is important to note that, as with some opportunistic pathogens in this patient population, antifungal treatment merely reduces the signs and symptoms of infection and thus produces a transient clinical response by lowering the quantity of organisms in the affected area. it is extremely difficult to fully eradicate candida from the mucosal surfaces of patients who are immunocompromised, especially those who are hiv - positive. 8,18,23 the combination of frequent clinical relapses and increased antifungal utilization are frequently associated with antifungal resistance.24 this is reflected, clinically, by the ineffectiveness of antifungals to which a patient has not previously been exposed. thus, we are becoming increasingly aware that choosing the appropriate agent, even in the early stages of disease, is extremely important because of the future repercussions of these selections in advanced stages of infection. classes of agents used in the treatment of mc in hiv includes the polyenes (eg, amphotericin b, nystatin), pyrimidine synthesis inhibitors (flucytosine), azoles (miconazole, clotrimazole, ketoconazole, itraconazole, fluconazole, voriconazole, posaconazole), and, more recently, the echinocandins (caspofungin, micafungin, anidulafungin).17,22,24 common dosage regimens of these agents are listed in tables 1 and 2. the key trials evaluating antifungals in the treatment of opc and ec are summarized in table 3. most controlled studies to date have evaluated the azole antifungal agents and in general, clinical response rates appear to be similar. studies of antifungal treatment in mc can be problematic to evaluate, particularly in the hiv - positive population. limitations often include a small number of patients, open label design, and a short follow - up time. additional studies are particularly needed among patients with low cd4 t lymphocyte counts, as this population tends to have lower clinical and mycological response rates. a recent meta - analysis suggested that larger studies using more consistent outcome measures and reporting would be helpful in applying future research to current clinical practice.25 additionally, these authors also suggested that future investigations should include less expensive antifungal interventions and, at the same time, evaluate symptom - free periods, quality of life, survival, and the development of clinical and in vitro resistance. prior to the development of the polyenes and the azoles, topical therapy consisted of gentian violet applications that were reasonably effective in localized mc, but were extremely messy because of the purple color.24,26 while topical therapies have historically been effective in less severe disease and are relatively inexpensive compared to some of the systemic therapies, their use has diminished due to poor tolerability and poor adherence. topical therapy of opc can be accomplished with a multitude of antifungal agents including nystatin, amphotericin b, clotrimazole, and, more recently, miconazole (table 1).17,22,24,2631 nystatin is available in several formulations, including pastilles and suspension. limitations of topical agents such as nystatin include a bitter taste, gi - adverse effects, and frequent dosing, all of which may contribute to reduced adherence.24,27 importantly, nystatin has not demonstrated significant efficacy in severely immunocompromised patients, such as those with advanced hiv infection.27,28 for instance, in hiv - positive patients, nystatin exhibited lower rates of both clinical cure (52% vs 87%) and mycological cure (6% vs 60%) when compared to oral fluconazole. additionally, the 28- day relapse rate was found to be greater with nystatin (44% vs 18%) when compared to fluconazole.27 amphotericin b is also an option for topical therapy and is available in suspension, lozenge, and tablet form.1,22 neither nystatin, nor amphotericin b is absorbed from the gastrointestinal tract so administration must be frequent (four times daily) to provide adequate drug exposure to the infected mucosal tissues.1,22 prospective, comparative studies evaluating amphotericin b oral solution against other antifungals are limited. topical azole antifungal agents, such as clotrimazole 10 mg troches, administered five times daily, provide another option for opc patients. topical clotrimazole has been used successfully in treating mild - to - moderate opc during the early stages of hiv disease.2830 the newest topical antifungal is the mucoadhesive buccal tablet (mmbt) containing 50 mg of miconazole (loramyc).31 this novel formulation of miconazole has been approved in europe since 2008 for the treatment of opc in immucocompromised hosts. the formulation is unique, because 50 mg of miconazole is contained in each mucoadhesive tablet and is applied to the mucosa of the upper gum over the canine fossa, once daily, for 714 days. the mmbt adheres to the gum surface because of the milk protein concentrate composition of the tablet. this interaction leads to a rapid and prolonged adhesion to the mucosa due to an adsorption mechanism, followed by a protein - protein interaction. in pharmacokinetic studies, 50 mg of mmbt provides a maximum salivary concentration of 15 g / ml, up to seven hours after the application of the tablet.31 three separate clinical studies have evaluated the use of mmbts for the treatment of opc in the hiv - positive population and in patients with head and neck cancers.3234 in a phase iii, double - blind, double - dummy, multicenter trial evaluating 578 patients with hiv and opc, mmbt treatment was compared to clotrimazole troches (10 mg, five times daily, for a period of 14 days).32 the results at the primary endpoint of test of cure (toc) in both the intent - to - treat (itt) population and in the per - protocol population (pp) demonstrated that the once daily administration of a mmbt was as effective as the five times daily clotrimazole treatment. clinical cure rates at toc in both the itt (61% vs 65%) and pp (68% vs 74%) populations demonstrated no inferiority to clotrimazole. in addition, secondary endpoints such as safety and tolerability were similar between both treatment groups. the use of topical antimycotic agents has been replaced with systemic azole antifungals such as ketoconazole, fluconazole, itraconazole, and more recently posaconazole (table 1).17,24 part of the reason for this is that, although clinical cure rates may be similar, microbiologic cure and long term efficacy are not. in one example, a study comparing systemic oral fluconazole with clotrimazole troches in hiv - positive adults found clinical efficacy to be similar (98% vs 94%). however, microbiological cure rates were greater in patients treated with fluconazole (65% vs 48%) and clinical response sustained through a 2-week follow up was also greater (82% vs 50%).30 the currently available systemic azoles include ketoconazole, fluconazole, itraconazole, and posaconazole. ketoconazole was the first available oral systemic imidazole antifungal agent with high rates of efficacy in opc.35,36 however, in comparative trials, ketoconazole was found to be less efficacious then fluconazole in both clinical and mycological cure rates.37 in addition, the use of ketoconazole is further limited by potentially severe adverse reactions including hepatotoxicity, poor oral bioavailability, and a host of drug drug interactions.36 ketoconazole is a very potent inhibitor of cytochrome p450 3a4 and is relatively contraindicated with some hiv - protease inhibitors.38 because the drug s absorption is dependent upon an acidic ph, there are also concerns that systemic absorption may be inadequate in patients receiving acid - suppressive therapy or with aids - related hypochlorhydria.39 in view of the lower clinical efficacy rates and the associated adverse event profile (ie, hepatotoxicity), ketoconazole is not widely used anymore. in contrast to ketoconazole, other azoles such as fluconazole, itraconazole, voriconazole, and posaconazole have demonstrated improved efficacy, as well as excellent safety profiles, and have thus become the drugs of choice for opc, especially in hiv - positive patients (table 3).1,22,24,30 fluconazole is the most commonly used antifungal in the treatment of mc in hiv - infected patients. fluconazole is more readily absorbed than other oral azoles without being affected by either food or gastric acidity. the clinical efficacy of fluconazole has been established in many well controlled clinical trials, such that it has become the standard comparator in clinical trials of novel agents.24,30,37,4042 most studies of fluconazole have used an initial loading dose of 200 mg followed by 100 mg daily, but clinical success has been achieved with doses as low as 50 mg / day.40 clinical response is usually apparent within 10 days with 50 mg / day, and 5 to 7 days for doses of 100200 mg / day.17,40 of the available azoles, fluconazole is associated with the fewest drug drug interactions because it has less affinity for the cyp3a4 enzyme.38,43 the pharmacological interactions with fluconazole include concomitantly administered phenytoin, rifampin, rifabutin, cyclosporin a, and possibly some of the protease inhibitors.38,39,4345 fluconazole is generally well tolerated, however, as with any other azole being used long term, periodic surveillance of liver enzymes to monitor for hepatotoxicity is useful.24,43 itraconazole is supplied in a cyclodextrin oral solution or capsule ; the parenteral formulation is no longer manufactured in the united states.1,24,46 like ketoconazole, itraconazole exhibits a strong potential for drug drug interactions through the cpy3a4 enzyme system.38,46,47 the itraconazole oral solution has greater bioavailability than the capsule and absorption is further enhanced by postprandial administration. 40,4648 one prospective, randomized trial in hiv - positive and aids patients with opc, found itraconazole oral solution to have similar efficacy and safety as fluconazole (clinical response 97% vs 87%).49 however, approximately 50% of the patients in both groups experienced relapses at the 1-month follow - up evaluation. posaconazole is the newest triazole on the market and is approved for the treatment of acute opc and antifungal - refractory mc.5052 it is an oral extended - spectrum triazole with potent in vitro activity against pathogenic yeast and moulds, including fluconazole- and itraconazole - resistant candida strains.50 as with other azoles, posaconazole inhibits lanosterol 14--demehylase. it appears that mutations near the heme cofactor of cyp51 reduce the binding affinity of compact azoles, such as fluconazole and itraconazole, and may lead to azole crossresistance.50 however, 3-dimensional binding models suggest that the long side chain of posaconazole may result in tighter binding affinity.53,54 thus, posaconazole may be less susceptible than some azoles to the development of secondary azole resistance. posaconazole is absorbed in an oral suspension.50 it is important to note that posaconazole absorption is enhanced by coadministration with food, especially high fatty meals, or with a nutritional supplement such as boost plus (nestl, fremont, mi).55,56 when food intake is limited, dividing the daily dose from bid (twice daily) to qid (four times daily) also increases the plasma concentration. unlike itraconazole and voriconazole, posaconazole is not primarily metabolized by fungal cytochrome p450 enzymes.57 during several drug interaction trials evaluating the effects of posaconazole on the cyp450 enzymes, posaconazole did demonstrate an inhibitory effect on cyp3a4, but did not influence the other isoenzymes. in a large multicenter, randomized clinical trial, a 100 mg daily dose of posaconazole was compared to a 100 mg daily dose of fluconazole in hiv - positive patients with opc.58 clinical success was reported in 92% of the posaconazole recipients compared with a success rate of 92% for those patients that received fluconazole. the only difference was in the long - term follow - up, where clinical relapses occurred more frequently in the fluconazole group compared to the posaconazole group (38.2% vs 31.5%). several concerns have been raised about the widespread use of the more potent oral azoles, which may offer only minor advantages for patients. these concerns include drug interactions, side effects, increased expense, and risk of developing antifungal resistance. an increased frequency of c. glabrata isolation has been described by several investigators of hiv - positive patients receiving prolonged courses of fluconazole.5962 in addition, although azole resistance in c. albicans is rare, several reports describe both clinical failure and in vitro resistance in both non - hiv patients and in hiv - infected patients on long - term azoles.24,63 systemic antifungal therapy using oral or parenteral fluconazole has been the mainstay in the management of ec for over a two decades (tables 2 and 3).1,17 topical antifungals such as nystatin, clotrimazole, and miconazole are of minimal value in ec.22,64 the initial step in the management of ec should always be to attempt to minimize all possible predisposing factors, such as corticosteroids, chemotherapeutic agents, and antimicrobials.1,17,22 oral fluconazole has an excellent safety profile when compared to ketoconazole, demonstrates excellent gastric absorption, and can also be given intravenously when necessary. similar to the observations in opc research, studies comparing fluconazole with either clotrimazole or ketoconazole for ce demonstrate cure rates that are superior to those with other imidazoles. moreover, fluconazole demonstrated a more rapid onset of action and quicker resolution of symptoms. 6567 itraconazole has also been shown to be effective in the treatment of ec.6870 patients treated with itraconazole oral solution (100200 mg / day) had clinical response rates comparable to those of patients treated with fluconazole tablets (100200 mg / day) of 94% and 91%, respectively, without significant adverse effects in either group.68 the mycological cure rates were also similar at 92% and 78%, respectively. voriconazole, another broad spectrum systemic triazole is also approved for the treatment of ec at a dose of 200 mg bid for 1421 days. in vitro, it has been shown to be 10- to 500-fold more potent than fluconazole against a wide array of yeast and moulds, including many isolates that have demonstrated in vitro itraconazole- and/or fluconazole - resistance. 71 in a double - blind, randomized, multicenter study, voriconazole 200 mg bid was compared to fluconazole 200 mg daily in ec.72 the overall success rates were comparable at 98.2% for voriconazole vs 95% for fluconazole. as with most azoles, voriconazole has an effect on the cyp450 enzymes, so caution regarding drug - drug interactions is warranted.36,71 a common side effect with voriconazole includes photopsia (visual abnormalities) that may occur in 20%30% of patients. other side effects may include elevations in transaminases (~10%) and skin rashes (~10%), with rare cases of photodermatitis in patients who are exposed to direct sunlight.71 prior to the availability of azole antifungal agents, amphotericin b deoxycholate was used extensively. however, it is now rarely used in any patient and is generally reserved for antifungal - refractory cases of candida esophagitis that do not respond to azoles or echinocandins.73 if necessary, low dose amphotericin b (0.150.3 mg / kg / day or 1020 mg / day for 1014 days) is sufficient for moderate to severe disease.1,17,73 however, because of the improved adverse event profiles of the lipid formulations of amphotericin b, they have also become popular. oral flucytosine (100150 mg / kg / day in divided doses), although effective, is rarely used because of the tendency for resistance to develop during therapy and the well described frequency of bone marrow suppression and transaminase elevation.1,74 furthermore, in one comparative clinical trial, clinical efficacy and mycological cure rates were lower for flucytosine when compared to fluconazole.74 the echinocandin class of antifungals, which includes caspofungin, micafungin, and anidulafungin are a novel class of antifungal agents with a completely different mechanism of action.75 all three agents have demonstrated excellent in vitro activity against a broad array of candida species, including those that are resistant to fluconazole, itraconazole, or voriconazole.75 there have been a total of five clinical trials evaluating the efficacy of the three echinocandins in treating ec in hiv - positive patients. in all studies, the echinocandins are compared to either fluconazole or amphotericin b. in a clinical trial comparing caspofungin to amphotericin b in both opc and ec in a population that was predominantly hiv - positive, clinical success rates for caspofungin ranged from 74% to 91%, which was numerically greater than the success rate for amphotericin b (63%).76 in a related study, 177 patients with ec were stratified to receive either fluconazole 200 mg / day or caspofungin 50 mg/ day. caspofungin was found to have similar response and relapse rates as fluconazole.77 in another clinical trial, the treatment of ec among hiv - positive patients was also found to be similar for micafungin 150 mg / day when compared to fluconazole 200 mg / day.78 in a separate multicenter clinical study in hiv - positive patients (n = 601, 75% with aids) evaluating anidulafungin versus fluconazole in patients with ec found this echinocandin to have similar clinical response rates when compared to fluconazole, in terms of both efficacy and safety.79 so, while the echinocandins are not considered first line therapy for ec, they are effective antifungals for the treatment of mc in hiv - positive patients. their use however, is limited because of the lack of an oral formulation. clinical relapse is not uncommon, especially in patients with persistent underlying immunodeficiency (eg, untreated and advanced hiv infection). relapse appears to depend on the duration of antifungal therapy and degree of immunosuppression and may occur sooner following clotrimazole and ketoconazole therapy than after itraconazole, fluconazole, or posaconazole therapy.30,71,80,81 after several recurrences of symptomatic opc in patients with aids, clinicians may consider maintenance (secondary) prophylaxis.80 several dosages of fluconazole as the primary prophylaxis have been evaluated. although most studies documented a reduction in the frequency of mc in treated patients with aids, the regimens did not provide complete protection and occasional breakthrough infections occurred.82,84 powderly compared fluconazole 200 mg / day and clotrimazole troches 10 mg five - times / day prophylactically in hiv - positive patients.83 overall, fluconazole reduced the frequency of mc, superficial fungal infections, and cryptococcal disease when compared to clotrimazole.83 the benefit was greatest in patients with less than 50 cd4 t cells / mm. unfortunately, in this study, the incidence of in vitro resistance and the effect fluconazole had on fungal flora was not evaluated. in addition, although the incidence of fungal infections was reduced, the survival rate was similar in both groups. in this setting, one must weigh the benefits against the cost of daily drug administration, not only in the financial sense, but also the influence of the agent on the patient s mycoflora. schuman evaluated fluconazole 200 mg / week vs placebo in hiv - infected women with cd4 t cell counts < 300 cells / mm.85 the study concluded that weekly fluconazole was effective in preventing symptomatic opc and vaginal candidiasis while the rates of clinical and in vitro resistance were low. additionally, women receiving fluconazole had a reduction in the colonization rates of c. albicans, but had an increased isolation of non - albicans candida species. in a recent multicenter, randomized clinical trial evaluating the role of prophylaxis in hiv - positive patients, goldman showed that the number of episodes of opc and other invasive fungal infections was statistically lower in hiv - positive patients with cd4 t counts < 150 cells / mm, when receiving continuous (three times a week fluconazole) when compared to those patients only receiving episodic treatment with fluconazole for opc recurrences.84 the study also demonstrated that the incidence of clinically significant resistance was no higher in the group receiving continuous therapy than in the group using episodic administration of fluconazole, provided the patients were on highly active antiretroviral therapy. in general, when making the clinical decision to initiate secondary prophylaxis the physician should consider several key points : the impact of excessive recurrences on the patients well being and quality of life, the need for prophylaxis of other fungal infections, financial costs, adverse event profiles, and drug - drug interactions. the clinical impact of antifungal resistance in patients with aids has been demonstrated in patients who failed conventional antifungal therapy for mc.24,86 after the development of fluconazole - resistant opc, patients were noted to have a median survival of approximately 184 days. moreover, after the onset of clinical resistance to parenteral amphotericin b, patients were found to have an astonishing 83-day median survival rate.24,86 although mc per se is not fatal, clinical failure is probably a comorbid factor associated with the rapid demise of these patients. clinical failure may also be a marker of severe immunosuppression and a dysfunctional immune system. antifungal resistance can be divided into two categories, clinical and in vitro. clinical resistance signifies failure of the antifungal to eradicate the infection in the absence of in vitro resistance. such resistance may occur for a variety of reasons. in vitro resistance can also be subdivided into either primary (innate or intrinsic) or secondary (acquired) resistance.24,86 the usefulness of bacterial in vitro susceptibility testing is well established in the management of patients with infectious diseases. however, despite the fact that a subcommittee for in vitro antifungal susceptibility testing published approved guidelines, the utility of these results in managing fungal infections is still somewhat controversial.87,88 several studies have revealed a correlation between the in vitro susceptibility minimum inhibitory concentration [mic ] results of c. albicans and the clinical response to antifungal treatment in hiv - infected patients.60,87,88 investigators have published therapeutic antifungal successes and failures in patients with opc and candida isolates for which mics were both low and high. in fact, one group has reported several hiv - infected patients with documented in vitro fluconazole - resistant mc who were still able to respond to fluconazole therapy. initially, it was believed that the trigger for the emergence of resistance was primarily due to the use of low dose fluconazole for acute treatment as well as for prophylaxis.86,89,90 more recently, investigators have demonstrated that mc due to resistant isolates is seen in patients with low cd4 cell counts (< 50 cells / mm).91 conversely, another study indicated that a low cd4 cell count did not predict azoleresistant mc in patients with aids.61 investigators have be able to identify several key risk factors associated with the development of fluconazole - resistant mc in patients with aids, compared to those patients without evidence of fluconazole - resistant mc. these risk factors include : greater number of episodes of opc (6.1 vs 1.8), lower median cd4 cell count (11 vs 71 cells / mm), longer median duration of antifungal therapy (419 vs 118 days), and longer duration of systemic azoles (272 vs 14 days).91 when the authors used two controls matched by cd4 cell count, resistant cases continued to have a greater median exposure time to azoles (272 vs 88 days ; p = 0.005) as the significant risk factor.61,91 it is still not clear whether the total dose of antifungals, the duration of therapy, the type of antifungal, and/or the pattern of drug administration (continuous vs episodic) are the most important determinants in the development of antifungal - refractory fungal disease.24,84,90,91 more than likely, the etiology is multifactorial involving a combination of advanced immunosuppression, high fungal burdens, and prolonged exposure time to antifungals. the management of fluconazole - resistant mc is frequently unsatisfactory and the clinical response is short - lived with periodic and rapid recurrences. in some patients, the refractory candidiasis may respond to increases in the dose of fluconazole (table 4). for example, if patients fail fluconazole 200 mg / day, a dosage increase to 400800 mg / day will frequently produce a clinical response for a period of time. however, the response is generally transient and the disease recurs rapidly once patients have reached this stage. as noted, clinical and in vitro azole resistance is not uncommon in patients who fail to respond to fluconazole. occasionally, in some patients with fluconazole - refractory mc, fluconazole suspension may be beneficial.91 several reports describe improvement in these patients, possibly associated with increased salivary levels of fluconazole, which results when the suspension is taken with a swish - and - swallow technique.91 several clinical trials evaluating itraconazole oral solution have demonstrated promising results in patients with aids who failed fluconazole 200 mg / day.9295 a clinical cure or improvement occurred in 55%70% of these patients. as expected, mycological cure rates were low (< 30%) and relapses following treatment cessation were rapid, usually within 14 days. the two newly expanded spectrum triazoles, voriconazole and posaconazole, have been used successfully in patients with refractory mc.96,97 posaconazole is licensed in the united states for the treatment of refractory opc / ec. in the largest clinical trial evaluating antifungal agents for refractory opc / ec, posaconazole 400 mg suspension was given either qd (once daily) or bid and was evaluated in subjects with documented clinical resistance to either fluconazole or itraconazole.96 of the 176 subjects enrolled, 132 (75%) achieved a clinical response after 28 days of therapy with very few adverse events. only eight patients discontinued the study drug because of a side effect. as expected, during the 4-week follow up period the overall clinical relapse rate was 74%. it is important to note that with any fungal infection in patients with advanced hiv infection it is essential to continue suppressive antifungal therapy in an attempt to increase disease - free intervals and avoid morbidity and occasional mortality. these patients generally suffer from advanced - hiv disease and ultimately very low cd4 t cells (< 50) and high viral burdens along with uncontrolled hiv infection.81 it is important not to underestimate the significance of the underlying dysfunctional immune system in patients with aids and refractory fungal infections. although randomized clinical trials have not been done, the initiation of haart will frequently assist in the resolution of these recalcitrant infections. in fact, treatment with haart alone without antifungals have eradicated antifungal - refractory opc in patients with advanced hiv infection.98,99 the classic management of infections in the compromised host has depended upon antimicrobial agents, without taking into account host defects. several cytokines developed and produced by recombinant technology show promise in assisting the host response to fungal infection.100,101 there have been several reports describing the use of human recombinant granulocyte - macrophage colony stimulating factor (rhugm - csf) in patients with opc or ec refractory to fluconazole and amphotericin b.100,101 although no large studies have been published, case reports describe good response rates with rhugm - csf in patients with advanced hiv infection and refractory mucosal candidiasis.101,102 further studies evaluating these cytokines are certainly warranted. alternative therapeutic modalities using organic substances are also being administered empirically to combat refractory mc. one of these formulations is melaleuca alternafolia, or australian tea tree oil that has been formulated into an oral solution. a small, single center pilot study evaluating the melaleuca oral solution in 14 patients with aids and fluconazole - refractory opc has been completed.103 the results appear to indicate relatively good efficacy in these difficult - to - treat patients, demonstrating a good clinical response in 10 of 12 patients after four weeks of antifungal therapy. however, larger comparative studies are necessary to evaluate the role of this agent in refractory candidiasis. in conclusion, the impressive clinical trial results and the low grade side effect profile of the azole compounds continue to make them attractive choices in the management of fungal infections in any immunocompromised patient, especially hiv - positive patients. however, difficulties in managing these infections should remind us that we can not rely solely on antifungals. in addition, we must continue to develop more effective antifungal agents with different mechanisms of action and different modes of administration. numerous antifungal agents are available for the treatment of opc and ec in the hiv - positive patient (tables 1 and 2). in addition, guidelines for the management of mucosal candidiasis in hiv - infected patients have been published by the centers for disease control, national institutes of health, and the hiv medicine association of the infectious diseases society of america.22 however, several factors should be considered when selecting antifungal agents for patients with hiv infection. in hiv - positive patients, antifungals are frequently less efficacious than in patients with other immunodeficiencies such as cancer. similarly, the time to clinical response also tends to be delayed in this population.17,22,23 moreover, the relapse rate is higher in patients with hiv than in any other patient population. 1,17,22,23 for unknown reasons, a subgroup of hiv - positive patients experience recurrent episodes of opc and thus receive numerous courses of antifungals during their lifetime. as their hiv infection progresses they tend to experience shorter disease - free intervals between episodes of mucosal candidiasis and thus have greater antifungal exposure, which may ultimately lead to the development of clinical and in vitro antifungal resistance and its associated morbidity and mortality. it is important to note that, as with some opportunistic pathogens in this patient population, antifungal treatment merely reduces the signs and symptoms of infection and thus produces a transient clinical response by lowering the quantity of organisms in the affected area. it is extremely difficult to fully eradicate candida from the mucosal surfaces of patients who are immunocompromised, especially those who are hiv - positive. 8,18,23 the combination of frequent clinical relapses and increased antifungal utilization are frequently associated with antifungal resistance.24 this is reflected, clinically, by the ineffectiveness of antifungals to which a patient has not previously been exposed. thus, we are becoming increasingly aware that choosing the appropriate agent, even in the early stages of disease, is extremely important because of the future repercussions of these selections in advanced stages of infection. classes of agents used in the treatment of mc in hiv includes the polyenes (eg, amphotericin b, nystatin), pyrimidine synthesis inhibitors (flucytosine), azoles (miconazole, clotrimazole, ketoconazole, itraconazole, fluconazole, voriconazole, posaconazole), and, more recently, the echinocandins (caspofungin, micafungin, anidulafungin).17,22,24 common dosage regimens of these agents are listed in tables 1 and 2. the key trials evaluating antifungals in the treatment of opc and ec are summarized in table 3. most controlled studies to date have evaluated the azole antifungal agents and in general, clinical response rates appear to be similar. studies of antifungal treatment in mc can be problematic to evaluate, particularly in the hiv - positive population. limitations often include a small number of patients, open label design, and a short follow - up time. additional studies are particularly needed among patients with low cd4 t lymphocyte counts, as this population tends to have lower clinical and mycological response rates. a recent meta - analysis suggested that larger studies using more consistent outcome measures and reporting would be helpful in applying future research to current clinical practice.25 additionally, these authors also suggested that future investigations should include less expensive antifungal interventions and, at the same time, evaluate symptom - free periods, quality of life, survival, and the development of clinical and in vitro resistance. prior to the development of the polyenes and the azoles, topical therapy consisted of gentian violet applications that were reasonably effective in localized mc, but were extremely messy because of the purple color.24,26 while topical therapies have historically been effective in less severe disease and are relatively inexpensive compared to some of the systemic therapies, their use has diminished due to poor tolerability and poor adherence. topical therapy of opc can be accomplished with a multitude of antifungal agents including nystatin, amphotericin b, clotrimazole, and, more recently, miconazole (table 1).17,22,24,2631 nystatin is available in several formulations, including pastilles and suspension. limitations of topical agents such as nystatin include a bitter taste, gi - adverse effects, and frequent dosing, all of which may contribute to reduced adherence.24,27 importantly, nystatin has not demonstrated significant efficacy in severely immunocompromised patients, such as those with advanced hiv infection.27,28 for instance, in hiv - positive patients, nystatin exhibited lower rates of both clinical cure (52% vs 87%) and mycological cure (6% vs 60%) when compared to oral fluconazole. additionally, the 28- day relapse rate was found to be greater with nystatin (44% vs 18%) when compared to fluconazole.27 amphotericin b is also an option for topical therapy and is available in suspension, lozenge, and tablet form.1,22 neither nystatin, nor amphotericin b is absorbed from the gastrointestinal tract so administration must be frequent (four times daily) to provide adequate drug exposure to the infected mucosal tissues.1,22 prospective, comparative studies evaluating amphotericin b oral solution against other antifungals are limited. topical azole antifungal agents, such as clotrimazole 10 mg troches, administered five times daily, provide another option for opc patients. topical clotrimazole has been used successfully in treating mild - to - moderate opc during the early stages of hiv disease.2830 the newest topical antifungal is the mucoadhesive buccal tablet (mmbt) containing 50 mg of miconazole (loramyc).31 this novel formulation of miconazole has been approved in europe since 2008 for the treatment of opc in immucocompromised hosts. the formulation is unique, because 50 mg of miconazole is contained in each mucoadhesive tablet and is applied to the mucosa of the upper gum over the canine fossa, once daily, for 714 days. the mmbt adheres to the gum surface because of the milk protein concentrate composition of the tablet. this interaction leads to a rapid and prolonged adhesion to the mucosa due to an adsorption mechanism, followed by a protein - protein interaction. in pharmacokinetic studies, 50 mg of mmbt provides a maximum salivary concentration of 15 g / ml, up to seven hours after the application of the tablet.31 three separate clinical studies have evaluated the use of mmbts for the treatment of opc in the hiv - positive population and in patients with head and neck cancers.3234 in a phase iii, double - blind, double - dummy, multicenter trial evaluating 578 patients with hiv and opc, mmbt treatment was compared to clotrimazole troches (10 mg, five times daily, for a period of 14 days).32 the results at the primary endpoint of test of cure (toc) in both the intent - to - treat (itt) population and in the per - protocol population (pp) demonstrated that the once daily administration of a mmbt was as effective as the five times daily clotrimazole treatment. clinical cure rates at toc in both the itt (61% vs 65%) and pp (68% vs 74%) populations demonstrated no inferiority to clotrimazole. in addition, secondary endpoints such as safety and tolerability were similar between both treatment groups. the use of topical antimycotic agents has been replaced with systemic azole antifungals such as ketoconazole, fluconazole, itraconazole, and more recently posaconazole (table 1).17,24 part of the reason for this is that, although clinical cure rates may be similar, microbiologic cure and long term efficacy are not. in one example, a study comparing systemic oral fluconazole with clotrimazole troches in hiv - positive adults found clinical efficacy to be similar (98% vs 94%). however, microbiological cure rates were greater in patients treated with fluconazole (65% vs 48%) and clinical response sustained through a 2-week follow up was also greater (82% vs 50%).30 the currently available systemic azoles include ketoconazole, fluconazole, itraconazole, and posaconazole. ketoconazole was the first available oral systemic imidazole antifungal agent with high rates of efficacy in opc.35,36 however, in comparative trials, ketoconazole was found to be less efficacious then fluconazole in both clinical and mycological cure rates.37 in addition, the use of ketoconazole is further limited by potentially severe adverse reactions including hepatotoxicity, poor oral bioavailability, and a host of drug drug interactions.36 ketoconazole is a very potent inhibitor of cytochrome p450 3a4 and is relatively contraindicated with some hiv - protease inhibitors.38 because the drug s absorption is dependent upon an acidic ph, there are also concerns that systemic absorption may be inadequate in patients receiving acid - suppressive therapy or with aids - related hypochlorhydria.39 in view of the lower clinical efficacy rates and the associated adverse event profile (ie, hepatotoxicity), ketoconazole is not widely used anymore. in contrast to ketoconazole, other azoles such as fluconazole, itraconazole, voriconazole, and posaconazole have demonstrated improved efficacy, as well as excellent safety profiles, and have thus become the drugs of choice for opc, especially in hiv - positive patients (table 3).1,22,24,30 fluconazole is the most commonly used antifungal in the treatment of mc in hiv - infected patients. fluconazole is more readily absorbed than other oral azoles without being affected by either food or gastric acidity. the clinical efficacy of fluconazole has been established in many well controlled clinical trials, such that it has become the standard comparator in clinical trials of novel agents.24,30,37,4042 most studies of fluconazole have used an initial loading dose of 200 mg followed by 100 mg daily, but clinical success has been achieved with doses as low as 50 mg / day.40 clinical response is usually apparent within 10 days with 50 mg / day, and 5 to 7 days for doses of 100200 mg / day.17,40 of the available azoles, fluconazole is associated with the fewest drug drug interactions because it has less affinity for the cyp3a4 enzyme.38,43 the pharmacological interactions with fluconazole include concomitantly administered phenytoin, rifampin, rifabutin, cyclosporin a, and possibly some of the protease inhibitors.38,39,4345 fluconazole is generally well tolerated, however, as with any other azole being used long term, periodic surveillance of liver enzymes to monitor for hepatotoxicity is useful.24,43 itraconazole is supplied in a cyclodextrin oral solution or capsule ; the parenteral formulation is no longer manufactured in the united states.1,24,46 like ketoconazole, itraconazole exhibits a strong potential for drug drug interactions through the cpy3a4 enzyme system.38,46,47 the itraconazole oral solution has greater bioavailability than the capsule and absorption is further enhanced by postprandial administration. 40,4648 one prospective, randomized trial in hiv - positive and aids patients with opc, found itraconazole oral solution to have similar efficacy and safety as fluconazole (clinical response 97% vs 87%).49 however, approximately 50% of the patients in both groups experienced relapses at the 1-month follow - up evaluation. posaconazole is the newest triazole on the market and is approved for the treatment of acute opc and antifungal - refractory mc.5052 it is an oral extended - spectrum triazole with potent in vitro activity against pathogenic yeast and moulds, including fluconazole- and itraconazole - resistant candida strains.50 as with other azoles, posaconazole inhibits lanosterol 14--demehylase. it appears that mutations near the heme cofactor of cyp51 reduce the binding affinity of compact azoles, such as fluconazole and itraconazole, and may lead to azole crossresistance.50 however, 3-dimensional binding models suggest that the long side chain of posaconazole may result in tighter binding affinity.53,54 thus, posaconazole may be less susceptible than some azoles to the development of secondary azole resistance. posaconazole is absorbed in an oral suspension.50 it is important to note that posaconazole absorption is enhanced by coadministration with food, especially high fatty meals, or with a nutritional supplement such as boost plus (nestl, fremont, mi).55,56 when food intake is limited, dividing the daily dose from bid (twice daily) to qid (four times daily) also increases the plasma concentration. unlike itraconazole and voriconazole, posaconazole is not primarily metabolized by fungal cytochrome p450 enzymes.57 during several drug interaction trials evaluating the effects of posaconazole on the cyp450 enzymes, posaconazole did demonstrate an inhibitory effect on cyp3a4, but did not influence the other isoenzymes. in a large multicenter, randomized clinical trial, a 100 mg daily dose of posaconazole was compared to a 100 mg daily dose of fluconazole in hiv - positive patients with opc.58 clinical success was reported in 92% of the posaconazole recipients compared with a success rate of 92% for those patients that received fluconazole. the only difference was in the long - term follow - up, where clinical relapses occurred more frequently in the fluconazole group compared to the posaconazole group (38.2% vs 31.5%). several concerns have been raised about the widespread use of the more potent oral azoles, which may offer only minor advantages for patients. these concerns include drug interactions, side effects, increased expense, and risk of developing antifungal resistance. an increased frequency of c. glabrata isolation has been described by several investigators of hiv - positive patients receiving prolonged courses of fluconazole.5962 in addition, although azole resistance in c. albicans is rare, several reports describe both clinical failure and in vitro resistance in both non - hiv patients and in hiv - infected patients on long - term azoles.24,63 systemic antifungal therapy using oral or parenteral fluconazole has been the mainstay in the management of ec for over a two decades (tables 2 and 3).1,17 topical antifungals such as nystatin, clotrimazole, and miconazole are of minimal value in ec.22,64 the initial step in the management of ec should always be to attempt to minimize all possible predisposing factors, such as corticosteroids, chemotherapeutic agents, and antimicrobials.1,17,22 oral fluconazole has an excellent safety profile when compared to ketoconazole, demonstrates excellent gastric absorption, and can also be given intravenously when necessary. similar to the observations in opc research, studies comparing fluconazole with either clotrimazole or ketoconazole for ce demonstrate cure rates that are superior to those with other imidazoles. moreover, fluconazole demonstrated a more rapid onset of action and quicker resolution of symptoms. 6567 itraconazole has also been shown to be effective in the treatment of ec.6870 patients treated with itraconazole oral solution (100200 mg / day) had clinical response rates comparable to those of patients treated with fluconazole tablets (100200 mg / day) of 94% and 91%, respectively, without significant adverse effects in either group.68 the mycological cure rates were also similar at 92% and 78%, respectively. voriconazole, another broad spectrum systemic triazole is also approved for the treatment of ec at a dose of 200 mg bid for 1421 days. in vitro, it has been shown to be 10- to 500-fold more potent than fluconazole against a wide array of yeast and moulds, including many isolates that have demonstrated in vitro itraconazole- and/or fluconazole - resistance. 71 in a double - blind, randomized, multicenter study, voriconazole 200 mg bid was compared to fluconazole 200 mg daily in ec.72 the overall success rates were comparable at 98.2% for voriconazole vs 95% for fluconazole. as with most azoles, voriconazole has an effect on the cyp450 enzymes, so caution regarding drug - drug interactions is warranted.36,71 a common side effect with voriconazole includes photopsia (visual abnormalities) that may occur in 20%30% of patients. other side effects may include elevations in transaminases (~10%) and skin rashes (~10%), with rare cases of photodermatitis in patients who are exposed to direct sunlight.71 prior to the availability of azole antifungal agents, amphotericin b deoxycholate was used extensively. however, it is now rarely used in any patient and is generally reserved for antifungal - refractory cases of candida esophagitis that do not respond to azoles or echinocandins.73 if necessary, low dose amphotericin b (0.150.3 mg / kg / day or 1020 mg / day for 1014 days) is sufficient for moderate to severe disease.1,17,73 however, because of the improved adverse event profiles of the lipid formulations of amphotericin b, they have also become popular. oral flucytosine (100150 mg / kg / day in divided doses), although effective, is rarely used because of the tendency for resistance to develop during therapy and the well described frequency of bone marrow suppression and transaminase elevation.1,74 furthermore, in one comparative clinical trial, clinical efficacy and mycological cure rates were lower for flucytosine when compared to fluconazole.74 the echinocandin class of antifungals, which includes caspofungin, micafungin, and anidulafungin are a novel class of antifungal agents with a completely different mechanism of action.75 all three agents have demonstrated excellent in vitro activity against a broad array of candida species, including those that are resistant to fluconazole, itraconazole, or voriconazole.75 there have been a total of five clinical trials evaluating the efficacy of the three echinocandins in treating ec in hiv - positive patients. in all studies, the echinocandins are compared to either fluconazole or amphotericin b. in a clinical trial comparing caspofungin to amphotericin b in both opc and ec in a population that was predominantly hiv - positive, clinical success rates for caspofungin ranged from 74% to 91%, which was numerically greater than the success rate for amphotericin b (63%).76 in a related study, 177 patients with ec were stratified to receive either fluconazole 200 mg / day or caspofungin 50 mg/ day. caspofungin was found to have similar response and relapse rates as fluconazole.77 in another clinical trial, the treatment of ec among hiv - positive patients was also found to be similar for micafungin 150 mg / day when compared to fluconazole 200 mg / day.78 in a separate multicenter clinical study in hiv - positive patients (n = 601, 75% with aids) evaluating anidulafungin versus fluconazole in patients with ec found this echinocandin to have similar clinical response rates when compared to fluconazole, in terms of both efficacy and safety.79 so, while the echinocandins are not considered first line therapy for ec, they are effective antifungals for the treatment of mc in hiv - positive patients. clinical relapse is not uncommon, especially in patients with persistent underlying immunodeficiency (eg, untreated and advanced hiv infection). relapse appears to depend on the duration of antifungal therapy and degree of immunosuppression and may occur sooner following clotrimazole and ketoconazole therapy than after itraconazole, fluconazole, or posaconazole therapy.30,71,80,81 after several recurrences of symptomatic opc in patients with aids, clinicians may consider maintenance (secondary) prophylaxis.80 several dosages of fluconazole as the primary prophylaxis have been evaluated. although most studies documented a reduction in the frequency of mc in treated patients with aids, the regimens did not provide complete protection and occasional breakthrough infections occurred.82,84 powderly compared fluconazole 200 mg / day and clotrimazole troches 10 mg five - times / day prophylactically in hiv - positive patients.83 overall, fluconazole reduced the frequency of mc, superficial fungal infections, and cryptococcal disease when compared to clotrimazole.83 the benefit was greatest in patients with less than 50 cd4 t cells / mm. unfortunately, in this study, the incidence of in vitro resistance and the effect fluconazole had on fungal flora was not evaluated. in addition, although the incidence of fungal infections was reduced, the survival rate was similar in both groups. in this setting, one must weigh the benefits against the cost of daily drug administration, not only in the financial sense, but also the influence of the agent on the patient s mycoflora. schuman evaluated fluconazole 200 mg / week vs placebo in hiv - infected women with cd4 t cell counts < 300 cells / mm.85 the study concluded that weekly fluconazole was effective in preventing symptomatic opc and vaginal candidiasis while the rates of clinical and in vitro resistance were low. additionally, women receiving fluconazole had a reduction in the colonization rates of c. albicans, but had an increased isolation of non - albicans candida species. in a recent multicenter, randomized clinical trial evaluating the role of prophylaxis in hiv - positive patients, goldman showed that the number of episodes of opc and other invasive fungal infections was statistically lower in hiv - positive patients with cd4 t counts < 150 cells / mm, when receiving continuous (three times a week fluconazole) when compared to those patients only receiving episodic treatment with fluconazole for opc recurrences.84 the study also demonstrated that the incidence of clinically significant resistance was no higher in the group receiving continuous therapy than in the group using episodic administration of fluconazole, provided the patients were on highly active antiretroviral therapy. in general, when making the clinical decision to initiate secondary prophylaxis the physician should consider several key points : the impact of excessive recurrences on the patients well being and quality of life, the need for prophylaxis of other fungal infections, financial costs, adverse event profiles, and drug - drug interactions. the clinical impact of antifungal resistance in patients with aids has been demonstrated in patients who failed conventional antifungal therapy for mc.24,86 after the development of fluconazole - resistant opc, patients were noted to have a median survival of approximately 184 days. moreover, after the onset of clinical resistance to parenteral amphotericin b, patients were found to have an astonishing 83-day median survival rate.24,86 although mc per se is not fatal, clinical failure is probably a comorbid factor associated with the rapid demise of these patients. clinical failure may also be a marker of severe immunosuppression and a dysfunctional immune system. antifungal resistance can be divided into two categories, clinical and in vitro. clinical resistance signifies failure of the antifungal to eradicate the infection in the absence of in vitro resistance. can also be subdivided into either primary (innate or intrinsic) or secondary (acquired) resistance.24,86 the usefulness of bacterial in vitro susceptibility testing is well established in the management of patients with infectious diseases. however, despite the fact that a subcommittee for in vitro antifungal susceptibility testing published approved guidelines, the utility of these results in managing fungal infections is still somewhat controversial.87,88 several studies have revealed a correlation between the in vitro susceptibility minimum inhibitory concentration [mic ] results of c. albicans and the clinical response to antifungal treatment in hiv - infected patients.60,87,88 investigators have published therapeutic antifungal successes and failures in patients with opc and candida isolates for which mics were both low and high. in fact, one group has reported several hiv - infected patients with documented in vitro fluconazole - resistant mc who were still able to respond to fluconazole therapy. initially, it was believed that the trigger for the emergence of resistance was primarily due to the use of low dose fluconazole for acute treatment as well as for prophylaxis.86,89,90 more recently, investigators have demonstrated that mc due to resistant isolates is seen in patients with low cd4 cell counts (< 50 cells / mm).91 conversely, another study indicated that a low cd4 cell count did not predict azoleresistant mc in patients with aids.61 investigators have be able to identify several key risk factors associated with the development of fluconazole - resistant mc in patients with aids, compared to those patients without evidence of fluconazole - resistant mc. these risk factors include : greater number of episodes of opc (6.1 vs 1.8), lower median cd4 cell count (11 vs 71 cells / mm), longer median duration of antifungal therapy (419 vs 118 days), and longer duration of systemic azoles (272 vs 14 days).91 when the authors used two controls matched by cd4 cell count, resistant cases continued to have a greater median exposure time to azoles (272 vs 88 days ; p = 0.005) as the significant risk factor.61,91 it is still not clear whether the total dose of antifungals, the duration of therapy, the type of antifungal, and/or the pattern of drug administration (continuous vs episodic) are the most important determinants in the development of antifungal - refractory fungal disease.24,84,90,91 more than likely, the etiology is multifactorial involving a combination of advanced immunosuppression, high fungal burdens, and prolonged exposure time to antifungals. the management of fluconazole - resistant mc is frequently unsatisfactory and the clinical response is short - lived with periodic and rapid recurrences. in some patients, the refractory candidiasis may respond to increases in the dose of fluconazole (table 4). for example, if patients fail fluconazole 200 mg / day, a dosage increase to 400800 mg / day will frequently produce a clinical response for a period of time. however, the response is generally transient and the disease recurs rapidly once patients have reached this stage. as noted, clinical and in vitro azole resistance is not uncommon in patients who fail to respond to fluconazole. occasionally, in some patients with fluconazole - refractory mc, fluconazole suspension may be beneficial.91 several reports describe improvement in these patients, possibly associated with increased salivary levels of fluconazole, which results when the suspension is taken with a swish - and - swallow technique.91 several clinical trials evaluating itraconazole oral solution have demonstrated promising results in patients with aids who failed fluconazole 200 mg / day.9295 a clinical cure or improvement occurred in 55%70% of these patients. as expected, mycological cure rates were low (< 30%) and relapses following treatment cessation were rapid, usually within 14 days. the two newly expanded spectrum triazoles, voriconazole and posaconazole, have been used successfully in patients with refractory mc.96,97 posaconazole is licensed in the united states for the treatment of refractory opc / ec. in the largest clinical trial evaluating antifungal agents for refractory opc / ec, posaconazole 400 mg suspension was given either qd (once daily) or bid and was evaluated in subjects with documented clinical resistance to either fluconazole or itraconazole.96 of the 176 subjects enrolled, 132 (75%) achieved a clinical response after 28 days of therapy with very few adverse events. as expected, during the 4-week follow up period the overall clinical relapse rate was 74%. it is important to note that with any fungal infection in patients with advanced hiv infection it is essential to continue suppressive antifungal therapy in an attempt to increase disease - free intervals and avoid morbidity and occasional mortality. these patients generally suffer from advanced - hiv disease and ultimately very low cd4 t cells (< 50) and high viral burdens along with uncontrolled hiv infection.81 it is important not to underestimate the significance of the underlying dysfunctional immune system in patients with aids and refractory fungal infections. although randomized clinical trials have not been done, the initiation of haart will frequently assist in the resolution of these recalcitrant infections. in fact, treatment with haart alone without antifungals have eradicated antifungal - refractory opc in patients with advanced hiv infection.98,99 the classic management of infections in the compromised host has depended upon antimicrobial agents, without taking into account host defects. several cytokines developed and produced by recombinant technology show promise in assisting the host response to fungal infection.100,101 there have been several reports describing the use of human recombinant granulocyte - macrophage colony stimulating factor (rhugm - csf) in patients with opc or ec refractory to fluconazole and amphotericin b.100,101 although no large studies have been published, case reports describe good response rates with rhugm - csf in patients with advanced hiv infection and refractory mucosal candidiasis.101,102 further studies evaluating these cytokines are certainly warranted. alternative therapeutic modalities using organic substances are also being administered empirically to combat refractory mc. one of these formulations is melaleuca alternafolia, or australian tea tree oil that has been formulated into an oral solution. a small, single center pilot study evaluating the melaleuca oral solution in 14 patients with aids and fluconazole - refractory opc has been completed.103 the results appear to indicate relatively good efficacy in these difficult - to - treat patients, demonstrating a good clinical response in 10 of 12 patients after four weeks of antifungal therapy. however, larger comparative studies are necessary to evaluate the role of this agent in refractory candidiasis. in conclusion, the impressive clinical trial results and the low grade side effect profile of the azole compounds continue to make them attractive choices in the management of fungal infections in any immunocompromised patient, especially hiv - positive patients. however, difficulties in managing these infections should remind us that we can not rely solely on antifungals. in addition, we must continue to develop more effective antifungal agents with different mechanisms of action and different modes of administration. | mucocutaneous candidiasis is frequently one of the first signs of human immunodeficiency virus (hiv) infection. over 90% of patients with aids will develop oropharyngeal candidiasis (opc) at some time during their illness. although numerous antifungal agents are available, azoles, both topical (clotrimazole) and systemic (fluconazole, itraconazole, voriconazole, posaconazole) have replaced older topical antifungals (gentian violet and nystatin) in the management of oropharyngeal candidiasis in these patients. the systemic azoles, are generally safe and effective agents in hiv - infected patients with oropharyngeal candidiasis. a constant concern in these patients is relapse, which is dependent on the degree of immunosuppression commonly seen after topical therapy, rather than with systemic azole therapy. candida esophagitis (ce) is also an important concern since it occurs in more than 10% of patients with aids and can lead to a decrease in oral intake and associated weight loss. fluconazole has become the most widely used antifungal in the management of mucosal candidiasis. however, itraconazole and posaconazole have similar clinical response rates as fluconazole and are also effective alternative agents. in patients with fluconazole - refractory mucosal candidiasis, treatment options now include itraconazole solution, voriconazole, posaconazole, and the newer echinocandins (caspofungin, micafungin, and anidulafungin). |
quinones form the second largest class of antitumor agents approved for clinical use in the u.s., and several antitumor quinones are in different stages of clinical and preclinical development a common feature of quinone - containing drugs is their ability to undergo reversible redox reactions to form semiquinone and oxygen radicals. one - electron reduction of a quinone (q) gives the semiquinone radical (q or qh), whereas two - electron reduction gives the hydroquinone (qh2). the semiquinone can also be formed by a comproportionation reaction between a quinone and a hydroquinone (reaction 1 ; the opposite of reaction 1 is the semiquinone disproportionation reaction).1the catalytic enhancement of ascorbate (asch) oxidation by quinones has been previously observed. in addition, we have previously observed that quinones enhance the rates of ascorbate reduction of nitric oxide and s - nitrosothiols. in each of those works, the enhancement activity of quinones increases with its one - electron reduction potential within a certain range of one - electron reduction potential values. thus, the semiquinone, the one - electron - reduced species, is postulated to be the actual intermediate responsible for reducing those targets. furthermore, antitumor activity enhancement by quinones in the presence of ascorbate has been reported. hydrogen peroxide has important roles as a signaling molecule in the regulation of a variety of biological processes. hydrogen peroxide also plays an important role in aging and cancer. although quinone - enhanced ascorbate reduction reactions of the species mentioned above have been reported, very few works report the possibility of a semiquinone - mediated reduction of h2o2. in fact, koppenol and butler suggested that quinones with reduction potentials between 330 and 460 mv could be involved in metal - independent fenton reaction 2.2however, there has not been any study comparing the reactivities among quinones with regard to reaction 2 with their dependence on the quinone one - electron reduction potential. reaction 2 acquires more biomedical importance in view of the facts that parenteral administration of ascorbate generates h2o2 in tissues and that quinones enhance the antitumor activity of ascorbate. these observations suggest that quinones may be acting as h2o2 production promoters and/or as enhancers of hydroxyl radical production. the metal - independent production of hydroxyl radicals by the reaction of h2o2 with halogenated quinones in the absence of reducing agents has been reported. this type of reaction was not detected for non - halogenated quinones, indicating the need for electron - withdrawing substituents at the quinone ring for this reaction to occur. in this work, we have determined the relative extent of hydroxyl radical production in the presence and absence of non - halogenated quinones, using ascorbate as the reducing agent. hydroxyl radicals can be determined by spin trapping using nitrones, and the resulting nitroxide concentration can be measured by electron paramagnetic resonance (epr) spectrometry. however, nitroxides are prone to reduction to the epr - silent hydroxylamine in the presence of reducing agents. therefore, in this work, we have determined the relative ability of quinones to enhance the rate of ascorbate reduction of h2o2 by measuring the amount of methane produced from the reaction of hydroxyl radicals with dmso. a similar procedure for the selective detection of oh radical production, where dmso is the hydroxyl radical scavenger and the produced methane gas is detected by gas chromatography (gc), has been previously used in several works. the mechanism of this reaction has been previously determined using epr and radiolysis techniques. these quinones were selected because they have a wide range of one - electron reduction potentials, are commercially available, and were found to be active in the enhanced reduction of oxygen by ascorbate. methane (99.0% pure) was purchased in a gas lecture bottle from sigma - aldrich corp. stock solutions of quinones were prepared in water and used on the same day of their preparation. deionized and chelex - treated water was used in the preparation of all stock and sample solutions. chelex treatment of water was monitored using the ascorbate test, as described by buettner. stock solutions of ascorbic acid, phosphate buffer (ph 7.4), detapac, h2o2, dmso, and quinone were deareated by purging with high - purity n2 and equilibrated to 37 c in a temperature - controlled water bath. the reaction mixture most frequently used contained 10 m quinone, 100 m detapac, 100 m neocuproine, 10 mm ascorbate, and 2 mm h2o2 in a 1:3 dmso / phosphate buffer (20 mm, ph 7.4) (v / v) mixture. the reason for adding detapac and neocuproine was to further inhibit transition metal catalysis in addition to that mediated by chelex treatment of solutions. nitrogen - saturated amber 3.00 ml septum - capped bottles were used with continuous spinning bar stirring. the reaction bottles were placed in a water - jacketed beaker with a constant temperature of 37 c before addition of reagents. after the reaction, a single aliquot of 50 l was withdrawn from the gas phase on top of the reaction mixture and injected, using a gastight syringe, into a gas chromatograph for analysis. the chromatographic peak retention time of ch4 was established by using a ch4 standard. the chromatograph used was an agilent 6890 with thermal conductivity detection and an alltech 16226 porapak q column. chromatographic conditions were as follows : flow rate of 5.0 ml of he / min, column temperature of 35 c, and injector and detector temperatures of 120 and 150 c, respectively. alternatively, the detection of hydroxyl radicals was also done by measuring the hydroxylation products of salicylic acid. a reaction mixture containing 10 m phq or dq or no quinone, 100 m detapac, 100 m neocuproine, 10 mm ascorbate, 1 mm salicylic acid, and 2 mm h2o2 in 20 mm phosphate buffer, ph 7.4, was incubated for 40 min followed by hplc determination of 2,3- and 2,5-dihydroxybenzoic acid (2,3-dhb and 2,5-dhb, respectively) in the sample. the hydroquinone of phq was prepared, as described elsewhere, by reducing the quinone with nabh4. for this purpose, a n2-saturated hydroquinone aqueous stock solution was prepared by mixing a n2-saturated phq solution in methanol with solid nabh4. the solution was then dried with a n2 stream followed by addition of a n2-saturated water / dmso (1:1 v / v) mixture. the solution ph was decreased to 3 by hcl addition to destroy excess nabh4 and to stabilize the hydroquinone solution. half - wave reduction potentials were determined in nitrogen - purged acetonitrile solutions containing 1 mm quinone and 0.1 m tetra - n - butylammonium perchlorate (tbap) using differential pulse voltammetry (dpv). a bas cv 50w voltammetric analyzer using a glassy carbon working electrode was used for these determinations. an ag / agcl(sat) electrode was used as the reference electrode (e = + 0.22 v vs nhe), and a platinum wire was used as the counter electrode. differential pulse voltammograms were obtained in the potential range from 2.00 to 0.00 v, using a 50 mv pulse amplitude and 20 mv / s of scan rate. the reduction potential values were obtained from the dpv peak potential maxima. these are similar to the half - wave one - electron reduction potentials, e1/2, in normal polarographic measurements. hplc analyses were done using a hp zorbax c-18 (4.6 250 mm) column and eluted using a gradient from 100% ammonium phosphate (ph 3.5) to 100% methanol. an agilent 1100 analytical hplc system with absorption detection at 300 nm was used. the retention times of the corresponding phq, salicylic acid, and 2,3- and 2,5-dhb peaks were determined using commercial standards. all determinations were repeated at least three times, and the average of these determinations sd is reported. upon adding a n2-saturated millimolar ascorbate solution to a n2-saturated solution containing detapac, neocuproine, and h2o2 (millimolar) in a 1:3 dmso / phosphate buffer (20 mm, ph 7.4) (v / v) mixture and incubating that solution for 40 min, a gc peak with a retention time of 5.4 min was detected. the identity of that peak was established by injecting pure methane to the gc. if an identical sample was prepared that instead contained 10 m phq, then a methane gc peak was also detected that was 24 times more intense than that observed in the absence of quinone (figure 2). thus, phq strongly enhances the rate of hydroxyl radical formation, even at micromolar concentrations. gas chromatograms of the gaseous phase of samples after 40 min of incubation. samples contained (a) 100 m detapac, 100 m neocuproine, 10 mm ascorbate, and 2 mm h2o2 in 1:3 dmso / phosphate buffer (20 mm, ph 7.4) (v / v) ; (b) the same composition as in panel (a) but with 10 m phq ; and (c) 10 m fecl2, 10 mm ascorbate, and 2 mm h2o2 in 1:3 dmso / phosphate buffer (20 mm, ph 7.4) (v / v). as indicated earlier, the indirect selective detection of oh radical production, where dmso is the hydroxyl radical scavenger and the produced methane gas is detected by gas chromatography (gc), has been previously used in several works. the mechanism of this reaction has been previously determined using radiolysis and epr techniques. although ethane could be another observed product, its production is very limited at the high dmso concentration used in this work (ca. 3.5 m) and thus the major gaseous product is methane. if phq was replaced by other quinones, then production of hydroxyl radicals was also detected in a manner that depends on the quinone one - electron reduction potential (figure 3). incubation times of 4 to 20 h were previously used in determining henry s constants for methane in water. the incubation time of 40 min used in the present work was selected in order to detect a relatively strong gc signal as well as to discriminate reactivity differences among the quinones used. a time course for ch4 generation by a sample, using phq as the quinone, is shown in figure 4. although the gc ch4 peak areas do not correspond to the maximum areas that can be measured upon reaching methane solubility equilibrium, they reveal the catalytic effect of quinones on the production of hydroxyl radicals from h2o2 + ascorbate as well as their relative abilities to do so. relative ch4 concentrations determined after a 40 min incubation of n2-saturated samples containing 10 m quinone, 100 m detapac, 100 m neocuproine, 10 mm ascorbate, and 2 mm h2o2 in 1:3 dmso / phosphate buffer (20 mm, ph 7.4) (v / v). the symbols used to represent a blank sample without quinone, phqh2, and fe are for solutions without quinone, with 10 m phqh2, or with 10 m fecl2, respectively, and correspond to the methane concentration, but not to the one - electron reduction potential, indicated by the position of the symbol. the symbols ascribed to phq correspond to the same sample composition as that listed (), with 10 m dfo added (), or with 100 m bathophenanthroline disulfonate and 100 m ferrozine added (). time dependence of ch4 concentrations determined from the headspace of a n2-saturated sample containing 10 m phq, 10 mm ascorbate, 2 mm h2o2,100 m detapac, and 100 m neocuproine in 1:3 dmso / phosphate buffer (20 mm, ph 7.4) (v / v). in order to further evidence hydroxyl radical formation, another probe for hydroxyl radicals was used, i.e., the hydroxylation of salicylic acid to form 2,3- and 2,5-dhb. because the formation of 2,3-dhb is more prominent under our conditions, its chromatographic area was used as a measure of hydroxyl radical production. the ratio of the 2,3-dhb chromatographic peak area corresponding to a phq - containing sample to that of a dq - containing sample (4.0 0.2) was essentially the same as the ratio of the ch4 chromatographic peak area corresponding to a phq - containing sample to that of a dq - containing sample (3.8 0.3). thus, the observed behavior between these 2 quinones is independent of the assay used to detect hydroxyl radical production. in the absence of ascorbate, the ch4 gc peak areas obtained for samples containing phq, ubq, mnq, and dmobq are essentially the same as that of a blank sample in the absence of quinone. this indicates that the reaction reported for halogenated quinones involving the nucleophilic addition of hoo to the quinone ring followed by hydroxyl radical production is not occurring. this is consistent with a previous report of the absence of hydroxyl radical production upon mixing h2o2 with dmbq or dq. varying chelating agent concentrations from 0 to 400 m in samples containing the same amounts of ascorbate, h2o2, and phq, respectively, produced only a variation of up to ca. furthermore, addition of 10 m of the iron chelator deferroxamine (dfo) or 100 m of each of the nonhydroxamate iron chelators bathophenanthroline disulfonate and ferrozine to the sample in the presence of 100 m detapac and 100 m neocuproine did not decrease the methane gc peak area compared to that in the absence of these chelators (figure 3). the concentration of dfo used should be able to chelate any trace iron present after chelex treatment of the water or solution. thus, the dependence of the extent of methane production on the quinone one - electron reduction potential and the absence of variations in methane production as a function of transition metal chelator concentration indicate that the observed catalytic activity of the quinones is not metal - mediated. substitution of phq with the same concentration of fecl2 as that of the quinone, but excluding metal chelators in the incubation sample, exceeds the reactivity of 10 m phq in producing hydroxyl radicals by 14% (figure 3), as detected from the methane gc peak area. the fenton reaction rate constant (fe + h2o2) is reported to be 2.0 10 m s in phosphate buffer. assuming instantaneous semiquinone formation (see below) and similar rate constants for reactions after the hydroxyl radical production step, we can estimate the rate constant for the phq - assisted fenton reaction (reaction 2) from the gc peak area obtained from the phq - containing sample, compared to that of the fe - containing sample, as k2 = (0.86) (2.0 10) = 1.7 10 m s. thus, phq reactivity is comparable to that of fe. because phq is a common toxin present in diesel combustion smoke, the possibility that phq - mediated catalysis of hydroxyl radical formation is similar to that of fe adds another important pathway to the modes in which phq can execute its toxicity. in an identical manner, values of k2 can be estimated for dmbq, mnq, and dq as 1.3 10, 7.4 10, and 4.6 10 m s, respectively. rate constants, k2, for the electron transfer from the semiquinones of dmbq, mnq, and dq to oxygen to form superoxide are 8.8 10, 2.4 10, and 2.2 10 m s, respectively. therefore, at equal concentrations of oxygen and h2o2, semiquinone reduction of oxygen is expected for these quinones instead of h2o2 reduction. thus, h2o2 reduction by semiquinones should acquire more importance in hypoxic tissues whenever h2o2 concentrations exceed that of oxygen by several orders of magnitude. relative ch4 concentrations determined after a 40 min incubation of n2-saturated samples containing (a) 10 m phq, 10 mm ascorbate, 2 mm h2o2, and various concentrations of detapac and neocuproine ; (b) 10 m phq, 100 m detapac, 100 m neocuproine, 2 mm h2o2, and various concentrations of ascorbate ; (c) 10 m phq, 100 m detapac, 100 m neocuproine, 10 mm ascorbate, and various concentrations of h2o2 ; and (d) 100 m detapac, 100 m neocuproine, 10 mm ascorbate, 2 mm h2o2, and various concentrations of phq in 1:3 dmso / phosphate buffer (20 mm, ph 7.4) (v / v). replacing 10 m phq with 10 m of its hydroquinone in a sample containing 100 m detapac, 100 m neocuproine, 10 mm ascorbate, and 2 mm h2o2 in a 1:3 dmso / phosphate buffer (20 mm, ph 7.4) (v / v) mixture and incubating the mixture for 40 min produced a ch4 peak area that is only 14 2% of that obtained in the phq - containing sample (figure 3). although this value is larger than that obtained in the absence of quinone, 2 1%, it is consistent with the semiquinone, not the hydroquinone, being largely responsible for the enhanced production of hydroxyl radicals. in order to obtain further evidence that the semiquinone is the actual h2o2-reducing species, a phq solution containing dmso, detapac, h2o2, neocuproine, and phosphate buffer (ph 7.4) (v / v) was mixed with an equal volume of a phqh2 solution in the absence of oxygen and ascorbate. final concentrations after mixing were 5 mm phq, 5 mm phqh2, 100 m detapac, 100 m neocuproine, and 2 mm h2o2 in 1:3 dmso / phosphate buffer (20 mm, ph 7.4) (v / v). the phqh2 concentration used contains the same amount of reducing equivalents as those involved in the one - electron oxidation of 10 mm ascorbate. the methane gc peak area obtained (93 8 arbitrary units) after incubation for 40 min was essentially the same as that obtained when h2o2 reduction occurs by reacting ascorbate with phq (97 4 arbitrary units, figure 3). because most para - semiquinones have pka values for their protonated form, qh, between 3 and 5 and those corresponding to ortho - semiquinones, between 4 and 5, the anion radical species is the most abundant species at physiological ph and thus is the carrier of electrons from ascorbate to h2o2. thus, it is very unlikely that michael addition of hoo to the quinones occurs at the ph used for this work because very low concentrations of this anion will be present. furthermore, the phq relative hplc peak area of a h2o2-containing sample and that of a sample with no h2o2 and no ascorbate added, both containing phosphate buffer, dmso, detapac, and neocuproine, after 40 min of incubation, was 103.4 0.5 and 100 1 relative units, respectively. the latter shows that phq is not being transformed to another type of molecule during the incubation time in the presence of h2o2. such a transformation would have made the dependence of the ch4 area on the e1/2 values of the quinones very erratic, as their identities in the reaction samples would have been different from those present during the e1/2 measurements. the rate of ch4 formation increases linearly with an increase in concentration of all reagents, i.e., phq, asch, and h2o2 (keeping the other reagents at constant concentrations) (figure 5), indicating first - order kinetics with respect to each of these species. the fact that this rate depends on h2o2 concentration is in contrast to the zero - order behavior of the ascorbate autoxidation rate with respect to o2. if the semiquinone reduction of h2o2 is postulated to be the slow step in the mechanism, a rate equation can be obtained which is first - order with respect to each species, phq, asch, and h2o2 (scheme 2). a constant steady - state concentration of the ascorbyl radical, asc, has been observed in previous works during the quinone - enhanced as well as in the iron- and methylene blue - catalyzed ascorbate oxidation reactions., quinones reduce h2o2 in the presence of ascorbate in a quinone redox - potential - dependent manner and independent of transition metal traces. the most active quinone in the studied series, phq, is only 14% less active than the classic fenton reagent cation, fe, at the same concentration. these observations are relevant to the antitumor - enhancing activity of quinones in the presence of ascorbate and to the toxic activity of environmental quinone contaminants. | the quinones 1,4-naphthoquinone (nq), tetramethyl-1,4-benzoquinone (dq), 2-methyl-1,4-naphthoquinone (mnq), 2,3-dimethoxy-5-methyl-1,4-benzoquinone (ubq-0), 2,6-dimethylbenzoquinone (dmbq), 2,6-dimethoxybenzoquinone (dmobq), and 9,10-phenanthraquinone (phq) enhance the rate of h2o2 reduction by ascorbate, under anaerobic conditions, as detected from the amount of methane produced after hydroxyl radical reaction with dimethyl sulfoxide. the amount of methane produced increases with an increase in the quinone one - electron reduction potential. the most active quinone in this series, phq, is only 14% less active than the classic fenton reagent cation, fe2 +, at the same concentration. since phq is a common toxin present in diesel combustion smoke, the possibility that phq - mediated catalysis of hydroxyl radical formation is similar to that of fe2 + adds another important pathway to the modes in which phq can execute its toxicity. because quinones are known to enhance the antitumor activity of ascorbate and because ascorbate enhances the formation of h2o2 in tissues, the quinone - mediated reduction of h2o2 should be relevant to this type of antitumor activity, especially under hypoxic conditions. |
controlled ovarian hyperstimulation (coh) with gonadotropins has improved success rates of in vitro fertilization (ivf) by increasing the number and opportunity for selection of embryos before transfer [13 ] as well as permitting the cryopreservation of supernumerary embryos for further fertility treatment [4, 5 ]. the basis of coh is to support the growth of multiple follicles to the preovulatory stage, a process achieved by bypassing physiological regulatory mechanisms. urinary - derived or recombinant follicle stimulating hormone (fsh) is administered to increase serum concentrations above the threshold required for dominant follicle selection, thus enabling the entire cohort of recruited follicles to develop and attain preovulatory status. luteinising hormone (lh) is often coadministered although, following pituitary downregulation, this is not essential for follicular development as remnant basal lh levels are sufficient to stimulate the theca cells. administration of a gnrh analogue (long protocol) or an antagonist (short protocol) that desensitizes the pituitary is primarily used to prevent premature lh surge as a consequence of supraphysiological serum oestradiol (e2) levels which, if it occurs, can lead to premature luteinisation and/or ovulation. with few exceptions, the last two decades have witnessed a mounting body of evidence indicating that ovarian stimulation has a detrimental effect on oogenesis, embryo quality, and endometrial receptivity [713 ]. demonstrated that retrieval of > 10 oocytes per woman adversely affected their quality based on oocyte / embryo morphology, fertilization, and implantation rates. more recently van der gaast. found 13 oocytes to be the optimum number retrieved in order to achieve a pregnancy using a long protocol, above which there was a fall in pregnancy rates. an extreme example of the negative impact of coh is the excessively high number of poor quality oocytes seen in ovarian hyperstimulation syndrome (ohss), which is putatively attributable to detrimental supraphysiological e2 levels. these observations in humans are supported by a number of rodent studies that investigated the impact of exogenous gonadotropin stimulation on oocytes and demonstrated a delay in embryo development [17, 18 ]. it has been suggested that gonadotropin stimulation may affect oocyte maturation and the completion of meiosis, thus leading to an increased risk of having aneuploid oocytes and/or embryos [10, 19 ]. as such, in vitro maturation (ivm) has been proposed as an alternative strategy since it reduces exposure to exogenous gonadotropin stimulation, but the process itself introduces a host of other variables / complications (e.g., disruption of the meiotic spindle) that do not allow a fair comparison of these approaches to be made. von wolff. recently demonstrated a varying endocrine follicular milieu together with the concentration of putative markers of oocyte quality, specifically anti - mllerian hormone (amh) between nc and coh ff, and suggest that this may be the cause for the lower oocyte quality following coh compared with naturally matured oocytes. there has also been some concern that suppressed lh concentrations in the late follicular phase may be detrimental through downstream perturbations in follicular steroid synthesis. consequently, stimulation protocols incorporating exogenous lh were developed, resulting in an increase in the percentage of diploid and good quality embryos obtained [22, 23 ]. by contrast, other investigators have reported a reduction in fertility and increased risk of miscarriage when incorporating exogenous lh into protocols [24, 25 ]. lh window below which e2 production is inadequate and above which lh may begin premature luteinisation and be detrimental to follicular development. postulate that the reduced levels of intrafollicular amh they demonstrated following coh compared with nc may be attributed to lh suppression, resulting initially in low follicular androgen concentrations, and subsequently to low amh production which in turn may be responsible for lower oocyte quality. natural cycle ivf (nc - ivf) has been proposed as an alternative treatment for older women and poor responders. indeed, there has been a resurgence of interest in nc - ivf for all patients in recent years because it avoids coh and its potential sequelae. moreover, this also supports the international drive to reduce multiple pregnancies rates with elective single embryo transfer and to minimise complications such as ohss [2830 ]. conducted a systematic review of 1,800 natural ivf cycles reported between 1989 and 2000 and concluded that nc - ivf has a pregnancy rate of less than 10% per cycle. more recent reports concur, presenting a similar 15.2% live birth rate per initiated cycle in all reported unstimulated ncs in women < 35 years (n = 795) in the united states (2006 - 2007). this method incorporates the use of low dose gonadotropin stimulation together with a gonadotropin releasing hormone (gnrh) antagonist aimed at generating fewer than eight oocytes per cycle. the term modified natural cycle ivf (mnc - ivf) is applied when drugs (e.g., human chorionic gonadotropin (hcg)) are used to induce final oocyte maturation whereby a gnrh antagonist is administered during a spontaneous cycle to reduce the risk of cancellation and/or where luteal support is provided. during folliculogenesis, follicular fluid (ff) composition exhibits dynamic changes as individual follicular cell types respond to gonadotropins by secreting different hormones and cytokines [34, 35 ]. as growth factors regulating all stages of folliculogenesis, cytokines have been shown to govern the development / function of somatic cells and the oocyte as well as the composition of ff [3642 ]. given that oocyte quality influences subsequent embryo viability, it has been suggested that the disruption in the balance of these intrafollicular mediators following coh may influence cycle outcome [4452 ]. the correct regulation of cytokine networks is essential to support normal physiology and this central role is underscored by the fact that inflammatory / immune dysfunctions underpin many pathological reproductive conditions, resulting in both local and systemic changes in cytokine profiles [5355 ]. for example, the levels of interleukin- (il-) 8, a chemotactic and angiogenic cytokine essential to folliculogenesis, have been found to rise from the midfollicular to the late follicular phase. these levels are comparable to those found during a coh cycle, implying that granulosa cell (gc) and theca cell (tc) il-8 secretion is a true physiological phenomenon associated with follicular growth / maturation rather than resulting from gonadotropin stimulation. in vitro enhancement of il-8 secretion by cultured gcs and tcs was evident following exposure to il-1 and il-1, but not tumour necrosis factor- (tnf-), suggesting that il-8 is both gonadotropin and cytokine - induced and may thus be involved in the hormonally regulated stages of folliculogenesis and ovulation. although cytokines are readily detected in ff, the complexity of their network regulation makes their study in isolation difficult to interpret. in view of their biological properties (pleiotropism, synergy, antagonism, functional redundancy, and differential sensitivity) [5860 ], cytokines should ideally be investigated in terms of their interrelationships as much as in terms of their absolute concentrations. whilst a recent study by bersinger. failed to demonstrate a difference in 13 ff cytokines between women undergoing nc and coh ivf, no specific attention was paid towards the complex interrelations within the cytokine networks. to date, there has been a paucity of studies focusing on minimal stimulation regimens and mncs, and comparisons of isolated cytokine concentrations (e.g., vascular endothelial growth factor (vegf) in coh and nc - ivf) have been inconsistent [62, 63 ]. therefore, this pilot study aimed at examining the impact of gonadotropins on the intrafollicular cytokine milieu in mnc and following coh cycles. from november 2008 to march 2009, ten women who required treatment with ivf / icsi due to unexplained or male factor infertility aged 2535 years with a body mass index (bmi) 1930 were selected to undergo mnc - ivf / intracytoplasmic sperm injection (icsi) at the assisted conception unit, st james 's university hospital, leeds, uk. they were required to be ovulatory (confirmed by transvaginal ultrasound scan (tvuss), progesterone levels, or commercial lh surge kits within the preceding three months) and have a normal endocrine profile (early follicular phase fsh < 8.0 iu / l and e2 50200 pmol / l), a negative infection screen (including negative serum chlamydia antigens), and normal pelvic anatomy confirmed by tvuss and laparoscopy. furthermore, they were required to have no risk factors for pelvic pathology (e.g., history of pelvic inflammatory disease, incomplete miscarriage, ectopic pregnancy, cervical dyskaryosis, and abdominal / pelvic / cervical surgery). women with coexisting morbidity (e.g., autoimmune diseases, inflammatory conditions, and diabetes mellitus) and those taking regular medications were also excluded. the study protocol was approved by national research ethics service, leeds (east) research ethics committee, and all participants provided written informed consent. all women underwent a baseline tvuss (aloka sssd 1700) in the early follicular phase. in the mnc cohort, a tvuss assessment was performed on alternate days from day 8 of the cycle, until the mean maximal diameter (mmd) measured in two planes (sagittal and transverse) of the dominant follicle measured 14 mm, after which they were performed daily. women were asked to use urinary lh kits twice daily (06:0008:00 and 18:0020:00) in order to identify the onset of lh surge prior to spontaneous ovulation. an injection of 5,000 iu hcg (pregnyl (organon, cambridge, uk)) was given when the mmd of the lead follicle measured 17 mm (17.018.1 mm). if the urinary lh kit was positive, ultrasound directed oocyte retrieval (udor) was performed the day after the surge was detected ; otherwise it was planned for 36 h after hcg. for subsequent analysis, women who had a spontaneous lh surge and women who were administered exogenous hcg were grouped together as the mnc cohort. pituitary downregulation was attained using leuprorelin acetate sr 3.75 mg (prostap (wyeth, maidenhead, berkshire, uk)) administered on the first day of the menstrual cycle. coh was achieved with 225 iu human menopausal gonadotropin (hmg) daily (menopur, ferring, slough, berkshire, uk). as with the mnc - ivf / icsi cycle, when one or more follicles had an mmd of 17 mm, 5,000 iu hcg (pregnyl (organon, cambridge, uk)) was administered 36 h prior to udor. all udors were performed between 09:00 and 11:00 to accommodate putative circadian variations in ovarian physiology. dead space (containing 1.5 ml 0.9% sodium chloride solution) within the oocyte harvesting needle and tubing was constant / uniform throughout such that the first 1.5 ml aspirated was checked and discarded. subsequent aspirate was considered to contain ff and, following oocyte assessment and retrieval, was subsequently labelled to ensure longitudinal tracking of the corresponding oocyte to its fate (for multifollicular cycles). follicles were flushed up to four times with culture medium (enhance htf culture medium with hepes ; conception technologies, san diego, california, usa) if no oocyte was obtained in the initial aspirate to minimise the risk of inadvertently aspirating a second follicle and collecting the oocyte from the previous one due to being contained within the dead space. all ultrasonically visible follicles were individually aspirated irrespective of their size. the aspiration pressure applied was uniform on all follicles (183185 mm / hg). whole blood and ff samples were centrifuged (2,000 rpm at 4c for 10 minutes) to isolate plasma and remove cell debris, respectively. all samples were frozen at 80c within one hour of retrieval until required for analysis. in the mnc cohort, only the ff from the single dominant follicle was analysed. in the coh cohort, ff analysis was performed on the follicles yielding the oocytes that generated transferred embryos (since double ets were performed in these cycles, the follicle selected for analysis was the one yielding the embryo with the highest morphological grading). in both cycles, routine procedures for fertilization with ivf / icsi were performed as previously described. a single et was performed in the mnc cohort, whilst a double et was performed in the coh cohort (as per acu protocols at the time). all women in the mnc cohort who underwent an et received 2,500 iu hcg (pregnyl) on the day of et and again 72 h later for luteal support. women in the coh cohort who developed < 15 follicles had an identical luteal support regimen, whereas those women with 15 follicles following coh were given daily intramuscular injection of 100 mg progesterone (gestone, nordic pharma, reading, berkshire, uk), which was continued throughout the first trimester of pregnancy. pregnancy tests were performed on first void urine 14 days after et with a commercial urinary kit. a clinical pregnancy was defined as one demonstrating a gestational sac with a fetal pole and a fetal heart or an ectopic pregnancy by tvuss at 6 - 7 weeks ' gestation. cytokine levels in both ff and plasma were measured by fluid - phase cytometric multiplex immunoassay (bio - rad laboratories, hercules, ca, usa) (bio - rad assays : human cytokine 27-plex assay m50 - 0kcaf0y ; human cytokine 21-plex assay mf0 - 005kmii) using a luminex 100 cytometer (luminex corporation, austin, texas, usa) equipped with bioplex 4.0 manager software (bio - rad laboratories, ltd. target cytokines included interleukin- (il-) 1 receptor antagonist (ra), il-2ra, il-3, il-6, il-7, il-8, il-9, il-10, il-12 (p40), il-12 (p70), il-13, il-15, il-16, il-18, leukaemia inhibitory factor (lif), granulocyte macrophage - colony stimulating factor (gm - csf), macrophage- (m-) csf, granulocyte- (g-) csf, stem cell factor (scf), interferon- (ifn-), ifn-, ifn- inducible protein- (ip-10), tnf-, tnf-, tnf related apoptosis inducing ligand (trail), vegf, platelet derived growth factor (pdgf), basic fibroblast growth factor (b - fgf), nerve growth factor- (ngf-), stem cell growth factor- (scgf-), growth regulated oncogene- (gro-), macrophage inflammatory protein- (mip-) 1, monocyte chemoattractant protein- (mcp-) 1, mcp-3, eotaxin, regulated upon activation of normal t cell expressed and secreted (rantes), stromal cell - derived factor- (sdf-) 1, cutaneous t - cell attracting chemokine (ctack), monokine induced by ifn- (mig), and macrophage migration inhibitory factor (mif). oocyte retrieval frequently results in disruption of the intraovarian vasculature such that blood (macroscopic or microscopic) contaminates the ff retrieved. furthermore, although the needle and tubing were primed with normal saline at the commencement of aspiration of each ovary, in between subsequent follicular aspirations, protein - free flush medium formulated with gentamicin (enhanced htf culture medium with hepes ; conception technologies, san diego, california, usa) was used, with potential dilution of the ff. this entailed cytokine, total protein (by lowry assay, bio - rad), and von willebrand factor (vwf ; by enzyme - linked immunosorbent assay ; r&d systems, abingdon, uk) measurement in both plasma and ff. since vwf is a large plasma multimeric glycoprotein that does not pass through the basement membrane and is not produced by follicular cells, it enabled accurate quantification of ff blood (and therefore circulatory cytokine) contamination (present authors, manuscript under review). the dilutional effect of the flush medium was instead accounted for by standardising both ff and plasma (the latter to enable a valid comparison with the former) cytokine concentrations to total protein (pg cytokine / mg protein). chi - squared, independent samples t - tests, or mann - whitney u tests were used to compare mnc and coh patient demographics, cycle details, ff, and plasma cytokines following tests for normal distribution by shapiro - wilk test (stata / se 11.1, texas, usa). in order to address the interrelationships between multiple cytokines and the impact that coh has upon these, heat maps were generated using r 2.7.0 software (r foundation for statistical computing, vienna, austria). correlations between the different cytokines were determined for mnc and coh data using kendall 's tau as a measure of correlation (stata / se 11.1). resulting p values were adjusted for multiple comparisons with holm 's correction (p < 0.05 was considered significant). in the mnc cohort, one patient had a positive lh surge and therefore did not receive exogenous hcg. at the time of udor 12 hours after the positive surge, spontaneous ovulation had occurred such that no ff was retrieved and an oocyte was not obtained. no statistically significant differences were noted in patient demographics (age, bmi, baseline endocrine profile (fsh, lh, and e2), and ethnicity), day of udor, follicular aspirate volume, oocyte maturity, and cycle outcome between the two groups (table 1). most ff cytokines (29 out of 40) were found to be at higher concentrations in the mnc group compared to the coh group (binomial test : p < 0.001). this relationship was statistically significant for lif (p < 0.01) and sdf-1 (p < 0.05) (figure 1). as with ff, the majority of circulatory cytokines in the mnc cohort were present at higher concentrations than in the coh group (figure 2), a relationship which was significant for 12 of these : il-2ra, il-3, il-12 (p40), lif, m - csf, ifn-, trail, ngf-, gro-, mcp-3, rantes, and sdf-1 (p < 0.05). conversely, plasma il-12 (p70) levels were present at significantly higher levels following coh (p < 0.05) (figure 2). heat maps were generated following correlation analysis using kendall 's tau to demonstrate ff cytokine interrelationships (figure 3). significantly more pairs of cytokines exhibited strong correlations in the mnc data compared to the coh data (binomial test) (p < 0.001). various relationship alterations were also noted ; for example, lif and tnf- demonstrated a weak negative correlation in the mnc group (kendall 's tau : 0.08) compared with a strong positive correlation following coh (kendall 's tau : 0.46). when plasma : ff cytokine ratios were compared between the mnc and coh cohorts, significantly higher concentrations of various cytokines including il-1, il-5, il-10, il-12, il-17, tnf-, and eotaxin have been recorded in endometrial secretions from stimulated cycles compared to ncs. similarly, in the ovary, it has been suggested that exogenous gonadotropins may influence the levels of cytokines such as il-1, il-6, and tnf- following earlier studies on ff.. demonstrated altered cumulus cell gene expression for leukocyte differentiation and t - cell activation and regulation following coh, which may in turn influence follicular cytokine profiles as highlighted by the present findings. to the best of our knowledge, ff has not previously been analysed for such an extensive range of cytokines in nc / mnc and coh cycles. in the present pilot study, ff concentrations of 29 out of the 40 cytokines analysed were found to be higher in the mnc cohort, significantly so for lif and sdf-1. lif is an embryotrophic cytokine whose secretion by gcs and stromal cells into ff has previously been shown to be stimulated by hcg [7173 ]. since both the mnc and coh cohorts in this study received identical doses of exogenous hcg, the elevated levels of lif in mnc ff may represent an enhanced response to hcg, whereas the likely perturbed cytokine response allied to coh may reflect a reduced sensitivity to hcg resulting in lower lif concentrations. coh also appeared to perturb relationships between cytokines, highlighted by the relative changes in intrafollicular lif and tnf- (where the latter modulates ovarian stromal cell secretion of the former). the heat map displays a strong positive correlation between lif and tnf- in the mnc cohort whereas this relationship is weakened following coh. interestingly, ff lif concentrations have also been correlated with e2 concentrations (possibly through its role in enhancing aromatase expression) which, in turn, relate to follicular maturity [74, 75 ]. although a comparable causal association has not been identified to date in the ovary, 17- oestradiol is known to induce lif synthesis in bovine oviduct epithelial cells. it is tempting to speculate that an analogous mechanism is at play in the ovary, where the supraphysiological e2 levels associated with coh may impair the induction of follicular lif synthesis, with a consequent impact on the ff milieu and oocyte quality. sdf-1 is a chemokine secreted by oocytes, which acts in a paracrine manner to prevent follicular activation, thereby controlling the entry of primordial follicles into the growing pool in ncs. furthermore, ff sdf-1 has previously been positively correlated with ff vegf levels in coh cycles, where it is believed to play a proangiogenic role in supporting follicular growth. our findings corroborate this correlation in the coh group (figure 3). by contrast, this relationship was much weaker in the mnc cohort, suggesting that it may thus in part be gonadotropin dependent. akin to what was noted for ff, plasma cytokine levels in the mnc group were higher than in coh cycles. the exception was circulatory il-12 (p70), which was measured at significantly higher concentrations following coh. furthermore, correlations between ff il-12 (p70) and other cytokines were markedly altered following stimulation. in the mnc cohort, most ff cytokines were positively correlated with il-12 (p70), whilst this relationship was reversed following coh. conversely, ifn- and tnf- were negatively correlated with il-12 (p70) in the mnc group and positively in the coh cohort. interestingly, an intricate triumvirate relationship between these particular cytokines has been identified in the regulation of inflammatory responses. il-12 (p70) induces ifn- production and, in turn, ifn- markedly augments il-12 (p70) production, thereby providing a key inflammatory amplifying mechanism. by contrast, tnf- is thought to inhibit ifn--induced il-12 (p70) production, as demonstrated by hodge - dufour., whose tnf mice exhibited a prompt inflammatory response which resolved spontaneously compared with a delayed, more vigorous, inflammatory response leading to death associated with elevated il-12 levels in tnf mice when injected with corynebacterium parvum. thus, tnf- is thought to contribute to the resolution of il-12- (p70-) driven inflammatory processes. in the mnc cohort, tnf- was negatively correlated with both il-12 (p70) and ifn-, thus suggesting that, in the absence of stimulatory gonadotropins, tnf- of follicular cell origin may play an analogous role via its capacity to regulate il-12 (p70) production. a positive correlation between these ff cytokines in combination with significantly elevated systemic il-12 (p70) levels suggests a disruption of this mechanism following coh. an analogous disruption in the ovary following coh could thus contribute to the detrimental sequelae of increased il-12 (p70) and ifn- levels despite the observed compensatory rise in modulatory tnf- levels. fluctuations in systemic white cell populations have been attributed to stimulation with exogenous gonadotropins such that the total number of circulatory leukocytes and neutrophils was increased on the day of hcg administration in coh compared to ncs. other studies have reported an increase in plasma white cell count and g - csf, but not in m - csf or il-6, during coh. furthermore, various cytokines have been found to be elevated in ff following coh, including il-1, il-6, and tnf-. the main difference between the present study and those cited is our administration of exogenous hcg in the mnc cohort. both lh and hcg induce il-1 and tnf-, both of which subsequently upregulate gm - csf expression. despite identical doses of hcg being used to trigger ovulation in both groups in the current study, gm - csf in particular was measured at higher (although not statistically significant) levels following coh in both ff and plasma. furthermore, following coh, there was a trend towards several other proinflammatory cytokines in both ff and plasma to be found at higher concentration, with il-12 (p70) levels in particular being significantly higher in coh plasma samples. this provides further evidence that coh can induce both local and systemic inflammatory network deregulations featuring perturbations in cytokine interrelationships which, speculatively, may potentially also impact oocyte viability and treatment outcome. unfortunately, the present pilot study was not powered to answer this particular question. in conclusion, this investigation demonstrated that coh not only alters ff cytokine profiles compared to mncs but also perturbs their circulatory levels and disrupts their interrelationships. given their central role in orchestrating normal follicular physiology, these changes have the potential to adversely affect follicular function and compromise oocyte viability. | background. the natural cycle is the prototype to which we aspire to emulate in assisted reproduction techniques. increasing evidence is emerging that controlled ovarian hyperstimulation (coh) with exogenous gonadotropins may be detrimental to oogenesis, embryo quality, and endometrial receptivity. this research aimed at assessing the impact of coh on the intrafollicular milieu by comparing follicular fluid (ff) cytokine profiles during stimulated in vitro fertilization (ivf) and modified natural cycle (mnc) ivf. methods. ten women undergoing coh ivf and 10 matched women undergoing mnc ivf were recruited for this pilot study. 40 ff cytokine concentrations from individual follicles and plasma were measured by fluid - phase multiplex immunoassay. demographic / cycle / cytokine data were compared and correlations between cytokines were computed. results. no significant differences were found between coh and mnc groups for patient and cycle demographics, including outcome. overall mean ff cytokine levels were higher in the mnc group for 29/40 cytokines, significantly so for leukaemia inhibitory factor and stromal cell - derived factor-1. furthermore, ff mnc cytokine correlations were significantly stronger than for coh data. conclusions. these findings suggest that coh perturbs intrafollicular cytokine networks, in terms of both cytokine levels and their interrelationships. this may impact oocyte maturation / fertilization and embryo developmental competence. |
a role for hpv in lung cancer has been proposed, although previous reports of hpv in lung tumor tissue have been inconclusive. paraffin - embedded tissue specimens from 450 lung cancer patients who were smokers or never - smokers enrolled in the environment and genetics in lung cancer etiology study were evaluated for hpv dna by real - time polymerase chain reaction to determine the hpv prevalence in tissues from lung cancer patients in a representative western population. two specimens were found to be hpv positive at low copy number but were negative on additional genotype - specific testing and on testing for a broad spectrum of hpv types. hpv is not associated with lung carcinogenesis in lung cancer patients from a representative western population. multiple tissue specimens were used from each patient for analysis and were not necessarily adjacent to or from the same paraffin block. also, the possible contamination of specimens during collection could not be determined. because of geographic variances in the prevalence of hpv reported in lung tumors, further studies in non - western populations should be completed. a role for hpv in lung cancer has been proposed, although previous reports of hpv in lung tumor tissue have been inconclusive. paraffin - embedded tissue specimens from 450 lung cancer patients who were smokers or never - smokers enrolled in the environment and genetics in lung cancer etiology study were evaluated for hpv dna by real - time polymerase chain reaction to determine the hpv prevalence in tissues from lung cancer patients in a representative western population. two specimens were found to be hpv positive at low copy number but were negative on additional genotype - specific testing and on testing for a broad spectrum of hpv types. hpv is not associated with lung carcinogenesis in lung cancer patients from a representative western population. multiple tissue specimens were used from each patient for analysis and were not necessarily adjacent to or from the same paraffin block. also, the possible contamination of specimens during collection could not be determined. because of geographic variances in the prevalence of hpv reported in lung tumors, further studies in non - western populations should be completed. eagle is a large population - based case control study with 2100 lung cancer patients that was designed to investigate the genetic and environmental determinants of lung cancer and smoking persistence (22). as previously described (22), individuals with newly diagnosed lung cancer were recruited from april 22, 2002, to february 28, 2005, from 13 hospitals in the lombardy region of italy (see notes). the study was approved by the institutional review board of each participating hospital and university in italy and by the national cancer institute, bethesda, md. lung cancer was diagnosed by standard clinical criteria and confirmed by pathology reports from surgery, biopsy, or cytology samples (approximately 95% of patients) or through clinical history and imaging (approximately 5% of patients) (22). tumor histology was classified by the 1999 world health organization histological typing of lung and pleural tumors (23). the 13 italian hospitals treat approximately 80% of all patients with newly diagnosed lung cancer in the area, which includes more than 1.3 million people aged 3579 years from five cities and surrounding towns and villages. thus, participants in the eagle study should be broadly representative of lung cancer patients in western populations. we decided to test 450 adult male and female lung cancer patients, including all 30 never - smokers and a random sample of 420 smokers who had an adequate number of unstained tissue slides and at least one slide available that was stained with hematoxylin and eosin. these stained slides were reviewed by h. song to establish the presence and estimate proportion of tumor tissue in the sample, but these slides were not necessarily adjacent to the tissue that was tested for hpv dna. all 450 patients were tested for hpv16 and hpv18 dna because these are the most common types found in cervical cancer (24) and account for the overwhelming majority of noncervical hpv associated cancers (25). type - specific pcr for hpv16 and hpv18 was conducted in a laboratory at the ohio state university, as described below. for a subset of 100 patients including all never - smokers and a random selection of smokers, a second tissue specimen was sent to ddl diagnostic laboratory (voorburg, the netherlands) for broad - spectrum hpv typing. all specimen receipt, processing, and pre - amplification analysis procedures were performed in ultraviolet light all equipment and reagents were specific to the pre - amplification analysis laboratory with strict one - way transport of specimens from this laboratory to a physically separate postamplification analysis laboratory. additional protocols to prevent specimen contamination included sample processing in small batches, change of gloves after dna extraction of each sample, exclusive use of barrier pipette tips, weekly cleaning of all laboratory surfaces and equipment with 70% ethanol, and soaking of all racks and pcr plate holders in 10% bleach for a minimum of 10 minutes between each experiment. additionally, one negative control per seven experimental samples was included in each column of each 96-well pcr plate. dna was extracted from 450 formalin- or ethanol - fixed paraffin - embedded tissue samples by means of paraffin removal with octane, proteinase k digestion, phenol chloroform extraction, and ethanol precipitation. briefly, after octane exposure, the sample was washed with 100% ethanol, resuspended in 200 l of digestion buffer (50 mm tris hcl at ph 8.5, 1 mm edta, and proteinase k at 20 g / ml), and incubated for 48 hours at 55c. if tissue remained, 10 l of proteinase k (400 g / ml) was added, and digestion was continued for an additional 2448 hours. chloroform by use of qiagen maxtract tubes (qiagen, germantown, md) and ethanol precipitated in the presence of glycogen (0.02 mg / ml). pellets were washed once in 70% ethanol, dried at 37c in a dry incubator, and resuspended in 50 l of diethylene pyrocarbonate - treated water. dna quantity and purity (calculated by use of the ratio of the absorbance at 260 nm to that at 280 nm [260/280 ratio ]) were measured with the nanodrop spectrophotometer (thermo fisher scientific, inc, wilmington, de). an estimate of the number of cells analyzed in each pcr was made by custom taqman real - time pcr assays targeting a single - copy human gene on chromosome 7, human endogenous retrovirus 3 (erv3) (26). in a pilot study of 19 specimens, dna integrity was evaluated through amplification of erv3 sequences with primers generating an amplicon of 58, 135, or 354 base pairs (bp) in size (supplementary table 1, available online). primer and probe sequences for the 58-bp amplicon were as follows : forward primer, 5-gataatttcacactaaccgcc-3 ; reverse primer, 5-agatgctctgacttgatggt-3 ; and the probe sequence, 5-56-fam - ctcttccctcgaacctgc-3bhq1 - 3 (where 56-fam is 56-carboxy - fluorescein and 3bhq1 is 3-black hole quencher-1). primer and probe sequences for the 135-bp amplicon were as follows : forward primer, 5-catgggaagcaagggaacta-3 ; reverse primer, 5-cccagcgagcaatacagaattt-3 ; and probe, 5-56-fam - tcttccctcgaacctgcaccatcaagtca-3bhq1 - 3. primer and probe sequences for the 354-bp amplicon were as follows : forward primer, 5-catgggaagcaagggaactaatg-3 ; reverse primer, 5-gcccacagatccagtagagg-3 ; and probe were the same as for the 135-bp amplicon above. briefly, purified genomic dna (2 l) from lung cancer specimens was analyzed. a standard curve was generated in duplicate from a fivefold dilution series (from 150 000 to 1.92 cells) of a diploid human cell line, ccd-18lu (catalog number ccl-205, atcc [american type culture collection ], manassas, va). results were reported as the number of human diploid genome equivalents of purified genomic dna from tumor samples that were evaluated for hpv16 and hpv18 dna by real - time pcr. all samples were tested once, and positive samples were retested in duplicate. although dna was degraded because of fixation, the mean yield was 54.5 ng / ml. this subset analysis indicated that dna integrity was sufficient to support amplification of targets with amplicon size between approximately 58 and 135 bp. dna specimens with at least 100 evaluable cell equivalents and a 260/280 ratio between 1.5 and 2.2 (27) were considered to have adequate dna quantity and quality for further analysis. type - specific taqman real - time pcr targeted to the oncogenic e6 region of the genome for hpv16 (amplicon size = 201 bp) and the e7 region of the genome for hpv18 (amplicon size = 137 bp) were performed in a 96-well plate format as previously described (28). to account for dna degradation in formalin - fixed and paraffin - embedded tissues, all samples were also evaluated by means of a taqman real - time pcr assay designed to amplify an 81-bp amplicon in the e6 region of the genome for hpv16. primers and probes were manufactured at idt technologies (coralville, ia) and sequences were : forward, 5-gagaactgcaatgtttcaggacc-3 ; reverse, 5-tgtatagttgtttgcagctctgtgc-3 ; and probe, 5-56-famcaggagcgacccagaaagttaccacagtt-3bhq1 - 3. standard curves were generated in duplicate by use of a fivefold dilution series from 2.5 10 copies of plasmid vector containing full - length viral genome (pgem hpv16) in a background of human placental dna (5 ng/l). reactions that were performed in an applied biosystems 7300 pcr system contained 2x taqman universal pcr master mix (applied biosystems, foster city, ca), 0.1 mol of probe, 0.2 mol of each primer, and 2 l of purified tumor dna. amplification conditions included a 12-minute incubation at 95c, followed by 50 cycles of 15 seconds at 95c and 60 seconds at 60c. the cycle threshold (ct) of unknown samples was determined from an equation derived from a linear regression through the log ct of the standard curve according to the manufacturer s recommendations. for positive samples, hpv viral load was normalized to erv3 copy number by real - time pcr, as a measure of viral copy number per cell analyzed. samples with any detectable hpv16 or hpv18 dna were tested in duplicate, with a mean viral load of greater than one viral copy per sample classified as real - time pcr positive. a mean viral load of greater than or equal to one viral copy per cell was considered indicative of a clonal viral specimens from all 30 never - smokers and 70 randomly sampled current and former smokers were selected to test for broad - spectrum hpv typing at ddl diagnostic laboratory. hpv typing used pcr with short pcr fragment (spf10) primers, which targets the hpv l1 gene, and a mixture of conservative probes that recognize at least 54 mucosal hpv genotypes. we used the dna enzyme immunoassay (deia) to determine hpv dna positivity by detecting amplimers, synthesized by biotinylated pcr primers, through hybridization to a mixture of hpv - specific probes, as described previously (29). stringent pcr contamination precautions that were similar to the type - specific testing for hpv16 and hpv18 conducted at the ohio state university were taken. irradiated laminar flow hoods that were separate from a postamplification analysis laboratory. during dna isolation, pcr amplification, and post - pcr analyses, specific negative (water blank) and positive (hpv dna extracted from the hpv18-positive hela cell line) controls were included. we monitored potential contamination of samples with negative controls and the sensitivity of the procedure with low - concentration positive controls. all reagents had been prepared in a separate reagent laboratory and subjected to strict quality control to confirm efficacy and the absence of contaminants. dna amplification by pcr with spf10 primers was followed by a reverse hybridization line - probe assay (lipa25, version 1 ; labo bio - medical products, rijswijk, the netherlands) for genotyping of 25 hpv types in spf10- and deia - positive samples. these primers produce very short pcr fragments of approximately 65 bp, making them ideal for amplification in formalin - fixed specimens (2931). eight specimens did not appear to have tumors in the tissue specimen received for testing. dna was extracted for 92 specimens (from 65 ever - smokers and 27 never - smokers) with identifiable tumor in the tissue specimen. for each specimen, half of the tissue section was removed from the slide with a swab after confirming the presence of tumor cells in that part of the section. this part of the tissue was transferred to a microtube (sarstedt, etten - leur, the netherlands) and digested with proteinase k buffer (45 mm tris hcl at ph 8, 0.9 mm edta, 0.45% tween 20, and proteinase k at 1 mg / ml). proteinase k solution (100 l) was added to each microtube and incubated for 1624 hours at 56c. resulting dna preparations were stored at 20c. during this process, negative and positive dna isolation controls were included. in these samples, dna quality and quantity were adequate as measured by real - time pcr for the human -actin gene in the same dna specimens as used for the spf10 pcr. for one patient (a current smoker) where additional investigation was warranted due to weak hpv16 positivity in the sample tested at the ohio state university, type - specific pcr for hpv16 was performed with a 92-bp amplicon as previously described (32). our goal in determining sample size was to be able to rule out a true hpv prevalence greater than 1.0%. using cii in stata (statacorp lp, college station, tx), we determined that zero hpv positives among 450 lung cancer patients would generate a one - sided 97.5% confidence interval (ci) of 0% to 0.82% and that one observed hpv - positive patient among 450 lung cancer patients would provide a two - sided 95% confidence interval of 0.0056% to 1.2% ; zero positives among 400 lung cancer patients would provide a one - sided 97.5% confidence interval of 0% to 0.92%, and one observed hpv - positive patient would provide a two - sided 95% confidence interval of 0.0063% to 1.4%. the chance of finding at least one positive when the true prevalence is 1.0% or more was 98.9% with a sample size of 450 patients ; thus, finding no positives with a sensitive assay rules out even a low prevalence of infection with high confidence. in addition to the sample size calculations described above, we used the cii command in stata (statacorp lp), version 9.0, to estimate hpv prevalence and corresponding confidence intervals. eagle is a large population - based case control study with 2100 lung cancer patients that was designed to investigate the genetic and environmental determinants of lung cancer and smoking persistence (22). as previously described (22), individuals with newly diagnosed lung cancer were recruited from april 22, 2002, to february 28, 2005, from 13 hospitals in the lombardy region of italy (see notes). the study was approved by the institutional review board of each participating hospital and university in italy and by the national cancer institute, bethesda, md. lung cancer was diagnosed by standard clinical criteria and confirmed by pathology reports from surgery, biopsy, or cytology samples (approximately 95% of patients) or through clinical history and imaging (approximately 5% of patients) (22). tumor histology was classified by the 1999 world health organization histological typing of lung and pleural tumors (23). the 13 italian hospitals treat approximately 80% of all patients with newly diagnosed lung cancer in the area, which includes more than 1.3 million people aged 3579 years from five cities and surrounding towns and villages. thus, participants in the eagle study should be broadly representative of lung cancer patients in western populations. we decided to test 450 adult male and female lung cancer patients, including all 30 never - smokers and a random sample of 420 smokers who had an adequate number of unstained tissue slides and at least one slide available that was stained with hematoxylin and eosin. these stained slides were reviewed by h. song to establish the presence and estimate proportion of tumor tissue in the sample, but these slides were not necessarily adjacent to the tissue that was tested for hpv dna. all 450 patients were tested for hpv16 and hpv18 dna because these are the most common types found in cervical cancer (24) and account for the overwhelming majority of noncervical hpv associated cancers (25). type - specific pcr for hpv16 and hpv18 was conducted in a laboratory at the ohio state university, as described below. for a subset of 100 patients including all never - smokers and a random selection of smokers, a second tissue specimen was sent to ddl diagnostic laboratory (voorburg, the netherlands) for broad - spectrum hpv typing. all specimen receipt, processing, and pre - amplification analysis procedures were performed in ultraviolet light all equipment and reagents were specific to the pre - amplification analysis laboratory with strict one - way transport of specimens from this laboratory to a physically separate postamplification analysis laboratory. additional protocols to prevent specimen contamination included sample processing in small batches, change of gloves after dna extraction of each sample, exclusive use of barrier pipette tips, weekly cleaning of all laboratory surfaces and equipment with 70% ethanol, and soaking of all racks and pcr plate holders in 10% bleach for a minimum of 10 minutes between each experiment. additionally, one negative control per seven experimental samples was included in each column of each 96-well pcr plate. dna was extracted from 450 formalin- or ethanol - fixed paraffin - embedded tissue samples by means of paraffin removal with octane, proteinase k digestion, phenol chloroform extraction, and ethanol precipitation. briefly, after octane exposure, the sample was washed with 100% ethanol, resuspended in 200 l of digestion buffer (50 mm tris hcl at ph 8.5, 1 mm edta, and proteinase k at 20 g / ml), and incubated for 48 hours at 55c. if tissue remained, 10 l of proteinase k (400 g / ml) was added, and digestion was continued for an additional 2448 hours. chloroform by use of qiagen maxtract tubes (qiagen, germantown, md) and ethanol precipitated in the presence of glycogen (0.02 mg / ml). pellets were washed once in 70% ethanol, dried at 37c in a dry incubator, and resuspended in 50 l of diethylene pyrocarbonate - treated water. dna quantity and purity (calculated by use of the ratio of the absorbance at 260 nm to that at 280 nm [260/280 ratio ]) were measured with the nanodrop spectrophotometer (thermo fisher scientific, inc, wilmington, de). an estimate of the number of cells analyzed in each pcr was made by custom taqman real - time pcr assays targeting a single - copy human gene on chromosome 7, human endogenous retrovirus 3 (erv3) (26). in a pilot study of 19 specimens, dna integrity was evaluated through amplification of erv3 sequences with primers generating an amplicon of 58, 135, or 354 base pairs (bp) in size (supplementary table 1, available online). primer and probe sequences for the 58-bp amplicon were as follows : forward primer, 5-gataatttcacactaaccgcc-3 ; reverse primer, 5-agatgctctgacttgatggt-3 ; and the probe sequence, 5-56-fam - ctcttccctcgaacctgc-3bhq1 - 3 (where 56-fam is 56-carboxy - fluorescein and 3bhq1 is 3-black hole quencher-1). primer and probe sequences for the 135-bp amplicon were as follows : forward primer, 5-catgggaagcaagggaacta-3 ; reverse primer, 5-cccagcgagcaatacagaattt-3 ; and probe, 5-56-fam - tcttccctcgaacctgcaccatcaagtca-3bhq1 - 3. primer and probe sequences for the 354-bp amplicon were as follows : forward primer, 5-catgggaagcaagggaactaatg-3 ; reverse primer, 5-gcccacagatccagtagagg-3 ; and probe were the same as for the 135-bp amplicon above. briefly, purified genomic dna (2 l) from lung cancer specimens was analyzed. a standard curve was generated in duplicate from a fivefold dilution series (from 150 000 to 1.92 cells) of a diploid human cell line, ccd-18lu (catalog number ccl-205, atcc [american type culture collection ], manassas, va). results were reported as the number of human diploid genome equivalents of purified genomic dna from tumor samples that were evaluated for hpv16 and hpv18 dna by real - time pcr. all samples were tested once, and positive samples were retested in duplicate. although dna was degraded because of fixation, the mean yield was 54.5 ng / ml. this subset analysis indicated that dna integrity was sufficient to support amplification of targets with amplicon size between approximately 58 and 135 bp. dna specimens with at least 100 evaluable cell equivalents and a 260/280 ratio between 1.5 and 2.2 (27) were considered to have adequate dna quantity and quality for further analysis. type - specific taqman real - time pcr targeted to the oncogenic e6 region of the genome for hpv16 (amplicon size = 201 bp) and the e7 region of the genome for hpv18 (amplicon size = 137 bp) were performed in a 96-well plate format as previously described (28). to account for dna degradation in formalin - fixed and paraffin - embedded tissues, all samples were also evaluated by means of a taqman real - time pcr assay designed to amplify an 81-bp amplicon in the e6 region of the genome for hpv16. primers and probes were manufactured at idt technologies (coralville, ia) and sequences were : forward, 5-gagaactgcaatgtttcaggacc-3 ; reverse, 5-tgtatagttgtttgcagctctgtgc-3 ; and probe, 5-56-famcaggagcgacccagaaagttaccacagtt-3bhq1 - 3. standard curves were generated in duplicate by use of a fivefold dilution series from 2.5 10 copies of plasmid vector containing full - length viral genome (pgem hpv16) in a background of human placental dna (5 ng/l). reactions that were performed in an applied biosystems 7300 pcr system contained 2x taqman universal pcr master mix (applied biosystems, foster city, ca), 0.1 mol of probe, 0.2 mol of each primer, and 2 l of purified tumor dna. amplification conditions included a 12-minute incubation at 95c, followed by 50 cycles of 15 seconds at 95c and 60 seconds at 60c. the cycle threshold (ct) of unknown samples was determined from an equation derived from a linear regression through the log ct of the standard curve according to the manufacturer s recommendations. for positive samples, hpv viral load was normalized to erv3 copy number by real - time pcr, as a measure of viral copy number per cell analyzed. samples with any detectable hpv16 or hpv18 dna were tested in duplicate, with a mean viral load of greater than one viral copy per sample classified as real - time pcr positive. a mean viral load of greater than or equal to one viral copy per cell was considered indicative of a clonal viral tumor relationship. specimens from all 30 never - smokers and 70 randomly sampled current and former smokers were selected to test for broad - spectrum hpv typing at ddl diagnostic laboratory. hpv typing used pcr with short pcr fragment (spf10) primers, which targets the hpv l1 gene, and a mixture of conservative probes that recognize at least 54 mucosal hpv genotypes. we used the dna enzyme immunoassay (deia) to determine hpv dna positivity by detecting amplimers, synthesized by biotinylated pcr primers, through hybridization to a mixture of hpv - specific probes, as described previously (29). stringent pcr contamination precautions that were similar to the type - specific testing for hpv16 and hpv18 conducted at the ohio state university were taken. irradiated laminar flow hoods that were separate from a postamplification analysis laboratory. during dna isolation, pcr amplification, and post - pcr analyses, specific negative (water blank) and positive (hpv dna extracted from the hpv18-positive hela cell line) controls were included. we monitored potential contamination of samples with negative controls and the sensitivity of the procedure with low - concentration positive controls. all reagents had been prepared in a separate reagent laboratory and subjected to strict quality control to confirm efficacy and the absence of contaminants. dna amplification by pcr with spf10 primers was followed by a reverse hybridization line - probe assay (lipa25, version 1 ; labo bio - medical products, rijswijk, the netherlands) for genotyping of 25 hpv types in spf10- and deia - positive samples. these primers produce very short pcr fragments of approximately 65 bp, making them ideal for amplification in formalin - fixed specimens (2931). eight specimens did not appear to have tumors in the tissue specimen received for testing. dna was extracted for 92 specimens (from 65 ever - smokers and 27 never - smokers) with identifiable tumor in the tissue specimen. for each specimen, half of the tissue section was removed from the slide with a swab after confirming the presence of tumor cells in that part of the section. this part of the tissue was transferred to a microtube (sarstedt, etten - leur, the netherlands) and digested with proteinase k buffer (45 mm tris hcl at ph 8, 0.9 mm edta, 0.45% tween 20, and proteinase k at 1 mg / ml). proteinase k solution (100 l) was added to each microtube and incubated for 1624 hours at 56c. resulting dna preparations were stored at 20c. during this process, negative and positive dna isolation controls were included. in these samples, dna quality and quantity were adequate as measured by real - time pcr for the human -actin gene in the same dna specimens as used for the spf10 pcr. for one patient (a current smoker) where additional investigation was warranted due to weak hpv16 positivity in the sample tested at the ohio state university, type - specific pcr for hpv16 was performed with a 92-bp amplicon as previously described (32). all specimen receipt, processing, and pre - amplification analysis procedures were performed in ultraviolet light all equipment and reagents were specific to the pre - amplification analysis laboratory with strict one - way transport of specimens from this laboratory to a physically separate postamplification analysis laboratory. additional protocols to prevent specimen contamination included sample processing in small batches, change of gloves after dna extraction of each sample, exclusive use of barrier pipette tips, weekly cleaning of all laboratory surfaces and equipment with 70% ethanol, and soaking of all racks and pcr plate holders in 10% bleach for a minimum of 10 minutes between each experiment. additionally, one negative control per seven experimental samples was included in each column of each 96-well pcr plate. dna was extracted from 450 formalin- or ethanol - fixed paraffin - embedded tissue samples by means of paraffin removal with octane, proteinase k digestion, phenol chloroform extraction, and ethanol precipitation. briefly, after octane exposure, the sample was washed with 100% ethanol, resuspended in 200 l of digestion buffer (50 mm tris hcl at ph 8.5, 1 mm edta, and proteinase k at 20 g / ml), and incubated for 48 hours at 55c. if tissue remained, 10 l of proteinase k (400 g / ml) was added, and digestion was continued for an additional 2448 hours. chloroform by use of qiagen maxtract tubes (qiagen, germantown, md) and ethanol precipitated in the presence of glycogen (0.02 mg / ml). pellets were washed once in 70% ethanol, dried at 37c in a dry incubator, and resuspended in 50 l of diethylene pyrocarbonate - treated water. dna quantity and purity (calculated by use of the ratio of the absorbance at 260 nm to that at 280 nm [260/280 ratio ]) were measured with the nanodrop spectrophotometer (thermo fisher scientific, inc, wilmington, de). an estimate of the number of cells analyzed in each pcr was made by custom taqman real - time pcr assays targeting a single - copy human gene on chromosome 7, human endogenous retrovirus 3 (erv3) (26). in a pilot study of 19 specimens, dna integrity was evaluated through amplification of erv3 sequences with primers generating an amplicon of 58, 135, or 354 base pairs (bp) in size (supplementary table 1, available online). primer and probe sequences for the 58-bp amplicon were as follows : forward primer, 5-gataatttcacactaaccgcc-3 ; reverse primer, 5-agatgctctgacttgatggt-3 ; and the probe sequence, 5-56-fam - ctcttccctcgaacctgc-3bhq1 - 3 (where 56-fam is 56-carboxy - fluorescein and 3bhq1 is 3-black hole quencher-1). primer and probe sequences for the 135-bp amplicon were as follows : forward primer, 5-catgggaagcaagggaacta-3 ; reverse primer, 5-cccagcgagcaatacagaattt-3 ; and probe, 5-56-fam - tcttccctcgaacctgcaccatcaagtca-3bhq1 - 3. primer and probe sequences for the 354-bp amplicon were as follows : forward primer, 5-catgggaagcaagggaactaatg-3 ; reverse primer, 5-gcccacagatccagtagagg-3 ; and probe were the same as for the 135-bp amplicon above. briefly, purified genomic dna (2 l) from lung cancer specimens was analyzed. a standard curve was generated in duplicate from a fivefold dilution series (from 150 000 to 1.92 cells) of a diploid human cell line, ccd-18lu (catalog number ccl-205, atcc [american type culture collection ], manassas, va). results were reported as the number of human diploid genome equivalents of purified genomic dna from tumor samples that were evaluated for hpv16 and hpv18 dna by real - time pcr. all samples were tested once, and positive samples were retested in duplicate. although dna was degraded because of fixation, the mean yield was 54.5 ng / ml. this subset analysis indicated that dna integrity was sufficient to support amplification of targets with amplicon size between approximately 58 and 135 bp. dna specimens with at least 100 evaluable cell equivalents and a 260/280 ratio between 1.5 and 2.2 (27) were considered to have adequate dna quantity and quality for further analysis. type - specific taqman real - time pcr targeted to the oncogenic e6 region of the genome for hpv16 (amplicon size = 201 bp) and the e7 region of the genome for hpv18 (amplicon size = 137 bp) were performed in a 96-well plate format as previously described (28). to account for dna degradation in formalin - fixed and paraffin - embedded tissues, all samples were also evaluated by means of a taqman real - time pcr assay designed to amplify an 81-bp amplicon in the e6 region of the genome for hpv16. primers and probes were manufactured at idt technologies (coralville, ia) and sequences were : forward, 5-gagaactgcaatgtttcaggacc-3 ; reverse, 5-tgtatagttgtttgcagctctgtgc-3 ; and probe, 5-56-famcaggagcgacccagaaagttaccacagtt-3bhq1 - 3. standard curves were generated in duplicate by use of a fivefold dilution series from 2.5 10 copies of plasmid vector containing full - length viral genome (pgem hpv16) in a background of human placental dna (5 ng/l). reactions that were performed in an applied biosystems 7300 pcr system contained 2x taqman universal pcr master mix (applied biosystems, foster city, ca), 0.1 mol of probe, 0.2 mol of each primer, and 2 l of purified tumor dna. amplification conditions included a 12-minute incubation at 95c, followed by 50 cycles of 15 seconds at 95c and 60 seconds at 60c. the cycle threshold (ct) of unknown samples was determined from an equation derived from a linear regression through the log ct of the standard curve according to the manufacturer s recommendations. for positive samples, hpv viral load was normalized to erv3 copy number by real - time pcr, as a measure of viral copy number per cell analyzed. samples with any detectable hpv16 or hpv18 dna were tested in duplicate, with a mean viral load of greater than one viral copy per sample classified as real - time pcr positive. a mean viral load of greater than or equal to one viral copy per cell was considered indicative of a clonal viral tumor relationship. specimens from all 30 never - smokers and 70 randomly sampled current and former smokers were selected to test for broad - spectrum hpv typing at ddl diagnostic laboratory. hpv typing used pcr with short pcr fragment (spf10) primers, which targets the hpv l1 gene, and a mixture of conservative probes that recognize at least 54 mucosal hpv genotypes. we used the dna enzyme immunoassay (deia) to determine hpv dna positivity by detecting amplimers, synthesized by biotinylated pcr primers, through hybridization to a mixture of hpv - specific probes, as described previously (29). stringent pcr contamination precautions that were similar to the type - specific testing for hpv16 and hpv18 conducted at the ohio state university were taken. irradiated laminar flow hoods that were separate from a postamplification analysis laboratory. during dna isolation, pcr amplification, and post - pcr analyses, specific negative (water blank) and positive (hpv dna extracted from the hpv18-positive hela cell line) controls were included. we monitored potential contamination of samples with negative controls and the sensitivity of the procedure with low - concentration positive controls. all reagents had been prepared in a separate reagent laboratory and subjected to strict quality control to confirm efficacy and the absence of contaminants. dna amplification by pcr with spf10 primers was followed by a reverse hybridization line - probe assay (lipa25, version 1 ; labo bio - medical products, rijswijk, the netherlands) for genotyping of 25 hpv types in spf10- and deia - positive samples. these primers produce very short pcr fragments of approximately 65 bp, making them ideal for amplification in formalin - fixed specimens (2931). eight specimens did not appear to have tumors in the tissue specimen received for testing. dna was extracted for 92 specimens (from 65 ever - smokers and 27 never - smokers) with identifiable tumor in the tissue specimen. for each specimen, half of the tissue section was removed from the slide with a swab after confirming the presence of tumor cells in that part of the section. this part of the tissue was transferred to a microtube (sarstedt, etten - leur, the netherlands) and digested with proteinase k buffer (45 mm tris hcl at ph 8, 0.9 mm edta, 0.45% tween 20, and proteinase k at 1 mg / ml). proteinase k solution (100 l) was added to each microtube and incubated for 1624 hours at 56c. resulting dna preparations were stored at 20c. during this process, negative and positive dna isolation controls were included. in these samples, dna quality and quantity were adequate as measured by real - time pcr for the human -actin gene in the same dna specimens as used for the spf10 pcr. for one patient (a current smoker) where additional investigation was warranted due to weak hpv16 positivity in the sample tested at the ohio state university, type - specific pcr for hpv16 was performed with a 92-bp amplicon as previously described (32). our goal in determining sample size was to be able to rule out a true hpv prevalence greater than 1.0%. using cii in stata (statacorp lp, college station, tx), we determined that zero hpv positives among 450 lung cancer patients would generate a one - sided 97.5% confidence interval (ci) of 0% to 0.82% and that one observed hpv - positive patient among 450 lung cancer patients would provide a two - sided 95% confidence interval of 0.0056% to 1.2% ; zero positives among 400 lung cancer patients would provide a one - sided 97.5% confidence interval of 0% to 0.92%, and one observed hpv - positive patient would provide a two - sided 95% confidence interval of 0.0063% to 1.4%. the chance of finding at least one positive when the true prevalence is 1.0% or more was 98.9% with a sample size of 450 patients ; thus, finding no positives with a sensitive assay rules out even a low prevalence of infection with high confidence. in addition to the sample size calculations described above, we used the cii command in stata (statacorp lp), version 9.0, to estimate hpv prevalence and corresponding confidence intervals. among the 450 lung cancer patients, 246 (54.7%) had adenocarcinoma and 137 (30.4%) had squamous cell carcinoma (table 1). most patients had a history of tobacco smoking : 220 patients (48.9%) were current smokers, 198 patients (44.0%) were former smokers, and 30 patients (6.7%) were never - smokers. the median age at diagnosis of lung cancer was 67.6 years (range = 35.479.9 years), and the median body mass index was 25.3 kg / m (range = 15.960.8 kg / m). among smokers, the median duration of smoking was 45 years (range = 170 years), the median average smoking intensity was one pack per day (range = 03 packs per day), the median pack - years was 43.8 (range = 0.1192 pack - years), and the median age at initiation of smoking was 16 years of age (range = 645 years). the majority of patients had less than a high school education (72.5% or 319 patients), drank alcohol (84.3% or 365 patients), were married or cohabitating (82.2% or 356 patients), had abnormal respiratory function (51.7% or 186 patients), and had stage iib disease or less (71.3% or 321 patients) (table 2). characteristics of 450 lung cancer patients from the population - based case control environment and genetics in lung cancer etiology study, including histology, smoking status, and human papillomavirus (hpv) status pcr = polymerase chain reaction. results from 399 specimens with adequate dna of 450 selected for testing. results from 92 specimens with tumor present in the sample of 100 selected for testing. includes mixed type (adenosquamous, n = 10), synchronous (two different lung cancers, n = 6), non - small cells (n = 2), and other (including poorly differentiated, epithelial, neuroendocrine, mucoepidermoid, non - small cell, pleomorphic, sarcomatoid, n = 6). descriptive characteristics of 450 lung cancer patients from the population - based case control environment and genetics in lung cancer etiology study copd = chronic obstructive pulmonary disease. the median number of cells evaluable for the hpv16 and hpv18 pcr as measured by erv3 real - time pcr was 3680 cells (range = 1.9399 750 cells). combining these data, 399 (88.7%) of 450 specimens had adequate dna quantity and quality for analysis (ie, 100 or more cell equivalents and a 260/280 ratio between 1.5 and 2.2). on review of the 450 slides that were stained with hematoxylin and eosin, 412 specimens contained tumor cells. of those tumor - positive specimens, 73.8% (n = 304 specimens) contained greater than 60% tumor. we included all specimens with adequate dna (n = 399 specimens) in the prevalence estimate. among the 399 specimens with adequate dna, one female former smoker with adenocarcinoma was positive for hpv16 on initial testing. given the very low viral load in this tumor (average = 0.4 per 1000 cell equivalents across three tests of the same sample), we tested another tumor tissue specimen from this patient. eosin slide from this specimen consisted of greater than 80% cancer cells, indicating there was adequate tumor available to detect any hpv dna if it were present. this patient was diagnosed with stage ib lung cancer at 72 years of age and had smoked for 48 years at an average intensity of 0.75 packs per day (36 pack - years). pcr analysis targeting an 81-bp amplicon in the hpv16 e6 region confirmed the prior positive test (average of two viral copies per 1000 cell equivalents) in the first sample, and the second tumor tissue specimen from this patient was again negative. another patient who was initially negative for hpv16 tested positive with this assay but at low copy number (131 per 1000 cell equivalents). this second patient was a male with squamous cell carcinoma diagnosed at 64 years of age who had smoked for 43 years at an average intensity of one pack per day (43 pack - years). eosin slide from this patient also contained greater than 80% cancer cells. in the subset of 92 specimens tested with the broad - spectrum assay spf10-deia - lipa25 (including those from the two patients who tested positive with low viral load, as described above), no specimens were positive for hpv16 dna or for any hpv type. ddl diagnostic laboratory conducted type - specific testing for hpv16 as a follow - up test for the male patient identified as being hpv positive by pcr analysis targeting an 81-bp amplicon in the hpv16 e6 region. these results indicate that the prevalence of hpv dna is 0.0% (table 1) with an upper 97.5% confidence interval of 0.92% for the 399 specimens with adequate dna, including 370 ever - smokers (upper 97.5% confidence bound = 0.99%) and 27 never - smokers (upper 97.5% confidence bound = 12.8%). in this study, which is to our knowledge the largest study of hpv in lung tumor tissue from a western country, we found no evidence that hpv is associated with lung carcinogenesis. extensive laboratory efforts to avoid dna contamination and state - of - the - art, highly sensitive hpv dna detection assays were performed. the two tumors that tested positive had a very low viral load of less than one copy of hpv16 per cell, despite being composed of greater than 80% tumor cells. one of these patients tested negative in a separate tissue specimen, and the other was negative on additional type - specific hpv16 testing in a separate sample. none of the subset of 92 patients was positive for any other hpv type by the very sensitive spf10-deia - lipa25 testing method. hpv - related carcinogenesis at other anatomical sites indicates that hpv should be present in every tumor cell if it truly contributed to the development of that tumor (25). thus, whereas low - level hpv positivity in one tissue sample and complete lack of hpv in a separate sample may be possible, such tumor heterogeneity is unlikely to reflect a truly causal association. on the basis of these results, the prevalence of hpv in lung tumor tissue from this population was essentially 0%. a limitation of this study was that the tissue specimens were not necessarily adjacent or from the same block, although as described above, lack of uniform hpv results throughout the tumor would suggest that hpv was not associated with tumorigenesis. because tissue was collected for diverse purposes, it was not possible to address contamination in specimen collection. we also used viral load to assess whether detected hpv dna was present at a meaningful level, as described above. although dna degradation may occur in paraffin - embedded tissues, dna quality was confirmed by satisfactory 260/280 ratio and real - time pcr for the human actin gene and/or erv-3 in 399 of the 450 specimens. these results indicate that the contamination precautions used during pcr analysis were largely sufficient to avoid contamination and that the dna from the paraffin - embedded tissues was adequate for hpv dna detection. a particular strength of this study was that two independent laboratories extracted dna (at the ohio state university using phenol chloroform extraction and at ddl diagnostic laboratories using crude extraction methods, as described in materials and methods) from different tissue specimens from the same patient and conducted separate pcr assays for hpv dna. all 450 specimens were tested for the e6 and e7 oncogenes of hpv16 and hpv18, the two types most strongly associated with cancer outside the cervix (25), which circumvented concerns about false - negative results because of loss of the l1 gene through integration. a substantial proportion (92 specimens from 450 lung cancer patients) was tested for a broad range of hpv types with the l1-based spf10-deia - lipa25 system. the spf10-deia - lipa25 system is the gold standard for hpv dna testing in paraffin - embedded tissue because of its short pcr product (31) and ensured that specimens were tested for all carcinogenic and many noncarcinogenic hpv types. geographic differences in the prevalence of hpv in lung tumor tissue may be associated with variation in smoking habits, sexual behaviors, or other factors related to environmental exposures, culture, topography, or genetics (10). asian studies typically report higher pcr - based hpv prevalences of hpv in lung tumors than european studies, with a summary meta - estimate of 11.6% (95% ci = 9.5% to 14.2%) for hpv16 and 8.8% (95% ci = 6.0% to 12.8%) for hpv18 in lung tumor tissues from asia compared with 3.5% (95% ci = 2.3% to 5.3%) for hpv16 and 3.6% (95% ci = 2.3% to 5.7%) for hpv18 in lung tumor tissues from europe (10). the largest pcr - based asian studies found hpv dna in 42.0% (n = 92 specimens, 95% ci = 35.6% to 48.6%) of 219 paraffin - embedded lung tumor tissues (33,34) and 44.1% (n = 138 specimens, 95% ci = 38.5% to 49.8%) of 313 fresh - frozen lung tumor tissues (10,35). similar to our study, the largest previous european pcr - based study from france found a very low prevalence of hpv dna in 218 fresh - frozen lung tumor tissues (1.8% prevalence, 95% ci = 0.7% to 4.8%, n = 4 positive samples) (10,36). a study of lung cancer in taiwan found that nonsmoking female lung cancer patients were more likely to have hpv - positive lung tumor tissue than male lung cancer patients, who were more likely to smoke (37). environmental tobacco exposure is unlikely to account entirely for the increased risk in never - smokers. non smoking - related factors must therefore contribute to lung cancer among never - smokers. given the high upper confidence limit of our prevalence estimate in never - smokers (12.8%), further study of hpv in lung tumor tissue from never - smokers may be warranted. for example, the pcr - based prevalence of hpv in lung tumor tissues from japan ranges from 0.0% to 78.3% (10). although we found essentially no hpv in lung tumor tissues from italy, others have reported 12.8%21.1% hpv dna positivity in italian lung cancer patients (10). such disparities within the same geographic region emphasize the importance of taking precautions to avoid pcr contamination and prevent false - positive results (38) when using sensitive hpv dna detection assays to avoid false - negative results. our study used such precautions in two independent laboratories, verified dna quality before hpv detection, and used multiple sensitive methods for hpv dna detection. although a few previous asian and european studies have found 0% prevalence of hpv in lung tumor tissues, these studies included less than 100 patients. with nearly 400 patients evaluated, we had a sufficient sample size to detect any true prevalence of hpv greater than 1%. our study was twice as large as the next largest study in europe (n = 218 specimens). in conclusion, using multiple state - of - the - art methods to evaluate the presence of hpv dna in resected lung cancer tumors from a representative western study population, we found no evidence that hpv is associated with the development of lung cancer. although we detected essentially no hpv in specimens from the eagle study, evaluation in a larger population of never - smokers, in which the attributable risk of non smoking - related risk factors is necessarily higher, may be informative. differences in smoking habits could potentially account for the higher prevalence of hpv dna detected in lung tumor tissue from asian countries. for western populations, however, this study found no data to support that hpv is associated with lung carcinogenesis. this work was supported by an intramural research award from the division of cancer epidemiology and genetics, national cancer institute at the national institutes of health, and a national cancer institute director s innovation award, national cancer institute at the national institutes of health to j.k. ; general funds from the intramural research program of the national cancer institute at the national institutes of health ; and the cancer prevention fellowship program, office of preventive oncology, national cancer institute at the national institutes of health, bethesda, md. control study and cleared the article for publication but had no role in the analysis, decision to publish, or preparation of the article. | backgroundlung cancer kills more than 1 million people worldwide each year. whereas several human papillomavirus (hpv)associated cancers have been identified, the role of hpv in lung carcinogenesis remains controversial.methodswe selected 450 lung cancer patients from an italian population based case control study, the environment and genetics in lung cancer etiology. these patients were selected from those with an adequate number of unstained tissue sections and included all those who had never smoked and a random sample of the remaining patients. we used real - time polymerase chain reaction (pcr) to test specimens from these patients for hpv dna, specifically for e6 gene sequences from hpv16 and e7 gene sequences from hpv18. we also tested a subset of 92 specimens from all never - smokers and a random selection of smokers for additional hpv types by a pcr - based test for at least 54 mucosal hpv genotypes. dna was extracted from ethanol- or formalin - fixed paraffin - embedded tumor tissue under strict pcr clean conditions. the prevalence of hpv in tumor tissue was investigated.resultsspecimens from 399 of 450 patients had adequate dna for analysis. most patients were current (220 patients or 48.9%) smokers, and 92 patients (20.4%) were women. when hpv16 and hpv18 type specific primers were used, two specimens were positive for hpv16 at low copy number but were negative on additional type - specific hpv16 testing. neither these specimens nor the others examined for a broad range of hpv types were positive for any hpv type.conclusionswhen dna contamination was avoided and state - of - the - art highly sensitive hpv dna detection assays were used, we found no evidence that hpv was associated with lung cancer in a representative western population. our results provide the strongest evidence to date to rule out a role for hpv in lung carcinogenesis in western populations. |
the electronic database of a tertiary university - affiliated medical center was searched for all patients who underwent nd : yag laser capsulotomy from january 2011 to july 2014 following uneventful phacoemulsification surgery. exclusion criteria were the presence of ocular pathologies other than senile cataract (e.g., corneal disease, uveitis, open or closed angle glaucoma, pseudoexfoliation syndrome, retinitis pigmentosa, proliferative diabetic retinopathy, exudative age macular degeneration, and high myopia), history of intraocular surgery and evidence of intraoperative complications (e.g., capsulorhexis rim tear, incomplete continuous curvilinear capsulorhexis, zonular rupture, and posterior capsule rupture) or postoperative complications (e.g., iris synechiae, toxic anterior segment syndrome). all cataract surgeries were performed at our medical center, by either senior surgeons (attending ophthalmologists) or 5-year ophthalmology residents (junior surgeons), with a standard phacoemulsification, aspiration and implantation of an intraocular lens. thorough rotation of the nucleus following hydroprocedures and thorough polishing before iol implantation were performed in every cataract surgery, as it is a common practice in our institute. the type of lens selected for implantation between the two types available at our center, namely, the hydrophilic seelens or the hydrophobic acrysof, was left to the discretion of the attending surgeon. patients were encouraged to return to our medical center for examination if vision deteriorated at any time after surgery. pco was diagnosed on the basis of a subjective patient complaint of blurred vision and clinical findings on ophthalmological examination, including opacity in the central 3 mm area of the iol optic. positive findings of pco and best - corrected visual acuity (bcva) < 6/9 served as the basis for the decision to treat. patient- and procedure - related data were retrieved retrospectively from the medical files, as follows : patients ' age and sex, laterality, ocular comorbidities, date of cataract surgery, relevant details from the operative report completed by the surgeon immediately after surgery (including the name of the surgeon, lens type, and intraoperative complications), and findings on postoperative follow - up. details from nd : yag laser, capsulotomy report was retrieved as well, including date of capsulotomy, bcva, funduscopic examination with pupil dilatation, and slit - lamp assessment of pco. this is a retrospective analysis and therefore, for this type of study, formal consent was not required. statistical analysis was performed with the spss statistical package, version 17 (spss inc., continuous variables were expressed as a mean and standard deviation, minimum and maximum and categorical variables, as percent distribution. pearson 's chi - square test was used to compare the mean values of categorical variables. backward elimination regression analysis was performed to determine predictors of time from cataract surgery to laser capsulotomy. for this purpose, snellen visual acuity values were converted to logmar notation for analysis. the value of p < 0.05 was considered statistically significant. the study group included 222 patients (255 eyes), 107 (48%) male and 115 (52%) female, of mean age 73 8 years at the time of laser capsulotomy. a comparison between the hydrophilic (seelens) and hydrophobic (acrysof) iol groups is depicted in table 1. the seelens group was generally older (72.04 8.55 versus 67.38 7.59, p = 0.003) and a lower percentage of these lenses implanted by experienced surgeons (83% vs. 100%, p = 0.006) than the acrysof group. there were no significant differences between both lenses in terms of gender or bcva at presentation. comparison of seelens af and acrysof sa60at parameters the mean interval between cataract surgery and laser capsulotomy was 24 13 months (range 270 months). there was no significant difference between experienced and nonexperienced surgeons in terms of the time interval (24.1 13.7 versus 23.6 12.1, p = 0.81). the time interval was significantly shorter for surgeries with seelens (23.3 13.5 months) than acrysof implantation (28.2 13.0 months, p = 0.04) [fig. 1 ]. in backward elimination regression analysis with age at cataract surgery, with surgeon 's experience and lens type as independent variables and time from cataract surgery to laser capsulotomy as the dependent variable, only lens type remained statistically significant (p = 0.04). histogram of number of patients per time interval between cataract surgery and laser capsulotomy for the two types of intraocular lenses, including means of each lens this study compares the time interval to nd : yag laser capsulotomy following uneventful cataract surgery with implantation of one of two commonly used hydrophilic or hydrophobic acrylic posterior chamber iols. to the best of our knowledge, there are no published studies comparing this single - piece acrylic hydrophilic iol (seelens af) with other iols. the results showed that compared to the single - piece acrylic hydrophobic lens (acrysof sa60at), the widely used hydrophilic lens (seelens af) was associated with a statistically significant shorter time span between uneventful cataract surgery and laser capsulotomy. the overall mean time elapsed between cataract surgery and laser capsulotomy was 24 months (range 270 months), which is in - line with the follow - up time reported in previous prospective studies. importantly, the mean interval was significantly shorter, by almost 5 months, for implantations of the hydrophilic lens compared to the hydrophobic lens [fig. 1 ]. since we used a retrospective study design, the real - life interval between cataract surgery and laser capsulotomy was unlimited, as opposed to prospective studies in which the follow - up time is predefined. this statistically significant difference in the time interval between the two lenses may also be related to higher laser capsulotomy rates found in previous studies after implantation of hydrophilic versus hydrophobic iols. the lack of a statistically significant difference in bcva before laser capsulotomy by type of lens implanted suggests the severity of pco was similar between the two groups. in this study, therefore, our results may be generally applicable to other public tertiary medical centers. at the same time, this factor may act as a possible confounder ; since the surgeries were performed by multiple surgeons, analysis of the outcome of surgery may not be free of bias. to account for possible bias, we divided the operating surgeons into two groups by expertise and adjusted the results accordingly. in contrast to the study of fong. that found a correlation between surgical experience and laser capsulotomy rates, we did not find a correlation between surgical experience and time from cataract surgery to capsulotomy regardless of the type of iol used. the main value of this study lies in the finding of a significantly shorter time interval from cataract surgery to laser capsulotomy with the acrylic single - piece seelens hydrophilic lens, versus the acrysof hydrophobic lens, a relationship that has not been previously investigated. as described in an earlier retrospective study by johansson, the quality and reliability of the data depend on the functionality of the public health - care system within which the study is performed and the possibility of over- or under - treatment. our public health system is appropriate for this study, with a relatively short waiting list for capsulotomy (maximum 3 months), long follow - up, no - cost patient treatment, and no incentives for the treating ophthalmologists. further randomized prospective studies are needed to evaluate the long - term impact of using these iols as opposed to other types of lenses in clinical practice in terms of the risk of pco development. an additional limitation of this study was that pco was assessed by a clinical examination of the treating ophthalmologist without the use of a classification system. however, in all patients, pco caused significant enough visual disturbances that prompted individuals to seek medical attention and was deemed clinically significant by the treating ophthalmologist. the design of this study did not allow for the comparison of risk factors and evaluation of laser capsulotomy rates in all the patients in our medical center. in addition, because laser capsulotomy was the primary endpoint, we were unable to track every patient who underwent laser capsulotomy after cataract surgery. therefore, patients with pco who attended other medical centers or did not seek medical help were not detected. nevertheless, it is plausible that the majority of patients with pco causing significant visual disturbances would seek medical care and return to their providing medical center. furthermore, following cataract surgery at our medical center, patients are routinely instructed to return for examination if at any time they find their vision has deteriorated. systemic factors, such as diabetes, were not incorporated into this study ; however, patients with ocular pathologies other than senile cataract were excluded from the study. furthermore, the current study 's findings apply only to the specific lenses tested and may not apply to other hydrophilic or hydrophobic lenses. duration from uneventful cataract surgery to laser capsulotomy in patients with no ocular comorbidities is significantly shorter with implantation of the acrylic hydrophilic iol, seelens af, than with the acrylic hydrophobic iol, acrysof sa60at. the unique finding of this study is the shorter time from cataract surgery to laser capsulotomy after implantation of the hydrophilic lens, in comparison to the hydrophobic lens, a finding that was not reported before. future prospective studies comparing pco grading of different lenses and seelens af with masked observers before performing laser capsulotomy may be of interest to explore additional characteristics of these lenses. there are no conflicts of interest. | purpose : the aim of this study is to compare the length of time from uneventful cataract surgery using one of two common posterior chamber intraocular lenses (iols) (hydrophilic versus hydrophobic acrylic) to laser capsulotomy.materials and methods : retrospective analysis of all patients who underwent neodymium : yttrium - aluminum - garnet laser capsulotomy between 2011 and 2014 following uneventful phacoemulsification surgery at a tertiary university - affiliated medical center. medical records were reviewed for demographics, ocular comorbidities, operative details, postoperative follow - up, and findings of the precapsulotomy ophthalmologic examination. parameters, including age, sex, laterality, visual acuity, surgeon 's experience, and time from cataract surgery to capsulotomy, were compared between patients who received hydrophilic (seelens af, kibbutz hanita, israel) or hydrophobic (acrysof sa60at, alcon laboratories, fort worth, tx, usa) iols.results:the cohort included 222 patients (255 eyes), of which, 107 were male and 115 female, of mean age 73 8 years. mean interval from cataract surgery to laser capsulotomy was 24 months (range 270) and was significantly shorter in patients with seelens (23 13 months) than acrysof iol implantation (28 13 months, p = 0.04). lens type remained significant in multivariate analysis after including surgeon 's experience and age as potential confounders (p = 0.04).conclusion : the hydrophilic seelens iol is associated with a significantly shorter time interval from cataract surgery to laser capsulotomy than the hydrophobic acrysof iol. |
female pattern hair loss (fphl) is one of the most common complaints among female patients referred to dermatology clinics. it is the most common cause of alopecia involving 612% of women aged 2030 years and over 55% of women over 70 years of age. fphl is clinically characterized by diffuse nonscarring hair loss without obvious hair thinning in frontal, central, and parietal lobes of the scalp. a similar pattern is observed in men with miniaturized follicles and is known as androgenic alopecia. fphl designation is preferred in women over androgenic alopecia since the role of androgens in women is not still clear. fphl pathophysiology is not well understood yet, and it is likely to be a multifactorial genetic trait. androgen - independent mechanisms may contribute to this phenotype in addition to androgen - dependent ones. fphl is also seen in women without elevated androgen levels, raising the likelihood of interference of androgen - dependent mechanisms, and explaining the lack of response to treatment with androgen inhibitors in some fphl patients. serum levels of vitamin d are a factor recently considered in approaching patients with hair loss complaint. in a recent study, it has been suggested to measure vitamin d level in patients complaining from hair loss in addition to tests such as complete blood count (cbc), thyroid stimulating hormone, ferritin, testosterone, and dehydroepiandrosterone sulfate. the function of vitamin d3 receptor is critical in a population of keratinocyte stem cells remaining in the bulge region of hair follicles. defective vitamin d function leads to defective stem cell renewal and loss of hair follicle cycle. it has been suggested that an optimum concentration of vitamin d3 is essential to delay aging and hair loss, and a possible link has been proposed between the lack of vitamin d in serum and fphl. to confirm this hypothesis, a study was conducted in egypt in 2013 by rasheed. to investigate the role of vitamin d in women with chronic telogen effluvium or fphl serum level of vitamin d was considerably lower in women with chronic telogen effluvium as well as women with fphl relative to the control group (p 25.1. in control group, 5 patients (11.1%) had bmi 018, 32 patients (71.1%) had bmi 18.125, and 8 patients (17.8%) had bmi > 25.1. chi - square test showed no significant difference in bmi between the two groups (p = 0.33). according to ludwig classification, 28 patients (66.7%) were in mild group, 12 (28.6%) were in moderate group, and 2 patients (4.8%) were in severe group [table 1 ]. participant characteristics factor in case and control groups mean bmi in patient and control group was 22.94 3.27 and 22.78 3.07, respectively with no significant difference (p = 0.69). mean (sd) serum vitamin d3 level in patient and control group was 13.45 (8.40) and 17.16 (8.96), respectively. t - test showed a significant difference between the two groups in terms of serum vitamin d3 levels (p = 0.04). moreover, after classification of vitamin d level into three categories ([019.9 ], [2029.9 ], and [30150 ]), the evaluation of frequency distribution of vitamin d levels showed that 36 patients (80%) had vitamin d 019.9, 5 patients (11.1%) had vitamin d 2020.9, and 4 (8.9%) had vitamin d 30150. in control group, 32 patients (71.1%) had vitamin d 019.9, 6 (13.3%) had vitamin d 2020.9, and 7 (15.6%) had vitamin d (30150). pearson chi - square test showed no significant difference between the three categories of vitamin d in both groups (p = 0.56). the relationship between serum levels of vitamin d in fphl patients with participant characters has been shown in table 2. there was no significant correlation between serum levels of vitamin d3 and age (r = 0.08, p = 0.61), bmi (r = 0.06, p = 0.69), and fphl duration (r = 0.04, p = 0.77). furthermore, there was no significant relation between serum vitamin d3 level and family history of fphl, menstrual disorder, hirsutism, ludwig score of alopecia severity, and skin type of the patients. relationship between serum levels of vitamin d in female pattern hair loss patients with other variables in this study diffuse reduction in density of the scalp hair is associated with thinning of hair with complete or nearly complete maintenance of frontal hairline. bitemporal hair recession is observed in 13% and 37% of women before and after menopause, respectively. follicles are not lost but hair is miniaturized, and the space between hairs is increased so that the head skin is revealed over time. fphl pathophysiology is still not well known, and it is probably a genetically multifactorial trait. androgen - dependent mechanisms, as well as androgen - independent mechanisms, may contribute to this phenotype. in addition to sex hormones, fphl can be associated with insulin resistance, microvascular, and inflammatory disorders. insulin resistance reduces sex hormone - binding globulin in circulation that leads to early - onset androgenetic alopecia in men. miniaturization of hair follicles together with diffuse hair loss in frontal lobe can be seen in women without increased levels of androgens, which justifies the lack of response to androgen inhibitors in some women with fphl. on the other hand, fphl cases in patients with complete androgen insensitivity serum level of vitamin d is one of the factors recently considered in approach to patients with complaints of hair loss so that a recent study recommended the measurement of serum vitamin d level as well as androgen, thyroid hormone assay, and cbc. so far, except for a single study, the relationship between fphl and vitamin d3 deficiency has not been established. in the study of rasheed. in 2013 in egypt, serum vitamin d3 level in fphl was significantly lower than the control group. according to this study, low level of vitamin d3 is associated with hair loss in women with fphl. a screening test is useful to measure vitamin d3 level in women referring with hair loss, and dietary supplements can be useful to treat these patients. in the present study, mean level of vitamin d3 in patients with fphl was lower than healthy controls (p = 0.04). however, there was no significant different between the three categories of vitamin d (deficient, insufficient, and sufficient) in both groups (p = 0.56) and this could be due to the high prevalence of vitamin d deficiency in iran. in a study of 296 men with male pattern alopecia, no relationship was found between the extent and severity of alopecia with serum levels of vitamin d3. in another study on men with androgenic alopecia in 2012, no difference was found between vitamin d level in patients and the control group. considering these two studies and the lack of difference between vitamin d levels and androgenic alopecia in men, it seems that unlike men, vitamin d deficiency is involved in the development of androgenic alopecia or fphl through androgen - independent mechanisms. alopecia in some families with vitamin d - dependent rickets raises the likely important role of vdr in hair biology. vdr gene is a negative regulator of a number of genes and reduced suppressor activity of this gene by unliganded vdr leads to derepression of these genes, which may eventually lead to alopecia in these patients. histological evidence of hair follicles after treatment with vitamin d3 analogs in the skin biopsies of beigo / nude / xid mice afflicted with congenital alopecia due to inborn lack of vdr emphasizes the role of vdr in alopecia. treatment with vitamin d analogs was associated with natural hair follicle formation and increased the expression of specific ha7, ha8, and hb3 keratins. a certain concentration of vitamin d is essential to delay aging and hair loss. in vitro studies mesodermal papillary cells and keratinocytes of outer root sheath epidermis express varying levels of vdr based on the stage of the hair cycle. in terminal anagen and catagen stages, extensive studies on animal models show that vdr plays an important role in the cycle of the hair follicle, especially in the anagen phase. it has recently been shown that 1.25(oh) 2 vitamin d, vdr, and -catein stimulate the differentiation of hair follicle. environmental factors such as longitude, season, weather conditions (e.g., cloudy) and air pollution affect vitamin d3 level in serum. in this study, we attempted to minimize the role of environmental factors by choosing the patients from a specific region in the country (northeast) during one season (autumn). individual variables are the factors affecting serum levels of vitamin d3, including age, weight, skin type, and exposure to the sun. since the two groups were matched in terms of age, bmi, skin type and exposure to the sun, the impact of these confounding factors on our results was negligible. in this study, among 45 patients, 60% were in the age group 1530 years, 37.77% in 3140 years age group, 0% in 4150 and 2.22% in the age group over 50 years with a mean age of 29.11 7.30 years. in the study of sarda. in 2015 on fphl patients, 68% were in the age group 1830 years, 14% in 3140 years age group, 8% in 4150 years age group, and 10% in the age group over 50 years. the mean age of fphl patients in the studies of sarda., zhang., and deloche. was 29.22 13.01, 34.4 10.6, and 34.9 11.1 years, respectively. the severity of hair loss was ludwig i in the majority of our patients (66.7%), which was similar to the study of sarda (66%). 27.3% of our patients had a family history of fphl, which was 38% in the study of sarda. in the study of zhang. and aktan., ludwig i pattern was the most common in fphl patients. according to this study, the risk of fphl was associated with decreased serum levels of vitamin d3, and it is recommended to evaluate serum d3 level along with other hormone assays to check the patient 's status. it is also suggested to evaluate the therapeutic effects of oral vitamin d3 supplements and topical compounds such as calcipotriol in the treatment of fphl. the limitation of this study was lack of a standard to assess diet in patients to match the control and patient groups in terms of the level of dietary d3 intake. | background : female pattern hair loss (fphl) is the most common cause of alopecia in women, characterized by diffuse nonscarring hair loss in frontal, central, and parietal areas of the scalp. pathophysiology of fphl is still not well known, and it is probably a multifactorial genetic trait. fphl is also observed in women without increased androgen levels, which raises the likelihood of androgen - independent mechanisms and explains the lack of response to antiandrogen treatments in some patients. vitamin d is a factor that has recently been considered in dealing with these patients. the purpose of this study was to evaluate the serum levels of vitamin d in patients with fphl and compare it with healthy controls.methods:in this case - control study, 45 women with fphl were evaluated as well as the same number of healthy women matched for age, hours spent under sunlight per day, and body mass index. serum 25(oh) d3 level was measured using elisa.results:60% of fphl patients were in 1530 years old age group with the mean standard deviation (sd) age of 29.11 (7.30) years. in the majority of patients (66.7%), severity of hair loss was ludwig i. mean (sd) serum vitamin d3 level in patient and control group was 13.45 (8.40) and 17.16 (8.96), respectively. t - test showed a significant difference between the two groups in terms of vitamin d3 serum levels (p = 0.04).conclusions : this study indicated the correlation between the incidence of fphl and decreased serum levels of vitamin d3. it is recommended to evaluate serum vitamin d3 levels as well as other hormone assays in these patients. |
appendicitis is the most frequent indication for urgent surgery in children. since 1894, when mc burney described the laparotomic technique for appendectomy, the same operation has been the gold standard for acute appendicitis for over a century. in 1983, semm described for the first time the standard three ports laparoscopic appendectomy and since then the minimally invasive approach has gained wide acceptance among the pediatric surgeons. different variations of the laparoscopic technique have been proposed, all aiming to better cosmetic results, reduction in costs, and charges for hospitals, while keeping the safety of the operation unchanged. the umbilicus as the unique site to gain access to the abdomen and to the appendix has been widely reported in the literature, both as a port to exteriorize the appendix and perform an extracorporeal operation [2, 3 ] and as the site to place all laparoscopic instruments and perform an intracorporeal appendectomy (sils ; single - site laparoscopic surgery) [4, 5 ]. the trans umbilical laparo - assisted technique (tulaa) merges together the advantages of both a good intraabdominal laparoscopic visualization and the safety and quickness of an extracorporeal traditional appendectomy. a large series of pediatric patients operated on with this technique was presented in 1999 by valla., but patients were selected for absence of complicated appendicitis. recently, ohno. presented a paper in which the tulaa procedure was used in 416 patients but without any perforated appendicitis or local abscesses in the series. we present the experience of our centre, in which the use of tulaa was firstly introduced in 2006, in a team where only one surgeon had used the technique before, and it was decided to perform it with every kind of appendicitis, with or without the suspect of complicated appendicitis. the charts of all patients admitted to our surgical department from january 2006 to december 2010, with a diagnosis of appendicitis based on clinical (migration of pain to right lower quadrant (rlq), fever, and rebound tenderness in rlq), laboratory (elevated wbc count, elevate c reactive protein (crp)), and ultrasound (us) findings were retrospectively reviewed for demographical data, surgical treatment, time for completing the operation, intraoperative finding, need for conversion, and surgical complications. before 2006, all suspected appendicitis, regardless of history and perforation status, were treated by open surgery, and antibiotic therapy was prescribed according to the preference of the surgeon. since 2006, a new protocol for the treatment of complicated and uncomplicated appendicitis was introduced in our surgical department. all patients with suspected nonperforated or perforated appendicitis but with a history of less than 72 hours and no ultrasound evidence of consolidated appendiceal mass are offered tulaa. all patients undergoing surgery are administered a single dose of ampicillin plus sulbactam (50 mg / kg / dose) as prophylaxis 30 before starting the operation. if there is no perforation, the therapy with the same antibiotic is continued for 24 hours and then stopped ; whenever perforation is found, a regimen of ceftriaxone (100 mg / kg / die in one administration) plus metronidazole (7.5 mg / kg / dose every 8 hrs) is administered as long as the patient is afebrile for at least 24 hours, inflammatory markers are diminishing, and full oral diet is restored. in case of discharge before 7 days of intravenous antibiotics, patients are put on oral amoxicillin (50 mg / kg / dose every 12 hrs) and metronidazole (7.5 mg / kg / dose every 8 hrs) to complete a whole week of therapy. all appendiceal masses (symptoms lasting for at least 72 hours before presentation and us confirming the presence of a consolidated appendiceal abscess) are admitted to the ward and treated conservatively with an antibiotic regimen of ampicillin plus sulbactam (50 mg / kg / dose every 8 hrs), metronidazole (7.5 mg / kg / dose every 8 hrs), and tobramicina (5 mg / kg / die in one administration). after 48 hours of antibiotics, the patients are evaluated clinically, and inflammatory markers (crp and wbc) are repeated : if laboratory and clinical improvements are observed, the antibiotic therapy is continued until the patients are afebrile for at least 48 hours, inflammatory markers are progressively diminishing, and oral diet is resumed. after 8 weeks, an interval tulaa is performed. if no improvements are seen after 48 hours of antibiotics, the patients are offered tulaa. appendiceal abscesses with us evidence of a fecalith are treated with immediate tulaa since the fecalith is a known risk factor for abscess persistence. patients are started on a liquid diet 12 hours after the operation and on semiliquid diet in the first postoperative day. criteria for discharge are patient afebrile for at least 24 hours, restoration of full oral diet, and decreasing inflammatory markers. the patient is placed in the supine position under general anesthesia and mechanical ventilation. no bladder catheterization is used since all patients are asked to void before entering the operatory theatre. a single - infraumbilical incision is performed, and an 11 mm balloon trocar is inserted under direct visualization. capnoperitoneum is maintained within a range of 8 to12 mmhg according to the bodyweight of the patient with insufflation of co2 at a rate of 1.5 l / min. a single - operative laparoscope (karl storz endoskope, hopkins optical devices) with a side - arm viewing is inserted through a single, transumbilical port (figure 1), and a grasper is used to identify the appendix and to dissect retroperitoneal adhesions : when the tip of the appendix is freed, it is exteriorized through the umbilicus. an extracorporeal appendectomy is performed by dividing and ligating the mesoappendix, suture ligation, and inversion with purse string of the appendiceal base. no endomechanical, one or two further additional 5 mm trocars for additional graspers or cautery hook might be introduced. at the end of the procedure, intraperitoneal local anesthetic drugs such as naropine 0.2% at a dose of 0.5 ml / kg are instilled in the peritoneal cavity through one of the trocars. postoperative analgesia is administered via an elastomeric intravenous pump with tramadol 28 mcg / kg / min for 24 hours plus repeated doses of paracetamol 10 mg / kg every 8 hours. nausea is controlled by ondansetron 0.15 mg / kg every 8 hours, and rescue analgesic therapy consists of ketoprofene 1 mg / kg every 8 hours. when the appendectomy is considered impossible to be safely completed with any laparoscopic technique, it is converted to an open access. a primary open access is chosen only when the performing surgeons are not trained in laparoscopy or abdominal distension is prominent. an expert tula surgeon is defined as a surgeon who has performed at least 30 procedures as first operator or is trained in laparoscopy. from january 2006 until december 2010, 203 patients (79 female and 124 male) with an average age of 10 years (range 317) were admitted to our surgical ward with a diagnosis of appendicitis. seven (3.4%) out of 203 patients presented with an appendiceal mass and were treated conservatively according to the protocol : none required urgent surgery, and they all underwent interval tulaa 8 weeks later. the remaining 196 patients (96.5%) underwent urgent surgery. in 15 out of 196 cases, a primary open access was chosen : in 3 cases for marked abdominal distension, in one case because the surgical team was not sufficiently trained in laparoscopy, and in 11 cases because of palpation of a mass at the induction of anesthesia, and neither surgeons was an expert operator. sixty - six percent of the primary open accesses were performed in the first two years of the study. the intraoperative tulaa finding (figure 2) was uninflamed appendicitis in 18 cases (10%), uncomplicated acute appendicitis (catarrhal / phlegmonous without signs of perforation) in 109 (60%) cases, 49 (27%) cases were either gangrenous or perforated appendicitis with local peritonitis, and 5 (3%) were diffuse peritonitis. the 7 elective cases operated on after antibiotic treatment showed an appendix with adhesions but no acute inflammation. none of these was converted, one required an additional trocar, and no complications were recorded. the mean operatory time for the elective procedure was 43. of all 181 urgent tulaa, 12 (6.6%) were converted : in 3 cases the intraoperative finding was nonperforated appendicitis with retrocaecal position, in 8 cases there was a perforation with local peritonitis, and one was a diffuse peritonitis. nine operations were converted by a team of nonexpert surgeons, and 3 by a team in which at least one surgeon was considered expert. of the 169 nonconverted tulaa procedures, 151 were carried out through the single umbilical port, 16 (9.4%) required a second trocar, and 2 (1.2%) required a third trocar. the mean operative time for single- port tulaa was 52 (47 when the first operator was an expert, 55 when the first was a nonexpert). among the 181 urgent operations, there were 5 wound infections (3.8%), of which one required a surgical revision, and 5 patients (3.8%) were diagnosed as having postoperative intraperitoneal abscess which were all managed conservatively with intravenous antibiotics. the tulaa technique was first reported in a large pediatric series by valla. in 1999. it was described as umbilical one - puncture laparoscopic - assisted appendectomy (uoplaa), and performed in 200 of preoperatively selected children, that showed no signs of advanced appendicitis or diffuse peritonitis. our choice of offering tulaa as the first choice operation to the whole spectrum of appendicitis (except local consolidated abscess without fecaliths) was dictated by the fact that this technique can be easily switched to a standard three - port laparoscopic appendectomy, which is widely reported in the literature to be feasible also in advanced form of appendicitis. in our series, only 10% of cases (16 urgent and one elective procedure) required an additional port, and only 2 cases (one perforated appendicitis with local peritonitis and one gangrenous retrocecal appendicitis) required the positioning of 2 additional trocars. the possibility to insert a second or a third trocar in a position that suites the intraoperative findings and the anatomy of the patient, rather than using the standard positions for the traditional laparoscopic procedure, can be of great help during the division of adherences and omentum especially in advanced cases. similar results in the number of additional ports were reported by stylianos. with 9.8% of 359 cases which required one or two additional ports, by valla. in 2006 reported a lower use of additional trocars in only 2 of 111 patients (2%). the latter report has also a lower rate of conversions (2%) than in our experience and this could be explained by the fact that when tulaa was first introduced in our hospital, the equipment was not well trained in laparoscopy : 75% of our conversions were made by nonexpert members of the staff, and 66% of cases were converted in the first two years of the protocol. this confirms the need of a period of learning curve and the possibility of using this operation as a starting training to acquire laparoscopic abilities. our operating time (52 minutes) seems longer than other reports : stylianos. 24 minutes, visnjic 33 minutes : these series, however, exclude perforated appendicitis while we include all stages of appendicitis. the only complication we exclude was us confirmed appendiceal abscess with a symptom duration longer than 72 hours, where a conservative management was carried on, according to the current literature. recently, numerous reports appeared in the literature describing the so - called sils (single - incision laparoscopic surgery) technique where a single umbilical trocar is used to introduce three or four instruments or, as an alternative, at the umbilical site a subcutaneous pocket is created and the natural umbilical fascial defect plus one or two other stab incisions are used to place cannulas (or only instruments) to perform an endocorporeal laparoscopic appendectomy [4, 5, 12 ]. however, this kind of approach results in longer operating times than standard multiport laparoscopic appendectomy because of the clashing of instruments [12, 13 ], and it does not have the remarkable reduction in costs that the single trocar operative scope have, compared to standard laparoscopic technique [9, 10 ]. in our series, 30% of cases were advanced stages of appendicitis but we feel that this is not a condition that should stop from starting the operation with a tulaa approach : the only real contraindication to tulaa is the intestinal loops ' huge distension that may exist in some diffuse peritonitis. the concern for umbilical infections due to exteriorization of a suppurative or ruptured appendix can be controlled if adequate skin gauze protection is secured around the umbilical opening when bringing the appendix out. our rate of wound infections (3.8%) matches perfectly the one calculated for standard three - port laparoscopic appendectomy in a recent meta - analysis comparing open and laparoscopic appendectomy, therefore, confirming that the extracorporeal operation does not endanger the umbilical scar. petnehazy. suggest that tulaa can be a simpler approach for appendectomy in obese children, and even if we did not stratify our population by weight in the present study, a single incision has proved to be a quick and effective approach for this kind of patients also in our hands. according to our experience, tulaa is a safe, minimally invasive approach to patients suffering for acute appendicitis, regardless of the perforation status. it is also a suitable operation for training laparoscopic abilities, and it has low instrumentation requirements. | the paper reports the results of a retrospective review of the medical charts of 203 patients admitted to a pediatric surgical unit with a diagnosis of acute appendicitis between january 2006 and december 2010 when a transumbilical laparoscopic - assisted appendectomy (tulaa) was introduced as a new surgical technique. among 203 admitted patients, 7 (3.5%) had a localized appendiceal abscess and were treated with antibiotics. all of them responded to antibiotics and underwent tulaa interval appendectomy 8 weeks later. 196 patients (96.5%) underwent immediate surgery. in 12/181 (6.6%) urgent cases, conversion to laparotomy was necessary, in 3 patients because of bowel distension and in 9 for retrocecal position of appendix. in all 181 tulaa completed procedures, one trocar was used in 151 cases (89.4%), two trocars in 16 (9.4%), and three trocars in 2 (1.2%). the mean operative time for single port tulaa was 52 complications included 5 wound infections and 5 intra - abdominal abscesses, all managed conservatively. in conclusion, tulaa is a safe, minimally invasive approach with acute appendicitis, regardless of the perforation status, and can be recommended in the pediatric urgical settings. |
encephalocele is a rare condition that consists of herniation of cerebral matter through openings of dura and skull the incidence of the congenital form is estimated to be 1 per 3,000 to 10,000 live births.1 2 many classification systems for encephalocele exist ; however, the most widely accepted is that from matson and ingraham,3 based on the location of the encephalocele : basal, sincipital, convexity, and atretic. convexity encephalocele, subdivided into frontal, parietal, occipital, and cervico - occipital, is the most common type.3 those lesions are usually located at midline, extending from the nasal region to the occiput, although off midline temporal regions have also been reported.4 5 a majority of the encephaloceles are congenital and manifest in childhood. here, we report a rare case of a traumatic intradiploic encephalocele (ie) presenting with contralateral hemiparesis several decades after an initial pediatric trauma. a 45-year - old caucasian, right - handed man presented to our emergency department (ed) with 3 to 4 weeks history of progressive gait disturbances and left lower extremity weakness. his pertinent medical history was negative for any recent history of head trauma, but was significant for a moderate traumatic brain injury (tbi) at the age of 9 years, where he fell off a tree and sustained a closed parietal skull fracture with no posttraumatic cognitive or neurologic deficits. he had no history of seizures, central nervous system infections, stroke, or brain tumor. his neurological exam at admission was significant for left hemiparesis (strikingly more pronounced on lower than upper extremity) and positive left pronator drift testing. he was initially seen in the ed where a magnetic resonance imaging (mri) showed ie of the right parasagittal parietal bone containing portions of the right precentral gyrus and surrounding brain parenchyma (fig. a 3-mm (a, b) coronal and (c) axial reformats in both (a) brain and (b) bone windows on (d) ct and coronal mri showing herniated brain parenchyma. ct, computed tomography ; mri, magnetic resonance imaging. noncontrasted computed tomography (ct) demonstrated a well - circumscribed, 4.3 3.7 cm area of osteolysis in the right parasagittal parietal bone, with internal soft tissue attenuation (fig. c), complete erosion of the inner table, and noncontiguous lytic changes of the outer table (fig. 1b, c). mri with and without gadolinium contrast administration demonstrated a right parasagittal dural defect with intradiploic herniation of the right precentral gyrus (fig. 1d). there was no restricted diffusion to suggest acute infarction (fig. (a) axial 5 mm se) t2 images, (b) axial 5 mm blood suppressed se t1 images, (c) axial 5 mm flair images, and (d) axial 5 mm postcontrast se t1 images (12 mm gadavist). ct, computed tomography ; mri, magnetic resonance imaging ; se, spin echo after initial neurosurgical evaluation in the ed, the patient was admitted to the neurosurgical ward and started on dexamethasone 4 mg intravenously every 6 hours. his left lower extremity weakness responded positively to the initial steroids therapy, with partial but transient improvement on his leg weakness to antigravity strength and ambulatory with a rolling walker. he was reluctant to proceed with surgical intervention initially, and elected to continue hospital observation for a few days. since no sustainable improvement on his strength was seen after 2 days of high - dose dexamethasone treatment, he decided to proceed with surgical intervention. under general anesthesia, and using ct - guided intraoperative frameless stereotactic navigation, the area correspondent to the encephalocele was identified and marked. the calvarium was exposed through a bicoronal incision. the bone overlying the encephalocele appeared to be mottled and thin. cortical brain matter was visualized directly underneath the most thinned portions of the right parietal bone. it appeared pale with no evidence of cortical vasculature, and the usual gyral anatomy had been lost. starting from the areas of bone dehiscence, the craniectomy was then extended centrifugally until identification of the normal inner table surrounding the whole encephalocele. once the margins of the encephalocele were completely identified and dissected, a large craniotomy incorporating the bone defect was completed using high - speed craniotome (fig. the pial - arachnoid plane surrounding that area was very sclerotic and firmly adherent to the encephalocele pedicle. due to the risk of vascular injury to an already friable but apparently still eloquent brain, we decided not to proceed with arachnoidal opening or resection of the encephalocele sac. the bone flap was replaced with a titanium mesh used for coverage over the craniectomy site. (a) intraoperative picture showing large craniotomy incorporating the bone defect. the pial - arachnoid plane surrounding that area was very sclerotic and firmly adherent to the encephalocele pedicle. the defect was shown in (b) and (c) for dorsal and ventral side, respectively. he tolerated the procedure well and was kept in intensive care unit for 24 hours without complications. postoperative noncontrasted ct head revealed no ischemic changes on the surrounding parenchyma and adequate reduction of the encephalocele (fig. (c) a 3-mm axial bone window and coronal reformats in both (b) brain and (a) bone windows on postoperative ct. on 2-month follow - up, his preoperative left leg weakness has greatly improved with near resolution of preoperative hemiparesis. a 45-year - old caucasian, right - handed man presented to our emergency department (ed) with 3 to 4 weeks history of progressive gait disturbances and left lower extremity weakness. his pertinent medical history was negative for any recent history of head trauma, but was significant for a moderate traumatic brain injury (tbi) at the age of 9 years, where he fell off a tree and sustained a closed parietal skull fracture with no posttraumatic cognitive or neurologic deficits. he had no history of seizures, central nervous system infections, stroke, or brain tumor. his neurological exam at admission was significant for left hemiparesis (strikingly more pronounced on lower than upper extremity) and positive left pronator drift testing. he was initially seen in the ed where a magnetic resonance imaging (mri) showed ie of the right parasagittal parietal bone containing portions of the right precentral gyrus and surrounding brain parenchyma (fig. a 3-mm (a, b) coronal and (c) axial reformats in both (a) brain and (b) bone windows on (d) ct and coronal mri showing herniated brain parenchyma. noncontrasted computed tomography (ct) demonstrated a well - circumscribed, 4.3 3.7 cm area of osteolysis in the right parasagittal parietal bone, with internal soft tissue attenuation (fig. c), complete erosion of the inner table, and noncontiguous lytic changes of the outer table (fig. mri with and without gadolinium contrast administration demonstrated a right parasagittal dural defect with intradiploic herniation of the right precentral gyrus (fig. (a) axial 5 mm se) t2 images, (b) axial 5 mm blood suppressed se t1 images, (c) axial 5 mm flair images, and (d) axial 5 mm postcontrast se t1 images (12 mm gadavist). ct, computed tomography ; mri, magnetic resonance imaging ; se, spin echo after initial neurosurgical evaluation in the ed, the patient was admitted to the neurosurgical ward and started on dexamethasone 4 mg intravenously every 6 hours. his left lower extremity weakness responded positively to the initial steroids therapy, with partial but transient improvement on his leg weakness to antigravity strength and ambulatory with a rolling walker. he was reluctant to proceed with surgical intervention initially, and elected to continue hospital observation for a few days. since no sustainable improvement on his strength was seen after 2 days of high - dose dexamethasone treatment, he decided to proceed with surgical intervention. after initial neurosurgical evaluation in the ed, the patient was admitted to the neurosurgical ward and started on dexamethasone 4 mg intravenously every 6 hours. his left lower extremity weakness responded positively to the initial steroids therapy, with partial but transient improvement on his leg weakness to antigravity strength and ambulatory with a rolling walker. he was reluctant to proceed with surgical intervention initially, and elected to continue hospital observation for a few days. since no sustainable improvement on his strength was seen after 2 days of high - dose dexamethasone treatment, he decided to proceed with surgical intervention. under general anesthesia, and using ct - guided intraoperative frameless stereotactic navigation, the area correspondent to the encephalocele was identified and marked. cortical brain matter was visualized directly underneath the most thinned portions of the right parietal bone. it appeared pale with no evidence of cortical vasculature, and the usual gyral anatomy had been lost. starting from the areas of bone dehiscence, the craniectomy was then extended centrifugally until identification of the normal inner table surrounding the whole encephalocele. once the margins of the encephalocele were completely identified and dissected, a large craniotomy incorporating the bone defect was completed using high - speed craniotome (fig. the pial - arachnoid plane surrounding that area was very sclerotic and firmly adherent to the encephalocele pedicle. due to the risk of vascular injury to an already friable but apparently still eloquent brain, we decided not to proceed with arachnoidal opening or resection of the encephalocele sac. the bone flap was replaced with a titanium mesh used for coverage over the craniectomy site. (a) intraoperative picture showing large craniotomy incorporating the bone defect. the pial - arachnoid plane surrounding that area was very sclerotic and firmly adherent to the encephalocele pedicle. the defect was shown in (b) and (c) for dorsal and ventral side, respectively. he tolerated the procedure well and was kept in intensive care unit for 24 hours without complications. postoperative noncontrasted ct head revealed no ischemic changes on the surrounding parenchyma and adequate reduction of the encephalocele (fig. (c) a 3-mm axial bone window and coronal reformats in both (b) brain and (a) bone windows on postoperative ct. on 2-month follow - up, his preoperative left leg weakness has greatly improved with near resolution of preoperative hemiparesis. a cephalocele is a protrusion of cerebral contents through a defect in the dura or skull. these lesions are classified according to their location and contents (meninges, brain parenchyma, ventricles, and/or vasculature). the presence of brain parenchyma characterizes an encephalocele, whereas those without brain parenchyma are called meningocele. the location of these lesions varies as they may involve the posterior fossa, occipital, basal, frontoethmoidal, and/or parietal regions.6 7 8 depending on the etiology, cephaloceles are further classified as primary or secondary. in the case of primary cephaloceles, a congenital defect exists in the dura or skull, usually at the site of a cranial suture, representing a midline closure defect of the neural tube. in secondary cephaloceles, a preceding injury such as infection, tumor, trauma, or surgery is usually the underlying cause. in this case report, we present an extremely rare phenomenon of a symptomatic posttraumatic parietal ie manifesting 36 years following traumatic head injury. medline and pubmed database literature search revealed only 11 cases previously reported in the literature. five had no history of trauma, four cases9 10 11 (including the present study) were found to be posttraumatic and/or iatrogenic, and one did not comment on any supposed etiology for the encephalocele.12 therefore, traumatic injuries to the skull were the reported etiology of the cephalocele in approximately 40% of the literature cases. the initial insult typically occurs in childhood with a delayed diagnosis decades later after manifestation of signs and symptoms, if at all.13 14 15 16 abbreviation : nr, not reported ; lle, left lower extremity ; lue, left upper extremity ; rle, right lower extremity ; rue, right upper extremity. patil and etemadrezaie9 reported a 64-year - old man had head trauma 8 months prior to experiencing an enlarging palpable scalp mass. lenthall and penney10 reported a 15-month - old infant sustained a head injury and presented with growing fracture with pulsatile mass 9 months later. our patent presented with progressive hemiparesis 36 years following pediatric traumatic head injury. depending on where the lesion is, symptoms may vary widely, from seizures to aphasia, numbness, hemiparesis, or even asymptomatic. of the case reports, the most common symptoms are hemiparesis / weakness11 12 17 (four patients including the present study), seizures5 17 (two patients), palpable mass9 10 (two patients) ; one was incidentally found.18 surgical repair with or without excision of the herniated brain parenchyma is generally performed in symptomatic patients. clinical response is generally favorable with minor improvement in symptoms to completely asymptomatic on follow - up. in order for an encephalocele to occur d'almeida and king19 postulated that if only the inner table and dura mater are affected by the fracture from tbi, the diploic space is opened and enlarged over time by pulsation of cerebrospinal fluid (csf). lenthall and penney10 stressed the importance of an outward driving force for enlargement of the diploic space secondary to normal brain growth, csf pulsatility, or increased intracranial pressure, as also seen in the coughing spell reported by loumiotis.12 patil and etemadrezaie9 proposed that low - velocity blunt injury may fracture only the inner table, which is thinner. the depression tears the dura and the recoil creates a negative pressure that forces the underlying arachnoid and brain into the diploic space. menk proposed that there could be congenital factors, although the article focused on intradiploic pseudomeningocele as opposed to ie. they proposed that intradiploic cyst develops through foveolae granulare which are anatomical and physiological defects that form a hollow space of the inner table. these granulations usually lie in the frontoparietal bone and near the superior sagittal sinus. in the setting of head trauma, dura and arachnoid membranes differential diagnosis includes epidermoid or dermoid cyst, cavernous hemangioma, eosinophilic granuloma, plasmacytoma, metastasis, and osteosarcoma.18 mri is necessary to establish a diagnosis. herniation of brain parenchyma through a dural defect into intradiploic space is what separates this lesion from the rest on the differential, as is seen in our case. however, definitive diagnosis still lies in surgical exploration and histopathological examination.13 20 21 surgical correction with craniotomy, reduction of herniated cortex, excision of herniated parenchyma, and cranioplasty have been reported as the optimal management strategy. if a patient remains asymptomatic, observation and regular follow - up may be adequate. surgical excision of the herniated brain parenchyma depends on the proximity to the location of the vital functional areas of the brain such as the motor cortex. in our case, because of the risk of vascular injury to an already friable but apparently still eloquent brain, we decided not to proceed with arachnoidal opening or resection of the encephalocele sac. functional mri, magnetic source imaging of evoked sensory potential, or intraoperative cortical mapping have been used to help with defining eloquent cortex.17 frameless neuronavigational systems as used in our case are recommended to enhance precision. regardless of the method, meticulous dissection must be exercised to maximize recovery and minimize complications. this case is unique in that the patient became symptomatic several decades after a pediatric head trauma. | encephalocele is a rare condition that consists of herniation of cerebral matter through openings of dura and skull. a majority of encephaloceles are congenital and manifest in childhood. we present a case of a 45-year - old man presenting with contralateral hemiparesis and found to have an extremely rare phenomenon of a symptomatic posttraumatic parietal intradiploic encephalocele (ie) manifesting 36 years following pediatric traumatic head injury. computed tomography and magnetic resonance imaging confirmed herniation of brain tissue into the intradiploic space. surgical treatment with reduction of the encephalocele achieved near resolution of preoperative hemiparesis on follow - up. the pathogenesis and a literature review of ie are discussed. |
energetic neutral atoms (enas) are produced by charge exchange processes between energetic ions and neutral gasses. as ions, these particles are constrained by the lorentz force to their local magnetic fields ; however, once they are neutralized they are not influenced by electromagnetic forces and travel on ballistic paths. imaging this ena emission from charged particlegas interaction regions is analogous to imaging photon emission from a glowing gas cloud and therefore a useful remote sensing technique for such regions. we motivate two specific scientific questions that a lowenergy, high angular resolution instrument would address : (1) to understand the spatial and temporal structure of the heliospheric boundary and (2) to understand the spatial structure of magnetospheric ion precipitation into the upper atmosphere. other topics such as imaging the terrestrial ring current and the subsolar magnetopause are of great interest and could be probed with this instrument, but we do not go into detail on these specific implementations here. the interstellar boundary explorer (ibex) mission [mccomas., 2009a ] and the voyagers produced breakthroughs in our understanding of the interaction between the sun and its local galactic neighborhood [e.g., stone. this new frontier of heliospheric physics is fundamental to our understanding of the influence of the solar wind on the local interstellar medium, and vice versa. the structure and dynamics of this interaction as imaged by ibexlo [fuselier., 2009 ], ibexhi [funsten., 2009 ], and the cassini magnetosphere imaging instrument (mimi) ion and neutral camera (inca) [krimigis., 2004 ] hint at the physical processes at play in this region. in situ measurements by the voyager 1 and 2 interstellar probes have revealed that this region is a wholly new plasma regime in which the nonthermal energetic particles dominate the pressure balance and define the structure of the interaction. the ibex and cassini inca ena images (e.g., figure 1) fundamentally challenged our preexisting picture of the interaction between the solar wind and the interstellar medium. for instance, the shape of the interaction had long been supposed to be simply an elongated, cometarylike interaction suggested by planetary magnetospheres as well as by remote observations of other astrospheres [e.g., mccomas., 2009c ]. however, an alternate configuration of a diamagnetic bubble was also proposed by parker and was invoked to interpret the ena observations of krimigis.. our best hopes for the resolution of this fundamental theoretical dichotomy lie in the orderofmagnitude improvement in observations that will span the energy ranges of the observations that underlie the ibex and inca measurements and better resolve the spatial, energy and temporal structure of this crucial interaction region. of special interest is the fine structure of the ribbon feature shown in the inset b of figure 1 (top) [mccomas., 2011 ] and the pronounced knot. this feature appears to have fine structures that lie beyond the ibex angular resolution ; in addition, their spectral indices are different [dayeh., 2011 ]. separating these features and their energy spectra will allow for better understanding of the underlying physics. heliospheric ena maps from (top) ibexhi and (bottom) cassini inca demonstrating the energy dependence of the spatial features. the second area of scientific interest for high angular resolution ena imaging is the region of lowaltitude emission (lae) in the earth 's magnetosphere. large numbers of energetic ions from the inner magnetosphere continuously precipitate and interact with the upper atmosphere through a complicated cascade of reactions (charge exchange, stripping, ionization, and excitation) that change their state from charged to neutral several hundreds of times. contrary to common belief, precipitating ions are not all lost to the atmosphere, but up to 30% or more escape upward as neutrals and ions through a particle albedo effect (figure 2) [e.g., bazell., this has fundamental consequences on how we understand the energy transfer to the atmosphere and how magnetospheric particles are lost. image hena and twins demonstrated that magnetospheric ion precipitation leads rather to an intense albedo of backscattered enas (or lae), than to an empty upward loss cone. the ena emissions come from a thin (~10 km) layer at about 300 km altitude [bazell., 2010 ]. there is a clear scientific need for new instrumentation that can better resolve the energy and angular distribution of enas. our answer to this scientific call is the lowenergy neutral imager (leni), which utilizes novel methods in ena imaging. from a sounding rocket, cubesat, small satellite, or spacecraft in orbit, leni would image the distribution of the bright lae of enas emitted as a result of magnetospheric ion precipitation into and backscattering from the upper atmosphere. show that this emission comes from a ~10 km layer at roughly 300 km altitude. to obtain 5 km resolution in this region with a 2 imager we would have to be within 140 km meaning that imaging from low earth orbit is possible. traditionally, ena measurements have used either charge conversion surfaces or thin foils to convert the neutrals into ions or produce electrons that can contribute to measuring the energy, composition, and/or velocity of the ena. with a charge conversion surface, incident enas graze the surface picking up or losing an electron in the process, while with carbon foils the ena traverses the foil releasing electrons from its surfaces. for very lowenergy enas it is not possible to use the carbon foil technique as the particle can not penetrate even the thinnest foil, hence the preference for charge conversion surfaces for very low energy (tens of ev) instruments. for highenergy enas, foils have generally been used, and their angular resolution has been satisfactory due to the modest angular scattering of these highenergy neutrals in the foils. however, for the mediumenergy (500 ev to 20 kev) range both ultrathin carbon foils and charge conversion surfaces have been used. charge conversion surfaces tend to be inefficient, with ionization efficiencies of 25% at 1 kev [e.g., fuselier., carbon foils degrade angular and energy resolution by scattering lowenergy enas through large angles (for a 0.6 ug / cm foil the half angle scattering at 1 kev is about 19 ; [funsten., 2009 ]). ibexhi successfully dealt with the large scattering angle by precollimation to ~4 and only analyzing the charged fraction exiting the foil (~7% at 1 kev) instead of the much more abundant neutral fraction (~93% at 1 kev) [funsten., figure 3 details the techniques used for four instruments that have been produced for ena detection for some portion of the 500 ev to 10 kev energy range. we propose to change the paradigm of low to mediumenergy (0.5 kev20 kev) ena imaging by tightly collimating the incoming ena beam and directly imaging the incoming particles as they traverse the start foil. the instrument concept is shown in figure 4 and the instrument capabilities are given in table 1. an ena enters the instrument through a single, zerobias angle microchannel plate (mcp) providing 2 2 or better collimation. immediately after exiting the entrance mcp, the ena traverses a ~0.5 g / cm ultrathin carbon foil producing both start and coincidence electrons from both sides of the foil. the start electron from the entrance side of the foil is then accelerated back through the entrance mcp where it produces an electron cascade that is imaged by a delay line anode on the start mcp (chevron stack). the coincidence electron from the back of the first foil is accelerated by a 1 kv potential through a second ~0.5 g / cm foil, which the ena also traverses producing a stop electron. due to the 1 kv acceleration of the coincidence electrons, they fall onto a region of the stop / coincidence mcp separate from the stop electrons produced on the second foil by the ena. stop electrons provide both timing and spatial coincidences that are used to efficiently remove background signals. the predicted leni instrument performance leni, like the jupiter icy moon explorer (juice) jovian energetic neutrals and ions (jeni) mitchell., cassini inca [krimigis., 2004 ], and image hena [mitchell., 2000 ] relies on acceleration and steering of secondary electrons by electrostatic fields to achieve both accurate timing and position resolution required for imaging enas. the leni electron optical design has the following advantages over previous generation ena imagers : the image is produced from the entrance to the first foil prior to angular scattering and energy straggling, reducing the effects of scattering in the image.narrow stop and coincidence timing windows result in a large reduction of the background.spatial coincidence between the start, stop, and coincidence signals for additional background rejection.particle detection does not rely on charge conversion through the carbon foils or onto a charge conversion surface. the image is produced from the entrance to the first foil prior to angular scattering and energy straggling, reducing the effects of scattering in the image. spatial coincidence between the start, stop, and coincidence signals for additional background rejection. particle detection does not rely on charge conversion through the carbon foils or onto a charge conversion surface. leni utilizes a variation of the mirrorfree electron optics developed and flown successfully for the hockey puck style ion detectors (new horizons pepssi [mcnutt., 2009 ], van allen rbspice [mitchell., 2013 ], juno jedi [mauk., 2013 ], mmseis [mauk., 2014 ]). this electron optical arrangement operates by placing a puller electrooptical element toward the mcp and a pusher element above as shown in the electron optical simion simulation in figure 5. the 1 kv stop foil accelerates the electrons from the backside of the start foil which are then detected on the same mcp as the stop electrons. the exit of the second foil is nearly identical to the region above the entrance mcp and has been modified slightly to accommodate both the electrons produced on the second foil and the accelerated electrons from the first foil. we note that the 1 kv accelerated electrons do not interact strongly with these thin foils and can therefore be efficiently accelerated through the foil with minimal losses. extrapolating the experimental data from kanter results in a critical energy (the energy required to transmit through the foil) of ~100 ev and an energy loss of under 10%. because ~1 kv electrons are much faster than any of the enas for which this instrument is designed, false stops generated by those few coincidence electrons that do generate secondaries at the exit surface of the stop foil will generate a tof too short for a valid ena, and the false event will be rejected. the electron optics design of leni is optimized for high angular and energy resolution imaging. black shows the path of the primary ena through the collimator and start and stop foils. the green shows the start and coincidence electrons derived from the back and front of the start foil, respectively. the inset shows how the collimator mcp doubles as an electron multiplier producing a large start signal. leni utilizes a novel approach to high angular resolution by collimating the enas as they enter the instrument. we note that though the am alpha has an energy of 5.486 mev, lower energy ions will behave similarly in this collimator. this collimator consists of a photonis 0 bias angle 50:1 mcp with 25 m pores in a 12 11 mm rectangular configuration (figure 7). enas, along with other unwanted signal producers (i.e., photons, ions) that strike the side walls of the mcp channels will produce secondary electrons in the mcp ; however, they will not produce the crucial stop and coincidence electrons from either of the foils. at angles slightly outside of the 2 mechanical collimation, some particles will forward scatter or produce knockon sputtering of water molecules on the mcp channel surface resulting in h atoms and ions that could masquerade as the original ena or ion and contribute to imaging artifacts [e.g., mbius. these contributions will result in potentially significant broadening of the angular response of the sensor. the primary advantage of using an mcp for the start collimator is that we can place this collimator directly on top of the start foil. since 2 2 collimation requires very high aspect ratio collimation, it is difficult to electrostatically pull electrons through a metallic collimator. this is because the secondary electrons will come off of the foil with a cosine distribution and will typically collide with the walls of this collimator and be lost to this interaction. however, with the mcp collimator, the electron interacts with the walls of the mcp and produces a cascade of electrons that can exit the collimator providing both a fast and amplified start signal that maintains the spatial resolution of the collimator. the collimator mcp design will not suffer from ion feedback due to the separation of the collimator from the start mcp ; ions produced in the mcp collimator will not travel to the start mcp due to the electron optics. measured transmission of 5.486 mev alphas from an am source through the leni mcp collimator as a function of incident beam angle, where = 0 corresponds to the boresight direction. the top left figure shows the test setup with the timing start and stop mcps. the bottom left figure shows the measured coincidence time between the start and stop mcp when tested using am alphas through the system (5.486 mev) with the black curve showing with the source is pointed down and the red curve with the source on the opposite side of the tof assembly pointed up through the instrument. leni is designed to use 0.5 g / cm amorphous carbon foils backing the collimator mcp for the start signal. these foils have been the standard workhorse for lowenergy plasma and ena measurements and have proven robust when handled and mounted carefully. these foils allow for the transmission of lowenergy enas (down to below 500 ev) and for the production of electrons from the surfaces for detection. they have demonstrated substantial reductions in angular scattering at low energies, especially for heavier species such as oxygen, as well as reductions in energy straggling when using these foils. if grapheme proves to be a superior choice to amorphous carbon foils, we will work toward incorporating graphene in the leni design. typical methods for rejecting unwanted charged particles include the use of a deflection system consisting of biased conducting plates in front of the collimator as was done on the inca [krimigis., 2004 ], due to the electric field between these deflection plates, charged particles in and slightly above the instrument energy range are forced into the plate surface, thus prohibiting them from entering the collimator and therefore reducing spurious counts. further methods used in reducing unwanted signal involve serrating the surface of the plates to inhibit forward scattering. note that the charged particle deflection system is not used for collimation and is purposely placed out of the leni field of view (fov). the leni charged particle deflection system is a reconfiguration of the same subsystem used effectively on the cassini inca and image hena sensors where they typically reduced ambient ion intensities by 4 orders of magnitude or more and ambient electron intensities by 2 to 3 orders of magnitude. the leni implementation employs two electrostatic charged particle deflection plates that are biased alternately at ground and up to + 6 kv. the charged particle deflection plates are placed outside of the leni fov such that iontoneutral conversion effects and forward scattering of ions into the collimator is minimized. a set of outer grounded plates provide a suitable emi / emc environment to the spacecraft and the hv plate is recessed such that the field protrusion is minimized allowing for accommodation near other fields and particles instrumentation. the voltage on the charged particle deflection system can be turned off to allow for direct ion observations. electrostatic simulations for the arrangement reject incoming charged particles with energies up to 200 kev / q, well outside of the leni energy range. previous ena sensors in the 1 kev range have reported artifacts in their data due to ion conversion on surfaces in front of the entrance aperture and electron impactgenerated ion beam effects ahead of the aperture. ion conversion is inevitable on the rejection plates as particles will interact with the surfaces producing either ions or neutrals as a result. if a resultant neutral particle is forward scattered with little energy loss, then it can be mistakenly identified as an unscattered ena. to minimize this contribution the charged particle deflection plate surfaces are serrated such that forward scattering is largely eliminated. previous ena sensors in this energy range have required light traps, electrostatic analyzers, or gold gratings to reject the ambient uv environment, at considerable cost to sensitivity. inca and hena used moderately thin, gridmounted foils in their entrance slits as well as covering the stop mcp. in hena and inca, these foils serve the dual purpose of producing secondary electrons as primary particles penetrate them, and reducing uv and visible light so that uvgenerated photoelectrons do not overdrive the mcp counting rates. for hena and inca, the start foils were designed to reduce the fuv and euvrelated counting rates to 10/s or less, which in the case of inca required a ~10 g / cm start foil thickness, whereas for hena, in the brighter earth environment, the start foil thickness was 15 g / cm. the stop foils in each were ~7 g / cm. these foils resulted in accidental rates consistent with the formula r acc = r start r stop w tof 10 10 10 10 s where r start and r stop are the start and stop uv rates and w tof is the window for valid time of flight. of course, these foils could have been made even thicker, eliminating even more of the uv photon flux, but the thickness of the foils, particularly the start foil, limits the minimum energy ion or ena that can penetrate both foils and reach the stop mcp. furthermore, those particles that are sufficiently energetic are still scattered in angle and lose some energy in transiting the start foil, so the thinner the foil, the lower the energy loss and the less the scattering. the jeni and leni approach to the start foils is to eliminate the requirement that they act as photon filters. therefore, the start foils, relieved of any requirement on uv suppression, may be made as thin as feasible, consistent with surviving launch and providing a source of secondary electrons. traditional plasma analyzers routinely use gridmounted carbon foils of 1 to 2 g / cm thickness. the ibexhi ena instrument employs foils ~0.6 g / cm covering its entire aperture of 156 cm, tiled in individual foils each ~10.5 cm. the ibexhi foils survived launch intact and continue to function nominally after 7 years in orbit. based on this experience as well as laboratory tests, leni will use ~0.5 1 g / cm start foils. the stop foil is also ~0.51 g / cm, to permit secondary emission from its exit side down to lowena incidence energy. because the foils are not relied upon as photon filters, our design is highly tolerant to loss of foil area to broken grid cells that may occur during launch, or in manufacture or handling. individual cell loss affects only sensitivity (sensor efficiency), so lost foil area on the order of 10% or even more has only minor impact on science. mission design and accommodation would be made such that the sun was kept out of the instrument fov, limiting the uv to such sources as earth shine (reflected or reemitted solar uv) and interstellar lyman alpha. experience with both inca and hena showed that the serrated charged particle deflection plate surfaces forward scattered very little light, reducing the keepout zone around the sun to < 5 from direct entry. the interstellar lyman alpha uv is roughly 1.5 kr at earth resulting in rates from uv of ~10/s per pixel for each foil surface. the accidental rate per pixel would be r acc = r start r coin r stop w tof w coin 10 10 10 10 10 1 10 s where r coin is the uvinduced rate from the back, coincidence side of the start foil, and r stop is the stop rate. we compare this expected noise rate with that of ibexhi, which was measured to be 5.8 10 hz for triple coincidence [funsten., the primary reason for the relatively small uv rate on the leni detectors comes from the small geometric factor per pixel of 10 cm sr. the highvoltage side of the mcp is facing out meaning that uv that strikes the top of the mcp or high in the pores will lead to very little or no electron multiplication. in addition, the charged particle deflection system blocks uv from a large swath of the sky. for a heliospheric mission the spacecraft would likely be a sunpointed spinning platform, in which case the instrument can be accommodated without allowing the sun (or solar wind) into the fov. for a magnetospheric mission keeping the sun out of the fov can be more difficult, but experience from image shows that it is possible with careful mission design. penetrators result in low rates of tofs, all shorter than those for foreground particles, and so recognized as invalid by the valid event processing. based on laboratory measurement as well as inflight performance of hena and inca, the serrated plates reduce ambient charged particle intensities by at least 4 orders of magnitude within the sensor energy range. the inca serrated plate performance was measured at ~200 kev, in the 0.5 to 20 kev range required here we expect similar or greater rejection efficiency due to increased forward scattering at higher energies. for a heliospheric imaging mission the instrument should be placed into an orbit with low penetrating radiation and the shielding would be minimal. for a magnetospheric mission careful instrument shielding could be applied to reduce counts from penetrators. leni, like the jupiter icy moon explorer (juice) jovian energetic neutrals and ions (jeni) mitchell., cassini inca [krimigis., 2004 ], and image hena [mitchell., 2000 ] relies on acceleration and steering of secondary electrons by electrostatic fields to achieve both accurate timing and position resolution required for imaging enas. the leni electron optical design has the following advantages over previous generation ena imagers : the image is produced from the entrance to the first foil prior to angular scattering and energy straggling, reducing the effects of scattering in the image.narrow stop and coincidence timing windows result in a large reduction of the background.spatial coincidence between the start, stop, and coincidence signals for additional background rejection.particle detection does not rely on charge conversion through the carbon foils or onto a charge conversion surface. the image is produced from the entrance to the first foil prior to angular scattering and energy straggling, reducing the effects of scattering in the image. spatial coincidence between the start, stop, and coincidence signals for additional background rejection. particle detection does not rely on charge conversion through the carbon foils or onto a charge conversion surface. leni utilizes a variation of the mirrorfree electron optics developed and flown successfully for the hockey puck style ion detectors (new horizons pepssi [mcnutt., 2009 ], van allen rbspice [mitchell., 2013 ], juno jedi [mauk., 2013 ], mmseis [mauk., 2014 ]). this electron optical arrangement operates by placing a puller electrooptical element toward the mcp and a pusher element above as shown in the electron optical simion simulation in figure 5. the 1 kv stop foil accelerates the electrons from the backside of the start foil which are then detected on the same mcp as the stop electrons. the exit of the second foil is nearly identical to the region above the entrance mcp and has been modified slightly to accommodate both the electrons produced on the second foil and the accelerated electrons from the first foil. we note that the 1 kv accelerated electrons do not interact strongly with these thin foils and can therefore be efficiently accelerated through the foil with minimal losses. extrapolating the experimental data from kanter results in a critical energy (the energy required to transmit through the foil) of ~100 ev and an energy loss of under 10%. because ~1 kv electrons are much faster than any of the enas for which this instrument is designed, false stops generated by those few coincidence electrons that do generate secondaries at the exit surface of the stop foil will generate a tof too short for a valid ena, and the false event will be rejected. the electron optics design of leni is optimized for high angular and energy resolution imaging. black shows the path of the primary ena through the collimator and start and stop foils. the green shows the start and coincidence electrons derived from the back and front of the start foil, respectively. the inset shows how the collimator mcp doubles as an electron multiplier producing a large start signal. leni utilizes a novel approach to high angular resolution by collimating the enas as they enter the instrument. we note that though the am alpha has an energy of 5.486 mev, lower energy ions will behave similarly in this collimator. this collimator consists of a photonis 0 bias angle 50:1 mcp with 25 m pores in a 12 11 mm rectangular configuration (figure 7). enas, along with other unwanted signal producers (i.e., photons, ions) that strike the side walls of the mcp channels will produce secondary electrons in the mcp ; however, they will not produce the crucial stop and coincidence electrons from either of the foils. at angles slightly outside of the 2 mechanical collimation, some particles will forward scatter or produce knockon sputtering of water molecules on the mcp channel surface resulting in h atoms and ions that could masquerade as the original ena or ion and contribute to imaging artifacts [e.g., mbius. these contributions will result in potentially significant broadening of the angular response of the sensor. the primary advantage of using an mcp for the start collimator is that we can place this collimator directly on top of the start foil. since 2 2 collimation requires very high aspect ratio collimation, it is difficult to electrostatically pull electrons through a metallic collimator. this is because the secondary electrons will come off of the foil with a cosine distribution and will typically collide with the walls of this collimator and be lost to this interaction. however, with the mcp collimator, the electron interacts with the walls of the mcp and produces a cascade of electrons that can exit the collimator providing both a fast and amplified start signal that maintains the spatial resolution of the collimator. the collimator mcp design will not suffer from ion feedback due to the separation of the collimator from the start mcp ; ions produced in the mcp collimator will not travel to the start mcp due to the electron optics. measured transmission of 5.486 mev alphas from an am source through the leni mcp collimator as a function of incident beam angle, where = 0 corresponds to the boresight direction. the apl leni breadboard test setup. the top left figure shows the test setup with the timing start and stop mcps. the bottom left figure shows the measured coincidence time between the start and stop mcp when tested using am alphas through the system (5.486 mev) with the black curve showing with the source is pointed down and the red curve with the source on the opposite side of the tof assembly pointed up through the instrument. leni is designed to use 0.5 g / cm amorphous carbon foils backing the collimator mcp for the start signal. these foils have been the standard workhorse for lowenergy plasma and ena measurements and have proven robust when handled and mounted carefully. these foils allow for the transmission of lowenergy enas (down to below 500 ev) and for the production of electrons from the surfaces for detection. have shown promising results using graphene foils. they have demonstrated substantial reductions in angular scattering at low energies, especially for heavier species such as oxygen, as well as reductions in energy straggling when using these foils. if grapheme proves to be a superior choice to amorphous carbon foils, we will work toward incorporating graphene in the leni design. typical methods for rejecting unwanted charged particles include the use of a deflection system consisting of biased conducting plates in front of the collimator as was done on the inca [krimigis., 2004 ], hena [mitchell., 2000 ], and mena [pollock charged particles in and slightly above the instrument energy range are forced into the plate surface, thus prohibiting them from entering the collimator and therefore reducing spurious counts. further methods used in reducing unwanted signal involve serrating the surface of the plates to inhibit forward scattering. note that the charged particle deflection system is not used for collimation and is purposely placed out of the leni field of view (fov). the leni charged particle deflection system is a reconfiguration of the same subsystem used effectively on the cassini inca and image hena sensors where they typically reduced ambient ion intensities by 4 orders of magnitude or more and ambient electron intensities by 2 to 3 orders of magnitude. the leni implementation employs two electrostatic charged particle deflection plates that are biased alternately at ground and up to + 6 kv. the charged particle deflection plates are placed outside of the leni fov such that iontoneutral conversion effects and forward scattering of ions into the collimator is minimized. a set of outer grounded plates provide a suitable emi / emc environment to the spacecraft and the hv plate is recessed such that the field protrusion is minimized allowing for accommodation near other fields and particles instrumentation. the voltage on the charged particle deflection system can be turned off to allow for direct ion observations. electrostatic simulations for the arrangement reject incoming charged particles with energies up to 200 kev / q, well outside of the leni energy range. previous ena sensors in the 1 kev range have reported artifacts in their data due to ion conversion on surfaces in front of the entrance aperture and electron impactgenerated ion beam effects ahead of the aperture. ion conversion is inevitable on the rejection plates as particles will interact with the surfaces producing either ions or neutrals as a result. if a resultant neutral particle is forward scattered with little energy loss, then it can be mistakenly identified as an unscattered ena. to minimize this contribution the charged particle deflection plate surfaces are serrated such that forward scattering is largely eliminated. previous ena sensors in this energy range have required light traps, electrostatic analyzers, or gold gratings to reject the ambient uv environment, at considerable cost to sensitivity. inca and hena used moderately thin, gridmounted foils in their entrance slits as well as covering the stop mcp. in hena and inca, these foils serve the dual purpose of producing secondary electrons as primary particles penetrate them, and reducing uv and visible light so that uvgenerated photoelectrons do not overdrive the mcp counting rates. for hena and inca, the start foils were designed to reduce the fuv and euvrelated counting rates to 10/s or less, which in the case of inca required a ~10 g / cm start foil thickness, whereas for hena, in the brighter earth environment, the start foil thickness was 15 g / cm. the stop foils in each were ~7 g / cm. these foils resulted in accidental rates consistent with the formula r acc = r start r stop w tof 10 10 10 10 s where r start and r stop are the start and stop uv rates and w tof is the window for valid time of flight. of course, these foils could have been made even thicker, eliminating even more of the uv photon flux, but the thickness of the foils, particularly the start foil, limits the minimum energy ion or ena that can penetrate both foils and reach the stop mcp. furthermore, those particles that are sufficiently energetic are still scattered in angle and lose some energy in transiting the start foil, so the thinner the foil, the lower the energy loss and the less the scattering. therefore, the start foils, relieved of any requirement on uv suppression, may be made as thin as feasible, consistent with surviving launch and providing a source of secondary electrons. traditional plasma analyzers routinely use gridmounted carbon foils of 1 to 2 g / cm thickness. the ibexhi ena instrument employs foils ~0.6 g / cm covering its entire aperture of 156 cm, tiled in individual foils each ~10.5 cm. the ibexhi foils survived launch intact and continue to function nominally after 7 years in orbit. based on this experience as well as laboratory tests, leni will use ~0.5 1 g / cm start foils. the stop foil is also ~0.51 g / cm, to permit secondary emission from its exit side down to lowena incidence energy. because the foils are not relied upon as photon filters, our design is highly tolerant to loss of foil area to broken grid cells that may occur during launch, or in manufacture or handling. individual cell loss affects only sensitivity (sensor efficiency), so lost foil area on the order of 10% or even more has only minor impact on science. mission design and accommodation would be made such that the sun was kept out of the instrument fov, limiting the uv to such sources as earth shine (reflected or reemitted solar uv) and interstellar lyman alpha. experience with both inca and hena showed that the serrated charged particle deflection plate surfaces forward scattered very little light, reducing the keepout zone around the sun to < 5 from direct entry. the interstellar lyman alpha uv is roughly 1.5 kr at earth resulting in rates from uv of ~10/s per pixel for each foil surface. the accidental rate per pixel would be r acc = r start r coin r stop w tof w coin 10 10 10 10 10 1 10 s where r coin is the uvinduced rate from the back, coincidence side of the start foil, and r stop is the stop rate. we compare this expected noise rate with that of ibexhi, which was measured to be 5.8 10 hz for triple coincidence [funsten., 2009 ]. the primary reason for the relatively small uv rate on the leni detectors comes from the small geometric factor per pixel of 10 cm sr. the highvoltage side of the mcp is facing out meaning that uv that strikes the top of the mcp or high in the pores will lead to very little or no electron multiplication. in addition, the charged particle deflection system blocks uv from a large swath of the sky. for a heliospheric mission the spacecraft would likely be a sunpointed spinning platform, in which case the instrument can be accommodated without allowing the sun (or solar wind) into the fov. for a magnetospheric mission keeping the sun out of the fov can be more difficult, but experience from image shows that it is possible with careful mission design. penetrators result in low rates of tofs, all shorter than those for foreground particles, and so recognized as invalid by the valid event processing. based on laboratory measurement as well as inflight performance of hena and inca, the serrated plates reduce ambient charged particle intensities by at least 4 orders of magnitude within the sensor energy range. the inca serrated plate performance was measured at ~200 kev, in the 0.5 to 20 kev range required here we expect similar or greater rejection efficiency due to increased forward scattering at higher energies. for a heliospheric imaging mission the instrument should be placed into an orbit with low penetrating radiation and the shielding would be minimal. for a magnetospheric mission careful instrument shielding could be applied to reduce counts from penetrators. this instrument is a novel solution to high angular resolution ena imaging in the energy range from 0.5 to 20 kev. the novel mcp collimator technique along with the use of ultrathin carbon foils will produce scatter free images with 2 angular resolution. our calculations of the expected noise rates and detection efficiencies have shown that leni could successfully image the heliospheric boundary at higher angular resolution than ibexhi or cassini inca and the magnetospheric lowaltitude emission and thus leni present a significant technological advancement to the current stateoftheart in ena imaging techniques. | abstractto achieve breakthroughs in the areas of heliospheric and magnetospheric energetic neutral atom (ena) imaging, a new class of instruments is required. we present a high angular resolution ena imager concept aimed at the suprathermal plasma populations with energies between 0.5 and 20 kev. this instrument is intended for understanding the spatial and temporal structure of the heliospheric boundary recently revealed by interstellar boundary explorer instrumentation and the cassini ion and neutral camera. the instrument is also well suited to characterize magnetospheric ena emissions from lowaltitude ena emissions produced by precipitation of magnetospheric ions into the terrestrial upper atmosphere, or from the magnetosheath where solar wind protons are neutralized by charge exchange, or from portions of the ring current region. we present a new technique utilizing ultrathin carbon foils, 2d collimation, and a novel electron optical design to produce high angular resolution (2) and highsensitivity (103 cm2 sr / pixel) ena imaging in the 0.520 kev energy range. |
the advent of next - generation sequencing and metagenomics has resulted in increasing numbers of ever - larger datasets describing the community structure and function of a variety of different environments, from the human gut (arumugam. 2011 ; david. 2014) to arctic peat soils (lipson. 2013) and deep - sea vents (xie. next - generation sequencing technologies have greatly reduced sequencing costs and speed, and researchers can now affordably study whole microbial communities and functions. prior to this, the focus was on community species composition, studied using 16s rrna targeted amplicon sequencing. amplicon sequencing does not require the dna coverage that metagenomic studies require and can accurately identify which species are present in a sample (woese and fox 1977 ; lane. 1985 ; hugenholtz 2002), but it does not provide the depth of information, such as gene function, that full metagenome sequencing and annotation provides. cost is no longer the primary limiting factor for undertaking metagenomic studies, rather it is now bioinformatics and processing power required to process the data produced. illumina 's hiseq platform, for example, can affordably sequence the most complex of microbial communities, and the challenge now is to interpret the data produced. (2014) provide an extensive directory of tools available for different tasks involved in a metagenomic project pipeline, related to a range of omics studies. megan (huson. 2007) is a popular graphical user interface program for analyzing and visualizing blast results to study the taxonomy of microbial communities. while megan typically analyses blast results in a few minutes, running blast searches against reference sequences in a database is computationally intensive and slow for metagenomes (desai. 2012 ; hunter. 2012 ; thomas, gilbert and meyer 2012). web - based servers are increasingly popular for processing large amounts of data. with an intuitive web interface and a variety of analytical tools to choose from, mg - rast (meyer. mg - rast allows users to upload raw sequence files that are processed through quality filters and annotated using a selection of user - defined parameters, such as reference databases, minimum identity cut - off values, maximum e - values or expect - values, and minimum alignment lengths. details of the processing procedure can be found in the mg - rast technical report (wilke. rapsearch2 (zhao, tang and ye 2012) translates nucleotide sequences and aligns them with annotated protein sequences, reporting to be c. 100-fold faster than blastx with only a 1.3%3.2% reduction in sensitivity (the proportion of sequences annotated). with pauda (huson and xie 2014) uses a similar approach and claims to be 10 000-fold faster than blastx, although with a significant reduction in sensitivity. diamond (buchfink, xie and huson 2015) purports to be both fast and accurate, with a 20 000-fold increase in processing speed compared to blastx. in sensitive mode, 99% of sequences are aligned, with a speed increase of 2000-fold compared to blastx. like blast with megablast, rapsearch2 and diamond offer fast and sensitive modes, each coming at the cost of the other. one codex is a web - based program that uses a different technique to blast and mg - rast to classify sequences (https://onecodex.com/). the program designers report that it runs 900 times faster than blast while maintaining similar genus - level sensitivity and precision (the proportion of annotated sequences that are correctly identified), taking hours rather than days to classify most metagenomes. one codex works by comparing k - mers (sequences of a set length) from a sequence to a reference database of k - mers ; the greatest number of 100% k - mer matches determines the classification. blast and mg - rast classify sequences by matching them with the most similar sequences in a database. unlike mg - rast, one codex does not annotate genes for function. the choice of database, minimum identity cut - off value (i.e. sequence match stringency), minimum alignment length cut - off value and minimum e - value limit (the probability a match has occurred by chance) all influence sequence annotation accuracy, which, in turn, affect the reproducibility and interpretation of the data. an inherent issue with metagenomic studies is that establishing the accuracy of sequence annotation for environmental samples is practically impossible, given that the quantities of organisms and genes are unknown. therefore, determining the most effective annotation method is fundamental to investigating environmental communities with confidence. there are a variety of different reference nucleotide and amino acid databases available for annotating gene or protein sequences (table s1, supporting information). the m5nr database (wilke. 2012) incorporates information from a selection of different databases (see table s1, supporting information), increasing the amount of reference data available for annotation. using a single reference database may be the best option in some cases, for example, where 16s rrna amplicons are used as a method to identify taxa, rather than other genes. whereas taxonomic nomenclature is universal, governed by international conventions, there are multiple approaches for functional classification. two popular methods include clusters of orthologous groups (cogs) (tatusov, koonin and lipman 1997) and the kyoto encyclopedia of genes and genomes (kegg) (kanehisa and goto 2000). cogs comprise orthologous functions that allow for functional description of poorly characterized genomes based on protein orthologs. cog descriptions are characterized under cellular processes, information storage and processing, metabolism and poorly characterized. kegg descriptions are characterized under cellular processes, environmental information processing, genetic information processing, human diseases and metabolism. due to the differences in characterization approaches, kegg operates on a subscription basis, and mg - rast uses the latest freely available version (updated in 2008). selecting a minimum identity cut - off value for metagenome analysis is challenging because interspecific sequence identity varies among genes. too high a value will accurately identify genes with highly conserved regions, such as 16s rrna or highly conserved coding genes with little synonymous substitution, but may fail to identify genes or non - coding regions that are highly variable. conversely, a value too low will allow for highly variable genes to be identified, but may also incorrectly identify an organism / function, thus providing false community / function profiles. the optimum identity cut - off point for species identification using the 16s rrna gene is widely accepted as 97% (stackebrandt and goebel 1994 ; rossell - mora and amann 2001 ; chun. 2007 ; richter and rossell - mra 2009 ; mende. 2013 ; vtrovsk and baldrian 2013), although this value has its limitations. some species, such as certain rickettsia spp., have a 16s rrna gene similarity greater than 97%, thus a cut - off value at this level would not differentiate between the species (fournier. 2003). stackebrandt and goebel (1994) suggest that a higher value may be more appropriate, but fewer sequences would be annotated due to sequencing errors and sequence mutations. typically, lower cut - off values are suitable for metagenomic studies as the multitude of genes that contain varying degrees of conservation are sequenced. the default value used by mg - rast, and used in many metagenomic studies (e.g. tatusov, koonin and lipman 1997 ; lipson. 2013), is 60%, as this allows for identification using less conserved genes and non - coding regions. a lower value allows shorter sequences to be annotated, although the chance of incorrectly annotating a shorter sequence is higher. a higher value will reduce this chance, but may also reduce the number of annotations overall. combining a low minimum alignment length with a strict minimum identity cut - off value allows shorter sequences to be annotated but with a high match criteria. setting maximum e - values and minimum alignment lengths allows stringency of annotations to be controlled. e - values denote the maximum probability that a sequence annotation has occurred by chance. lower maximum e - values will reduce the number of possible incorrect annotations, although this also reduces number of annotations retained for analysis. the aim of this study is to evaluate the accuracy of megan, mg - rast and one codex annotation methods while investigating how using different databases and parameters impact the annotation of metagenomes. to do this, a novel simulated metagenome was generated using the ncbi whole bacterial genome database and annotated using each pipeline and, for mg - rast, with different reference databases, minimum identity cut - off values, minimum alignment lengths and maximum e - values. using a simulated metagenome comprising known genome abundances allows the accuracy of annotation to be quantified. the simulated metagenome was also annotated using megablast, a faster variation of blast, to provide a control and so that megan, mg - rast and one codex could be compared to a standard in sequence annotation. comparing the megan, mg - rast and one codex annotations to the megablast annotations will quantify the accuracy of these programs for annotating sequences from organisms whose genomes are stored in the ncbi databases. a simulated metagenome, hereafter simmet, was created using nessm (jia. 2013), comprising the complete ncbi bacterial genome database (ftp://ftp.ncbi.nlm.nih.gov/genomes/bacteria/all.fna.tar.gz, may 2013 collection, accessed on 29/04/14). nessm creates synthetic metagenomes from input genomes based on user - defined parameters (e.g. sequence count, length and abundance distributions) that aim to simulate real sequencing data, including expected sequencing errors (i.e. substitutions, insertions and deletions) based on the chosen sequencing technology simulated (see step ii : error models and sequencing coverage bias estimation in jia. a total of 2400 000 sequences with a read length of 450 base pairs were designated for simulation, based on 454 pyrosequencing. one strain for each of the 1505 species in the ncbi bacterial genome database was randomly selected to be included in the simulation because certain species, e.g. model organisms and human pathogens such as escherichia coli, salmonella enterica, mycobacterium tuberculosis, bacillus cereus and staphylococcus aureus, have been extensively studied and are overrepresented in the databases. the species abundance distribution used for simulation was derived from the abundance distribution of a pasture soil metagenome (sequence count : 2378 586, mg - rast i d : 4554767.3) (see equation 1). (1)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation } y = - 2490\ln \left (x \right) + \ 19748 \end{equation}\end{document}where x is the randomly selected species rank. the sequences were processed with sickle (joshi and fass 2011) to trim low - quality ends, with the average threshold phred score set at 20 (a base call error rate of 1%). the simmet metagenome file was annotated with megablast (available from : http://blast.ncbi.nlm.nih.gov/blast.cgi?page_type = blastdocs&doc_type = download) as a control, using a reference database of the genomes used to create simmet. the ncbi nucleotide database (updated 17/11/2014) (ftp://ftp.ncbi.nlm.nih.gov/blast/db/) was also used to assess the annotation performance of megablast. the maximum e - value selected was 1-e and the minimum alignment length, 15 bases. megablast annotations using the simmet database will be referred to as control and those using the ncbi nucleotide database will be referred to as megablast. the blast results were uploaded to megan (version 5.2.3) and analyzed using the same parameters used in the blast. the databases investigated within mg - rast were genbank, greengenes, rdp, refseq, seed, swissprot and trembl. the m5nr and m5rna databases were excluded from individual sequence analysis, as individual sequence annotations were not available for download from mg - rast for these databases. for both the megablast and mg - rast annotations, which use a minimum sequence alignment match to annotated sequences, the minimum identity cut - off values tested were as follows : 40%, 50%, 60%, 70%, 80%, 90%, 95% and 97%. the minimum alignment lengths tested were as follows : 10, 15, 20, 25, 30, 25, 40, 45, 50, 55 and 60 bp. the maximum e - values tested were as follows : 1-e, 1-e, 1-e and 1-e. aside from testing, default parameters were used : 60%, 15 bp and 1-e, respectively, for minimum identify cut - off, minimum alignment length and maximum e - value. the sequence ids and annotations were extracted from the megablast results (https://github.com/sandyjmacdonald/blast_parser), and full taxonomic lineages were generated for each sequence using the ncbi taxonomy database (available from : ftp://ftp.ncbi.nlm.nih.gov/pub/taxonomy/ ; ncbi database version generated 13/05/2013). species level was excluded from analysis due to the high variation in annotated species nomenclature and the accepted caveats associated with microbial species classification (gevers. 2005 ; achtman and wagner 2008), e.g. horizontal gene transfer (gogarten and townsend 2005 ; bapteste and boucher 2009). discrepancies identified between databases for organism names were corrected for, such as ncbi using the old name chloroflexia (as of 17/11/14) and mg - rast using the new name chloroflexi for the same class. unidentified and those that were annotated but were either ambiguously annotated or not annotated at all taxonomic levels had the corresponding levels in the lineage replaced with unclassified. for megan and one codex, ncbi taxa ids were used to generate the lineages. the taxonomic lineage for each annotated sequence was compared to the lineage for the corresponding source sequence in simmet to determine the annotation sensitivity and precision at each taxonomic level. the effect that minimum identity cut - off values, minimum alignment lengths and maximum e - values had on annotation sensitivity and precision were established using megablast and mg - rast. the correlations between the relative abundances for each taxon in simmet and in the annotations were calculated using pearson 's product moment correlation coefficient. the natural logarithms of the relative abundance values were calculated for plotting, as the original distributions would not visually convey the variations in low abundance taxa. the taxa richness values for each taxonomic level were calculated. unlike investigating taxa, correct functional annotations can not be ascertained with 100% confidence. to investigate functional annotation performance, protein sequences associated with the sequences in simmet were extracted from genbank records and annotated using the kegg automatic annotation server (kaas) (moriya. both are web - based functional annotation tools independent of those investigated in this study. they did not contain sequencing errors and thus provided the best possible indication of the functional annotation accuracy, although the caveats associated with sequence annotation (e.g. possibly incorrectly assigning a function) are present. kegg orthology and cog ids were extracted from the kass and webmga results, respectively, for each sequence annotated and compared with the ids assigned by mg - rast. the parameters set for the taxonomic investigation were used, and the minimum identity cut - off values investigated were as follows : 40%, 50%, 60%, 70%, 80%, 90% and 95%. the minimum alignment lengths tested were as follows : 10, 15, 20, 25, 30, 25, 40, 45, 50, 55 and 60 base pairs. the maximum e - values tested were as follows : 1-e, 1-e, 1-e and 1-e. nessm produced 2399 077 sequences (length range : 195459 bp, median length 377 bp). s1, supporting information) and 98.7% of sequences are between 300 and 400 base pairs long (fig. s2, supporting information). of the 2399 077 sequences, kass annotated 1 341 362 (55.9%) sequences and webmga annotated 1945 674 sequences (81.1%). more stringent parameter values resulted in fewer sequence annotations but had a greater precision ; lower values resulted in more annotations being made, but these comprised increases in both correct and incorrect annotations. for example, with cut - off values of 95% and 40%, mg - rast refseq annotated 40.2% and 90.3% sequences, respectively, with incorrect annotation rates of 2.9% and 34.5% at the genus level. this was observed for all parameters tested and for both taxonomic and functional annotations (figs 16). as the taxonomic level moved up the taxonomic hierarchy, more sequences were correctly annotated (e.g. 0.5% and 10.2% for mg - rast refseq with cut - off values of 95% and 40%, respectively, at the class level). the effect of changing minimum identity cut - off value on the number of sequences correctly and incorrectly annotated across the taxonomic levels. the effect of changing minimum identity cut - off value on the number of sequences correctly and incorrectly annotated for functions. the effect of changing minimum alignment length on the number of sequences correctly and incorrectly annotated across the taxonomic levels. the effect of changing minimum alignment length on the number of sequences correctly and incorrectly annotated for functions. the effect of changing maximum e - value on the number of sequences correctly and incorrectly annotated across the taxonomic levels. the effect of changing maximum e - value value on the number of sequences correctly and incorrectly annotated for functions. the correlation coefficients between the taxa relative abundances in simmet and in the annotations decreased as parameter stringency increased (fig. 7, associated scatter plots in figs s3s5, supporting information). most databases achieved maximum correlations with a minimum identity cut - off value of 50%, a minimum alignment length of 30 bp and a maximum e - value of 1-e. greater decreases in correlation coefficients occurred with a minimum identity cut - off value above 70% and a minimum alignment length greater than 40 bp. the pearson 's product - moment correlation coefficients for the correlations between the genus relative abundances from simmet and those from various annotation methods using different (a) minimum identity cut - off values, (b) minimum alignment lengths and (c) maximum e - values. this produced the greatest number of correct annotations (99.4%) (table 1, fig. 8, table s2, supporting information, for all taxonomic levels). one codex annotated all of the sequences, but incorrectly annotated more sequences (5.8%) than megan (2.9%), megablast (2.5%) and the control (0.5%). megablast, megan and one codex correctly annotated 97.3%, 95.7% and 94.2% sequences respectively, significantly more than the next most successful methods : mg - rast refseq (55.9%), mg - rast trembl (54.9%) and mg - rast genbank (52.7%). mg - rast rdp and mg - rast greengenes, both rrna databases, annotated less than 1% of the sequences. this is consistent with the expected frequency of rrna genes within bacterial genomes (vtrovsk and baldrian 2013). as the taxonomic level increases, precision increases and becomes more similar across the different databases. the annotation sensitivity and number of sequences correctly annotated from a variety of methods and databases across the taxonomic levels investigated. the simmet taxonomic annotation statistics for each method and database at the genus level using default parameters. mg - rast kegg annotated 63.3% of the sequences and had a precision of 71.7%, with 45.4% of sequences correctly assigned a function and 17.9% incorrectly assigned a function. mg - rast cog annotated 50.5% of the sequences and had a precision of 91.1%, resulting in 46.0% of sequence being correctly assigned a function and 4.5% being incorrectly assigned a function (tables s3s5, supporting information). the portions of sequences correctly annotated by both methods were 81.5% for mg - rast kegg and 55.4% for mg - rast cog. megablast had the greatest genus - level correlation with simmet after the control (r = 0.95), while mg - rast seed had the weakest (r = 0.49). megan and one codex had genus - level correlations of r = 0.90 and r = 0.93, respectively. the greatest correlation achieved, aside from the control, was by megablast at the phylum level (r > 0.99) (fig. the pearson 's product - moment correlation coefficients for the correlations between the relative abundances from simmet and those from the annotation methods. mg - rast m5nr and mg - rast refseq generated 87 and 56 false positive class identifications, respectively (table 2). megan had only two false positive class identifications (unidentified and insecta) and one false negative identification (solibacteres). one codex also had a low abundance of false positive class identifications (eight) and no false negative class identifications. classes with many false positive identifications include eukaryotes, particularly fungi and bacteria such as spartobacteria. the greatest fold differences for classes can be found in table s6 (supporting information). false positive and negative class abundances. the false positive and negative classes from the mg - rast m5nr, refseq, one codex and megan annotations. six of the annotation methods underestimated the genus richness and six overestimated it (table 3, fig. the next closest estimate was achieved by megan (97.7%), followed by mg - rast swissprot (95.5%), mg - rast m5rna (95.2%), megablast (110.2%) and mg - rast refseq (118.2%). mg - rast m5nr produced the most incorrect richness value at 1244 genera (180.8%). the methods were inconsistent in response to the taxonomic level. with increasing taxonomic level, some estimates increased in accuracy while others decreased (fig. 10, table s7, supporting information). excluding the control and the domain level, where the number of taxa is low, megan achieved the most accurate richness value (101.2%) at the family level. mg - rast m5nr achieved the most inaccurate richness value (253.3%) at the order level. megablast and one codex achieved accurate results relative to other methods, but they still overstated taxa richness at every taxonomic level. the differences between annotated richness values and the actual richness value (dashed line) for each taxonomic level. the genus richness estimates and the differences from simmet for each annotation method. due to the low numbers richness values at all taxonomic levels can be found in table s7 (supporting information). in the study, we evaluated the performances of megan, mg - rast, one codex and megablast by determining their sequence annotation accuracies. all common taxonomic levels above species are studied, building on the work by lindgreen, adair and gardner (2016) who study several tools at the genus and phylum levels. by studying a range of taxonomic levels, we provide a guideline for researchers to establish the annotation accuracy costs of investigating lower taxonomic levels, allowing them to optimize their investigations depending on their requirements for taxonomic resolution. mg - rast and megablast use a selection of parameters to determine the stringency of matching a sequence with a reference sequence in a database. less stringent parameters (i.e. lower minimum identity cut - off values, lower minimum alignment lengths and higher maximum e - values) annotate more sequences, but more incorrect annotations are made, thus producing an incorrect community profile. more stringent parameters reduce the number of incorrect annotations, but many fewer annotations are made, resulting in much of the data being rejected. decreases in sensitivity generally occur from minimum identify cut - off values above 60%, a minimum alignment lengths greater than 30 bp or a maximum e - value below 1-e ; therefore, the default values used by mg - rast maximize sensitivity. according to carr and borenstein (2014), the impact of parameters such as e - value will vary depending on read length, something that should be considered in future evaluations as newer sequencing technologies produce longer reads (e.g. nanopore sequencing ; branton. the sensitivities and the number of sequences correctly annotated are relatively low for mg - rast at the genus and family levels. at the order level, the values are higher, suggesting that this would be the optimum taxonomic level to study, which maximizes the amount of data used without producing too many incorrect annotations. ultimately, there is a trade - off between taxonomic resolution and annotation accuracy, and this must be considered when determining methods for metagenomic studies. a marginal number of sequences were not annotated by the control and an even smaller number were incorrectly annotated. we can therefore conclude that 0.5% of intersample difference at the genus level may be attributed to sequencing error, an important consideration when interpreting data obtained from environmental samples using these methods. this is supported by hoff (2009) and carr and borenstein (2014), who found that increasing error rates decrease gene prediction accuracy. as the error rates of next - generation sequencing technologies improve, this effect will reduce. this is likely to be due to a combination of the kmer - based annotation method that it uses and that the simulated metagenome was created using the ncbi genome database, the primary reference source for one codex. other than the control, megablast correctly annotated the most sequences at the genus level (97.3%), although the sensitivity of this method was 0.2% less than one codex. megan had the second highest precision, annotating 98.6% of sequences, with 95.7% correct annotations. this suggests that megablast is the most reliable method for annotating sequences, and indicates that it is more conservative than one codex when assigning a sequence hit but also less likely to misidentify a sequence. megan 's performance was similar to megablast, which is expected as megan processed the megablast output. mg - rast refseq had the fifth greatest annotation sensitivity and the greatest of the mg - rast annotations (excluding mg - rast m5nr, for which sequence - specific annotation data were unavailable), although it also achieved the greatest number of misidentifications. at the genus level, 33.7% of sequences were misidentified and 55.9% were correctly identified, leaving the remainder unassigned despite the fact that all taxa in simmet are fully sequenced. this would suggest that investigating metagenomes at the genus level would be unreliable, generating many false positives and implying an incorrect community structure and composition. this supports garcia - etxebarria, garcia - garcer and calafell (2014), who found that more annotations are made at higher taxonomic levels and that discrepancies between known frequencies and annotations increase at lower taxonomic levels, and lindgreen, adair and gardner (2016), who report decreases in community annotation accuracy at the genus level compared to phylum. at the class level, the proportion of incorrect annotations is reduced to fewer than 10% for mg - rast refseq, with 80% being annotated correctly. while taxonomic resolution is reduced, it ensures that the confidence in the annotations remains high. mg - rast kegg correctly annotated a similar number of sequences to mg - rast cog, but incorrectly annotated many more. kegg offers a more descriptive annotation as it comprises specific gene and pathway annotations, whereas cog provides descriptions based on orthologous sequences. however, the specificity of kegg classifications may be the cause of the incorrect annotations as there are more annotations to be selected from and there may be more closely related functions, increasing the chance of misidentification. because kegg is now subscription based, and mg - rast uses the last free version (2008), it will not contain information added after that date. our results are in line with those produced by lindgreen, adair and gardner (2016), who also conclude that mg - rast 's functional annotation was accurate. the control, one codex, megablast and megan achieved the greatest correlation coefficients between simmet and annotation abundances at the genus level, all above 0.9. for all mg - rast annotations, the greatest correlation of all abundances was achieved at the order level by the m5nr database, closely followed by trembl and refseq. these correlations inform us about community - wide analyses, but they are not as sequence sensitivity and precision as correlating abundances values may occur from coincidental incorrect annotations. mg - rast overannotated many more classes than megan and one codex, for which the most abundant feature was the unidentified group. this supports the sensitivity and precision data in suggesting that one codex is more likely to categorize unknown sequences as unidentified, rather than incorrectly identifying them. the genus richness estimated by mg - rast m5nr was 81.0% greater that simmet 's actual richness, the highest overstatement, while megan achieved the most accurate genus richness value (2.3% lower) after the control (100.2%). this overstatement could be due to the greater number of sequences present in mg - rast m5nr. mg - rast m5rna produced a relatively accurate estimate of genus richness (95.2%) ; as m5rna is a 16s rrna database, it is unlikely to annotate non-16s rrna sequences, reducing the number of incorrect identifications. however, the taxa abundance correlations show that mg - rast m5rna achieved the second lowest correlation with simmet at the genus level, and the lowest at all other taxonomic levels. mg - rast refseq generated the fifth most accurate richness value, greater than one codex, although not as accurate as megablast and megan. combined with its high abundance correlation with simmet, this suggests that mg - rast refseq provides a relatively accurate representation of both the richness of a community and the abundance of organisms present. megan and one codex achieve more accurate taxa richness values and taxa abundance correlations than mg - rast refseq at the family level and above, suggesting they would be a viable alternative to mg - rast refseq. one limitation with evaluating annotations using organism nomenclature, rather than taxon ids (which were unavailable for mg - rast sequence - specific annotation data), is the lack of taxonomic metadata curation in some databases. some genomes in the ncbi database are stored with the abbreviated species name rather than complete name, thus amycolatopsis mediterranei would not automatically be identified as an amycolatopsis species. for example, the class chloroflexia has been renamed to chloroflexi, and is called this by mg - rast. however, ncbi is using the old name chloroflexia (as of 17/11/14), thus sequences identified as chloroflexi would not be correctly matched in simmet. these issues were corrected for during data processing ; however, there may be other cases of disparities in the plethora of organisms present in the analysis. a solution to this would be to use the taxon ids instead ; however, these were not available for sequence - specific annotations downloaded from mg - rast. in conclusion, we found that one codex, megablast and megan are suitable methods for annotating dna sequences that are located in the reference databases that they use for annotation, with one codex offering fast, web - based analyses and megan providing a user - friendly graphical user interface to analyze blast results. results appear to vary significantly depending on the program and parameters used, a conclusion also drawn by lindgreen, adair and gardner (2016). while mg - rast appears to have a greater rate of incorrect assignments, this is reduced when investigating higher taxonomic levels (e.g. with refseq : over 33% at the genus level compared to less than 15% and 10% at the order and class levels). the correlations between the annotated taxa abundances are greatest for mg - rast at the order level, using m5nr, trembl or refseq. in many of the tests, mg - rast m5nr proved to be a reliable database, but the diversity indices suggest that it is less reliable than mg - rast refseq ; at the class, order and family levels, mg - rast m5nr estimates more the double the actual richness values. therefore, we hypothesize that mg - rast m5nr would generate more false positive sequence annotations than mg - rast refseq. (2007), who evaluated different metagenomic processing methods using simulated metagenome developed from 113 isolated genomes, and by pignatelli and moya (2011), who used simulated data to study the performances of de novo short - read assembly programs. it should be noted that the performances of the methods discussed in this study are likely to differ from the reported results when annotating environmental sequence data ; a greater number of sequences are likely to be unidentified due to the multitude of uncultured microorganisms (streit and schmitz 2004) and non - sequenced microbial genomes (tringe. 2005) that are currently absent from the ncbi whole bacterial genome database. while this research focused on a selection of annotation methods, the overall conclusions drawn should be considered for any pipeline. in this study, we highlight and quantify the annotation errors for a selection of parameters and databases. we show that analysis pipelines are not equivalent and certain parameters can significantly reduce the confidence in results. these findings should be used as a guideline when determining methods for annotating metagenomic sequences and considered when interpreting metagenomic results. ultimately, the most appropriate balance between taxonomic resolution, annotation sensitivity and annotation precision needs to be identified for each study conducted. | the advent of next - generation sequencing has allowed huge amounts of dna sequence data to be produced, advancing the capabilities of microbial ecosystem studies. the current challenge is to identify from which microorganisms and genes the dna originated. several tools and databases are available for annotating dna sequences. the tools, databases and parameters used can have a significant impact on the results : nave choice of these factors can result in a false representation of community composition and function. we use a simulated metagenome to show how different parameters affect annotation accuracy by evaluating the sequence annotation performances of megan, mg - rast, one codex and megablast. this simulated metagenome allowed the recovery of known organism and function abundances to be quantitatively evaluated, which is not possible for environmental metagenomes. the performance of each program and database varied, e.g. one codex correctly annotated many sequences at the genus level, whereas mg - rast refseq produced many false positive annotations. this effect decreased as the taxonomic level investigated increased. selecting more stringent parameters decreases the annotation sensitivity, but increases precision. ultimately, there is a trade - off between taxonomic resolution and annotation accuracy. these results should be considered when annotating metagenomes and interpreting results from previous studies. |
two kinds of forces are involved in vitreoretinal traction diseases : tangential and anterior - posterior forces. when the posterior hyaloid partially detaches from the posterior pole and macula, keeping a strong attachment at a focal point, the anterior - posterior vector appears. these forces are less characterized and classified in literature compared to anterior - posterior ones, although they are responsible for retinal damages of the inner and outer layers. we will characterize the tangential vectors involved in the retinal changes : (i) anomalous posterior vitreous detachment and vitreoschisis ; (ii) epiretinal membranes (erms) ; (iii) vitreopapillary adhesion (vpa). the vitreous is an extended extracellular matrix that is composed mainly of water (98%) ; in youth, it is nonetheless a solid gel due to the intricate association of hyaluronan, collagen, and additional molecular components. in youth, this fine structure is strongly adherent to the retina in a fascial (as opposed to focal) manner, although the exact processes and cells responsible for the synthesis and adhesion of such macromolecules remain unidentified. the main function of the vitreous, however, is the maintenance of transparency within the eye (figure 1). this feature minimizes light scattering, allowing the unhindered transmission of photons to the retina for photoreception [5, 6 ]. during aging, changes in vitreous macromolecular interactions result in the formation of a liquid vitreous that consists primarily of hyaluronan and water as well as collagen fibrils that aggregate into bundles of parallel fibers. moreover, during aging, changes at the interface weaken vitreoretinal adhesion and promote vitreoretinal dehiscence in most individuals. these two processes must occur in concert and simultaneously to result in an innocuous posterior vitreous detachment (pvd). posterior vitreous detachment is characterized by synchysis (gel liquefaction) and syneresis (vitreoretinal dehiscence with collapse) of the posterior vitreous away from the retina. the effect of vitreous syneresis varies from negligible to significant, depending on the condition of the vitreoretinal interface. in diseases such as age - related macular degeneration and diabetic retinopathy, complete pvd is less frequent, but, if present, it protects against more advanced stages of disease. in a minority of cases, a clean separation of the posterior vitreous cortex (pvc) from the retina does not occur due to the presence of liquefaction without sufficient dehiscence at the vitreoretinal interface. five stages of pvd have been described : stage 0, absence of pvd ; stage 1, focal perifoveal pvd, with persistent attachment to the fovea, optic nerve head, and midperipheral retina ; stage 2, macular pvd with persistent attachment to the optic disc but without vitreofoveal adhesion ; stage 3, near - complete pvd with vpa only ; and stage 4, complete pvd [14, 15 ]. the sequelae of anomalous pvd vary depending on the position of the strongest retinal adherence of the pvc and greatest liquefaction of the gel. in the peripheral fundus, advanced gel liquefaction with firm vitreoretinal anomalous pvd can cause different vitreopapillopathies, and it can also play a role in promoting vitreous hemorrhage and neovascularization in ischemic retinopathies. at the macula, anomalous pvd can induce various pathologies, and it is relevant if the pvc that remains adherent to the retina is of partial thickness or full thickness. peripheral vitreoretinal separation with full - thickness vitreous cortex adherence to the macula can induce vitreomacular traction (vmt). in presence of symptomatic intraretinal changes, this condition is known as vitreomacular traction syndrome (vmts). in vmts, a broad, full - thickness posterior vitreous adhesion occurs to the margin of the fovea (macular adhesion size of ~ 1500 m). meanwhile, a more focal adhesion induces vitreofoveal traction syndrome (foveal adhesion size < 500 m). however, the smaller the diameter of the vitreofoveal adhesion is, the greater the tractional force that is exerted is, causing more serious foveal deformation. in these instances, the tractional forces are mainly anteroposterior, causing a central cyst in the vitreofoveal traction and retinal thickening with edema in the vmts. the pvc has a multilamellar structure, composed of densely packed collagen fibrils that run parallel to the retinal surface. it is comprised mostly of type ii collagen, but a hybrid of types v / xi and type ix is also present [13, 17 ]. the adhesion molecules such as fibronectin, laminin, and heparan sulfate keep these collagen fibers attached to the retina surface and interact with opticin in the vitreous gel [18, 19 ]. the packed collagen fibrils of the pvc are superficially inserted into the internal limiting membrane (ilm) of the retina. the pvc is 100300 m in thickness ; it is thinnest at the fovea, where collagen fibrils are more densely packed. hyalocytes, resident mononuclear phagocytes, are located in a single layer in the pvc approximately 50 m from the ilm of the retina [2022 ]. vitreoschisis results from anomalous pvd in presence of a firm vitreomacular adhesion (vma) that causes a splitting of the pvc during syneresis, in which the outermost vitreous layer remains attached to the macula, while the remaining vitreous collapses forward. studies using ultrasonography and histopathology have also resulted in the diagnosis of vitreoschisis in proliferative diabetic retinopathy. recently, combined optical coherence tomography / scanning laser ophthalmoscopy (oct / slo) led to the clinical diagnosis of vitreoschisis. in vitreoschisis, two membranous layers appear to join into one, forming the shape of the letter y or lambda () ; in other cases, this shape is not observed, but there is visible evidence of a membrane attached to the retina and a separate and distinct second membrane on the posterior side of the detached vitreous. indeed, studies conducted at the vmr institute using combined oct / slo imaging have identified vitreoschisis in 53% of the patients with macular holes (mhs) and in 43% of the patients with macular pucker (mp). these studies have suggested that it is important whether the split occurs anteriorly or posteriorly to the level of the hyalocytes [2022 ]. if the split occurs posteriorly to the layer of hyalocytes, a thin hypocellular membrane remains attached to the macula. if this membrane also maintains its attachment to the optic disc, it may cause an outward (centrifugal) tangential contraction, inducing a mh. if the split occurs anteriorly to the layer of hyalocytes, the remaining layer of vitreous attached to the macula will include the hyalocytes and will be relatively thick, hypercellular, and contractile. as mononuclear phagocytes of the reticuloendothelial cell system, sentinel hyalocytes can stimulate the migration of glial cells from the circulation to the retina and monocytes. contraction of this tissue induces inward (centripetal) tangential traction upon the underlying retina, causing a mp. recent studies have shown that cytokines can influence hyalocyte metabolism and may also induce further cell proliferation ; furthermore, other studies have demonstrated that hyalocytes can cause collagen gel contraction in response to platelet - derived growth factor (pdgf) and other cytokines. hence, hyalocytes are likely to be important in stimulating cell proliferation and in inducing tangential vitreoretinal contraction. the hypothesis that the pvc can split into layers is fundamental to the theory of anomalous pvd with vitreoschisis. the collagen fibrils within the pvc have a multilamellar organization, which has been confirmed in studies performed in monkeys [26, 27, 30 ], and these lamellae constitute a series of potential cleavage planes. were the first investigators to present in vivo intraoperative evidence of the occurrence of vitreoschisis. they noted that 80% of eyes with mp develop vitreoschisis during vitrectomy surgery. among those eyes in which vitreoschisis occurred intraoperatively, 58% had a visible hole in the pvc, while the remaining 42% had no visible hole. this finding suggests that vitreoschisis can occur at various levels within the pvc, consistent with the underlying multilamellar anatomy of the pvc. the aggressiveness with which surgeon searches for membranes during membrane peeling can be influenced by the knowledge that vitreoschisis can occur either preoperatively or during surgery. a higher index of suspicion can also be decisive in the choice to use or not to use surgical adjuncts that could function like particulate suspensions, such as triamcinolone, or colored stains, such as trypan blue and indocyanine green. myopia is associated with vitreous liquefaction excessive in relation to the degree of vitreoretinal adhesion, resulting in anomalous pvd and traction at vitreoretinal interface (figure 2). in high myopia, suggested that elongation and enlargement of the vitreous chamber might increase the vitreous and retinal shear stress exerted by the movement of the eye, playing an important role in the pathogenesis of pvd and retinal detachment (rd). this shear stress could be the origin of the disintegration of the collagen network and the consequent vitreous liquefaction and pvd. the vitreous, partially liquefied, is more rapidly disrupted in larger eyes than in normal eyes. this process leads to a significant reduction in the gel volume and a consequent increase in the liquid volume, which results in pvd or posterior vitreoschisis, whereas the increased rigidity of the preretinal vitreous might be the cause of undue tangential traction at the vitreoretinal interface either at the posterior pole or in the middle periphery [13, 32 ]. furthermore, in highly myopic eyes, when pvd occurs, sheets of residual cortical vitreous often remain attached to the inner surface of the retina. these sheets may contribute to a number of vitreoretinal diseases due to their subsequent contraction. in addition, in highly myopic eyes, an insufficient flexibility after axial length elongation could cause sclerotic retinal arteriole that determines a not distensible ilm. the rigidity of the ilm seems to be the primary component that generates an inward tangential traction on the retina along the arterioles and may be responsible for the pathogenesis of myopic vitreoretinal diseases, such as foveoschisis, macular hole formation, and paravascular retinal break formation [3436 ]. hypothesized that inflexibility of retinal vessels and the consequent tractional force in highly myopic eyes can lead to development of vascular microfolds. identified fibroglial cell debris and collagen fibers on the inner surface of the ilm peeled from eyes with myopic foveoschisis in 70% of cases. the origin of these cells was not exactly determined ; however, some cells such as astrocytes, abundant around retinal vessels, might migrate from the retina through small retinal pores in eyes with paravascular lamellar holes, produce collagen fibers, and initiate a proliferative response on the ilm. the rigid ilm, tightly attached to the posterior cortical vitreous, may cause the difficult differentiation between them on oct images. the panmetabolic disease of diabetes mellitus (dm) induces structural and biochemical changes in vitreous tissue (figure 3). the resulting diabetic vitreopathy plays an important role in the pathobiology of proliferative diabetic vitreoretinopathy. intravitreal glucose levels reflect blood glucose ones and can permit intravitreal nonenzymatic glycation reactions in patients with diabetes : high levels of early glycation products were found in patients with diabetes when compared with patients without diabetes who undergo vitrectomy. functionally, advanced glycation end (age) cross - links in the collagen fibrils of vitreoretinal interface cause reduced solubility, tissue rigidity, a decreased susceptibility of proteins to enzymatic digestion, aggregation of collagen fibers, and dissociation of collagen from hyaluronan, resulting in vitreous destabilization and alterations of pvc and hyalocytes [40, 42 ]. stitt. suggested that age - derived cross - links on the vitreous collagen network may cause earlier age - related vitreous degeneration in patients with diabetes than in those without diabetes and may cause anomalous pvd, vitreoschisis, and vitreoretinal traction [40, 43 ]. furthermore, proliferative diabetic retinopathy (pdr) alters the vitreous tissue by inclusion of fibrous tissue and vasogenic cells. structural changes at the vitreoretinal interface promote migration and proliferation of vasogenic cells in the vitreous and the consequent contraction can produce macular edema and vitreous hemorrhage. vitreoschisis in diabetics was first described by ultrasound and by histopathology in 80% of eyes with pdr. spectral oct - slo studies have furthermore detected vitreoschisis in half of eyes with macular hole and macular pucker. in a study of dme, 13 (57%) out of the 23 subjects with anomalous pvd had pdr. this is an important consideration, given the high prevalence of vitreoschisis in this group of patients. in eyes with pdr, the cortical vitreous is rarely detached ; rather there is a large, anterior, ring - like vitreoschisis, with multiple smaller ones, more posteriorly. induced pvd during pars plana vitrectomy (ppv) is a fundamental step to decrease the recurrence rate of rd. if the removal of pvc is incomplete, a thin but very adherent layer of the cortical vitreous remains attached to the retina and postoperative complications such as tractional redetachment and erm reproliferation may occur. indeed with an incomplete pvd mobile gel remains in the eye, with more room for it to exert dynamic traction, retinal shear stress, and redetachment ; the free fluid enters the original or newly formed break at the point of residual vitreoretinal adhesion. indeed, if only the anterior layer or inner wall of the vitreoschisis cavity is removed during surgery, the posterior layer, or outer wall of the vitreoschisis cavity, will remain on the anterior surface of the retina. in some studies of mh surgery, aggressive chromodissection has been associated with a lower rate of recurrent disease [29, 47 ], which is likely because aggressive chromodissection of the vitreoretinal interface results in definitive removal of the outer wall of the vitreoschisis cavity. other studies have reported a more favorable outcome by employing ilm peeling in mp surgery in comparison to those without aggressive membrane dissection. have proved that ilm peeling, compared with no - ilm peeling, is more effective in reducing the risk of reoperation in patients with idiopathic stage 2 or 3 full - thickness macular holes (ftmhs), removing completely the potential tangential ilm traction and any residual cortex on ilm. however, they did not find any significant differences in distance visual acuity after the two techniques. triamcinolone acetonide (ta) is most commonly used as an adjunct to visualize vitreous, posterior hyaloid, preretinal membrane, and ilm during vitrectomy or chromovitrectomy. the granules of ta adhered to the residual posterior hyaloid, making it more visible. this technique can disclose the residual hyaloid cortex pattern after surgical pvd and permit intraoperative visualization of vitreoschisis. indeed, diffuse posterior hyaloid cortex is frequently found in high myopia and diabetic retinopathy, and an island - like cortex is often left on the macula, which can lead to future macular pucker. ta - assisted vitrectomy facilitated removal of residual island - like cortex, thus ensuring a low reproliferation rate on vitreomacular interface. the aim of the use of ocriplasmin is to cleave vitreoretinal interface with an intravitreal injection, causing a complete pvd. recent studies have shown that intravitreal injection of ocriplasmin can induce vitreous liquefaction and its separation from the retina [5153 ], leading to closure of macular holes and resolution of vmt without causing serious adverse events [54, 55 ].. conducted two pivotal phase iii trials (tg - mv-006 and tg - mv-007) with a single injection dose of 125 g in patients with symptomatic vma / vmt. whereas ocriplasmin was generally well tolerated in these trials, recent studies showed some adverse effects, such as incomplete vmt release, retinal breaks, and visual impairment associated with subretinal fluid [5759 ]. it is possible that ocriplasmin may have an enzymatic effect not limited to areas of vma but diffuse on the retinal pigment epithelium or photoreceptors producing a phenomenon defined as lucency, characterized by the presence of subretinal fluid in macular area. rod photoreceptors seem to be more susceptible to the effects of ocriplasmin than cone photoreceptors. some variables may limit the success of ocriplasmin : lens status, broad versus focal vitreomacular attachments, multiple vitreomacular attachments, and patient 's age. vitreopapillary adhesion is defined as a prominent vitreous membrane attached to the borders of the optic disc, associated with the development of pvd (figure 4). dynamic vpa, which is associated with pvd stages 1 through 3, is occasionally severe enough to cause visual symptoms and anatomic changes [63, 64 ]. affected patients are usually asymptomatic or report transient photopsias and gaze - evoked amaurosis. visual acuity and automated perimetry rarely, a mild relative afferent pupillary defect may be transient [63, 64 ]. biomicroscopic signs often include fullness, elevation, and even subtle whitening of the peripapillary nerve fiber layer, which can simulate nontractional disc edema. intrapapillary and peripapillary hemorrhages may occur, but they are benign and self - resolving. although these hemorrhages can occur at any age, they are commonly associated with partial pvd development in young myopic patients. the strong and persistent adhesion of the posterior hyaloid to the optic nerve head results in traction and tenting of the papillary rim as well as elevation of the optic nerve head, which appears as a pseudopapilledema upon examination of the fundus [1, 68 ]. the presence of an elevated optic nerve head must be differentiated from several etiologies of disc edema, such as papillitis, papilledema, optic nerve head drusen, optic nerve infiltration, and optic nerve or orbital masses [63, 68, 69 ]. this condition can cause possible optic nerve dysfunction. indeed, tractional forces elongate the retinal nerve fibers and inflect the central retinal vessels. the stretching, thinning, and consequent deformation of the ganglion cell axons reduce the anterogradely or retrogradely axoplasmatic flow and account for a sensory blockade of neuroretinal signals ; the visual evoked potentials can result in alterations. mechanical restriction and feeding of the central retinal blood vessels also decrease prelaminar blood flow. vpa is also known to induce epiretinal traction and intraretinal changes such as cysts, mhs, mp, macular edema, and age - related macular degeneration (amd) and to promote retinal and optic disc neovascularization. while anomalous pvd may be the initial event, persistent adherence of the vitreous to the optic disc may influence the vectors of force that are exerted on the macular interface. after anomalous pvd with vitreoschisis, the outer layer of the splitted pvc remains attached to the macula. in the absence of vpa, inward (centripetal) tangential traction likely throws the underlying retina into folds, resulting in mp. if the vitreous is still attached to the optic disc, the vectors of force could be changed, resulting in outward (centrifugal) tangential traction that induces central retinal dehiscence, mh, and/or erm. the vectors of force that result from vpa may conceivably contribute to the perifoveal vitreous detachment proposed by johnson and associates as the primary pathogenic event in the formation of macular holes. sebag and colleagues have demonstrated that vpa is significantly more common in ftmhs than in lmhs, mp, dry amd, and age - matched controls. they also showed that vpa, when present in mhs and/or in mp, is highly associated with intraretinal cystoid spaces. indeed, in their study, cysts were found in 100% of the eyes with vpa and mh, 80% of the eyes with mp and vpa, 75% of the eyes with lmh and vpa, only 42.9% of the eyes with lmh without vpa, and 4.3% of the eyes with mp without vpa. therefore, vpa is far more common in mp and mh with cysts as compared to lmhs or mp without cysts. foveal cysts are believed to be the precursors of either ftmhs or lhs [75, 76 ]. wang and colleagues found that 100% of the eyes with mh and vpa were associated with intraretinal cysts. mhs and lmhs may have more cysts compared with mp because the inner retinal layers are severely disrupted by changing fluid dynamics. vpa is also more frequent in lmhs than in the presence of pseudomacular holes (pmhs) (figure 5). van newkirk and colleagues detected vitreous attachment to the peripapillary retina in 65 of 65 patients with stage 3 mhs. the results of these studies suggest that lmhs represent an intermediate stage in the process of macular hole formation and that foveal pseudocysts with partial pvd become lhs if the base is conserved and become ftmhs if the outer retinal layer is disrupted. these results have been confirmed by wang. who discovered vpa in 87.5% of the eyes with ftmhs as compared with 36.4% of the eyes with lmhs and 17.9% of the eyes with mp. vitreopapillary traction syndrome has also been described, but it is usually in conjunction with other manifestations of anomalous pvd and includes rhegmatogenous rd retinal detachment, mp, mhs, and proliferative diabetic vitreoretinopathy. vitreopapillary traction can occur in the absence of other forms of anomalous posterior vitreous detachment in diabetic vitreoretinopathy. the diagnosis of vpa is important because affected patients may inappropriately undergo ancillary testing, such as neuroimaging, or invasive procedures, such as lumbar puncture and referrals for retinal and neuroophthalmic evaluations. b - scan ultrasonography and oct are valuable diagnostic tools that can be used to confirm the presence of vitreopapillary traction and to distinguish it from other causes of optic nerve elevation. on b - scan ultrasonography oct can provide a precise diagnostic evaluation of the underlying etiology that may be challenging to establish with clinical observation and b - scan ultrasonography. oct image of peripapillary vitreoretinal traction is visualized as a partially detached vitreous band with continuous adherence to the optic nerve, thus causing elevation [69, 78 ]. the evaluation of oct is important not only to determine the prognosis but also to identify the requirement for surgical intervention with ppv. surgical release of vpa may significantly improve the anatomical and visual outcome [1, 73 ]. the tangential traction induced by vpa can cause damage to the outer retinal layers and the progression of a lmh. in presence of a lmh type 3 lmhs are the consequence of a vitreoschisis posteriorly to the level of hyalocytes and these holes can progress mainly in the presence of vpa. in presence of preoperative vpa, the induction of pvd during ppv should be performed with caution because removal of the adherent peripapillary membranes or posterior vitreous may lead to iatrogenic excision of axons that compromise visual acuity and the visual field. it is more commonly diagnosed in elderly people ; indeed, its prevalence is 2% in patients under the age of 60 years and 12% in those over 70 years of age. it has been shown that the prevalence of erm is lower in the asians than in caucasians [8083 ]. a recent study demonstrated that erm is significantly more common in the chinese compared to caucasians, blacks, and hispanics. the erm is a sheet of avascular fibrocellular tissue that can be formed on the ilm. this membranous tissue is usually deposited on the macula and then on the retinal periphery. erm is classified as idiopathic (ierm) when it is not associated with any ocular disease and as secondary when it occurs during ocular processes such as rd, intraocular inflammation, trauma, retinal vascular diseases, and retinal surgery. the term erm refers to a group of vitreoretinal diseases that includes the following : mp, cellophane maculopathy, and epiretinal fibrosis. however, many authors suggest that these diseases correspond to different stages of the same pathology. the difference between the various stages has been noted in terms of the type of collagen and the thickness : collagen type vi characterizes cellophane membrane, which is the thinnest ; types i and ii characterize epiretinal fibrosis, while type iv and laminin are ubiquitous. in a fundoscopic exam, erm can appear as a translucent, semitranslucent, or opaque membrane (especially in later stages) that is located on the inner surface of the retina. erms are composed of two main components : extracellular matrix (consisting of collagen, laminin, tenascin, fibronectin, vitronectin, and thrombospondin, among others) and retinal and extraretinal cells. a wide variety of cell types have been found in the membranes : glial cells (including microglia, mller cells, and fibrous astrocytes), epithelial cells from the retinal pigment epithelium (rpe) and ciliary body, hyalocytes, blood - borne immune cells, fibrocytes, and myofibrocytes [85, 89 ] histologically, two types of erm can be observed : a simple one and a complex one. the simple type, which grows directly on the ilm, is composed of a monolayer of retinal glial cells that produce type iv collagen (laminocytes). these cells for the first time described by foos like accessory glial cells migrating from the nerve fiber layer then were termed laminocytes by snead. in spite of their laminar arrangement, close association with the ilm, and evidence of novel ilm production. laminocytes of the simple type show positivity to glial fibrillary acidic protein (gfap), atypical protein of the astroglia, and the cytokeratin marker ae1/ae3 using immunocytochemistry. the expression of gfap indicates the ability of these cells to proliferate and migrate on the retinal surface. the complex or tractional type contains cells such as fibrous astrocytes, myofibroblasts, fibrocytes, hyalocytes, macrophages, and rpe cells in addition to glial cells [17, 9093 ]. this multilayer of cells is separated from the ilm by a layer of native vitreous (type ii) collagen that remains after incomplete pvd [92, 93 ]. the presence of type ii collagen positivity in these cells suggests an additional role in secreting collagen into the vitreous gel. simple erm is a noncontractile type and is associated with mild to no visual symptoms. however, the contraction of myofibroblasts within the more complex type has been proposed to exert a progressive tangential traction at the vitreoretinal interface, which can result in retinal puckering, radial wrinkles, thickening, folding, or detachment, together with vascular distortion and retinal edema or a pseudohole.. stated that mller cells and hyalocytes constitute the predominant cell type of the macular pucker. myofibroblasts, the major cell type in complex erm with glial cells, are considered to be derived from the transdifferentiation hyalocytes, rep cells, and glial cells. indeed, many new studies have demonstrated high levels of nerve growth factor (ngf) and transforming growth factor 1 (tgf 1) in the vitreous. these growth factors can cause fibroblast migration and deposition, differentiation into myofibroblasts, and contraction of the extracellular matrix [85, 98 ]. the transdifferentiated cells are characterized by a downregulation of gfap and cytokeratins, while proteins involved in motility and proliferation such as -smooth muscle actin are upregulated. therefore, the gfap content in epiretinal tissues has been shown to correlate inversely with the clinical contractility and directly with tractional forces. the retinal changes induced by tangential traction may appear on the fundus examination as follows : irregular wrinkling, nerve fiber layer dragging, ectopic fovea, winding corkscrew vessels surrounding the overlying erm, or major vessel straightening and crowding. fundus photography and, in particular, red - free or blue - reflectance imaging, can highlight the presence of erm. fundus autofluorescence shows hyperautofluorescent lines that indicate the original location of the retinal vessels, which have been displaced because of a tractional erm. these lines are called retinal vessel printings (rvp) and their visualization in eyes with erm may give useful information about the severity and direction of tangential traction (figure 6). recently, dell'omo and colleagues proved that the presence of rvp is associated with a higher degree of irregularity of the external limiting membrane (elm) and the junction between the photoreceptor inner segment and outer segment (is / os line) at the fovea and a higher average metamorphopsia score. the past theories have proposed that pvd certainly plays a critical role in the pathogenesis of this pathology through different possible mechanisms. among these, transient vitreoretinal traction during the development of pvd may cause breaks in the ilm through which glial cells and rpe cells can migrate and proliferate on the inner retinal surface [85, 102 ]. in addition, ierm may also result from the proliferation and transdifferentiation of hyalocytes contained within vitreous cortical remnants on the retinal surface following pvd [31, 103 ] and macrophages from subsequent inflammatory processes. indeed, partial or complete pvd has been found in 80% to 95% of eyes with idiopathic erm [96, 101 ]. actually, foos and bellhorn reported the presence of vitreous collagen fibrils in premacular fibrosis and within the erm in their studies [91, 104 ]. meanwhile, kishi and shimizu reported the presence of defects in detached posterior hyaloid membranes of patients with idiopathic preretinal fibrosis. this condition leads to a vitreoschisis and then to an erm, especially when the split occurs anteriorly to the level of hyalocytes [8, 17, 26 ]. in this way, hyalocytes attached to the retina stimulate glial cells to proliferate upon an intact ilm to form the scaffolding that allows the uptake of other cells into the membrane [85, 93 ]. pvd and chronic irritation of glial cells can induce a local release of factors that induce cell gliosis, cellular hypertrophy, and upregulation of gfap (simple static erm). found nestin-1 positivity, marker of progenitor cells of the retina, and sox2 positivity, marker of epithelial stem cells and of pluripotency potential, to indicate the origin of transdifferentiated cells of erm. when glial and pigment epithelial cells transdifferentiate to other cells, there is a reduction in cell - specific proteins such as gfap and cytokeratins. in addition to increasing age and pvd, other risk factors for this pathology are the presence of diabetes and hypercholesterolemia. the initial formation of this epiretinal tissue does not usually cause any clinically important reduction in vision ; however, the progression and contraction could be slow. therefore, advancement of the disease results in significantly reduced visual acuity. although the cellular mechanism underlying visual impairment in this pathology remains unclear, an increasing number of studies have suggested that it is related to abnormalities in macular morphology caused by erm traction. the contraction affects the outer segments with distortion or detachment of the retina and/or photoreceptors, and it disturbs the spatial arrangement of the cones. examined retinal changes in ierm and discovered that thickening was greater in the foveal region than in the extrafoveal regions and that the thickening of the external foveal retina to the inner plexiform layer was associated with visual impairment. tangential centripetal traction on the fovea results in a cleft between the inner retina and outer retina ; alternately, it may induce avulsion of the foveal tissue or the roof of a pseudocyst, leading to the formation of lamellar macular defects. oct is very useful in identifying the precise shape and size of erm, in confirming the relationship between pvd and erm, and in following its natural history (figure 7). using oct, it is quite easy to differentiate the posterior hyaloid, a minimally reflective signal, from an erm, which is highly reflective. however, so far, we do not have any classification of tangential traction, but only the classification of the morphology changes induced by anteroposterior vectors of traction. oct could also be shown to discriminate secondary from idiopathic types of abnormalities ; in contrast, secondary erm demonstrates focal adhesion points. the retinal structural changes can be resolved after surgical removal of erm, and some oct parameters, such as macular thickness, are associated with the surgical outcome. in recent studies, prolonged macular traction has been shown to cause irreversible photoreceptor cell loss and alignment disruption. outer retinal structures rarely return to normal after they are impaired, thus indicating a poor visual prognosis. falkner - radler and colleagues, using spectral - domain oct, identified the following prognostic factors in the erm surgery : baseline visual acuity, the is / os line integrity of the junction between the photoreceptor inner segment and outer segment, the ilm profile, and foveal contour - like. ppv with membrane peeling using vital dyes is the standard surgical procedure for patients with symptomatic erm. however, ierms recur in approximately 10% of cases, and reoperation is required in approximately 3% of cases. they also demonstrated that ilm peeling seemed to be the only factor preventing erm recurrence and that the use of staining dyes did not reduce the recurrence rate compared to ilm peeling in the absence of dyes. their study has shown that the formation of erms involves epiretinal glial proliferation with neuronal or glial cells on the retinal surface of the ilm and consequent intraretinal displacement. the adhesion of cells to the retinal surface of ilm might lead to focal force transmission into the retina during peeling with consequent retinal breakages and damage of mller cells. such iatrogenic retinal damage after erm - ilm peeling leads in patients with dme prominent cyst characteristics to the floor effect. such phenomena consist in the collapse of retinal layers, damage of outer retina, and worsening of visual acuity. looking through the literature we found a further classification of tangential tractions, on which we are working on, based on the area and the depth of traction, was necessary. the right interpretation of the forces involved in the epiretinal tangential tractions helps in a better definition of diagnosis, progression, prognosis, and surgical outcomes of many vitreoretinal diseases. | two kinds of forces are active in vitreoretinal traction diseases : tangential and anterior - posterior forces. however, tangential forces are less characterized and classified in literature compared to the anterior - posterior ones. tangential epiretinal forces are mainly due to anomalous posterior vitreous detachment (pvd), vitreoschisis, vitreopapillary adhesion (vpa), and epiretinal membranes (erms). anomalous pvd plays a key role in the formation of the tangential vectorial forces on the retinal surface as consequence of gel liquefaction (synchysis) without sufficient and fast vitreous dehiscence at the vitreoretinal interface. the anomalous and persistent adherence of the posterior hyaloid to the retina can lead to vitreomacular / vitreopapillary adhesion or to a formation of avascular fibrocellular tissue (erm) resulting from the proliferation and transdifferentiation of hyalocytes resident in the cortical vitreous remnants after vitreoschisis. the right interpretation of the forces involved in the epiretinal tangential tractions helps in a better definition of diagnosis, progression, prognosis, and surgical outcomes of vitreomacular interfaces. |
occasionally, however, it can be a sign of a more serious, often unrecognized condition. we report on a patient with carboplatin - induced bilateral papilledema resulting in only partially reversible visual impairment. despite the fact that carboplatin is widely used in the treatment of a variety of common tumors (including cancer of the lung, ovary, testis), this is the first report of carboplatin - induced papilledema following regular auc - determined dose. reported cases of papilledema after carboplatin administration include two cases after high - dose carboplatin (auc12), one report using carboplatin and cisplatin simultaneously, and two patients to whom carboplatin was given in a fixed standard dose (400 mg / m) rather than in a creatinine - clearance dependent dose (auc). a 70-year - old woman with stage iiic (figo) serous - papillary ovarian carcinoma started postoperative chemotherapy 5 weeks after optimal surgical debulking (tah+bso) in september 2005. concurrent medical conditions included stable coronary heart disease, controlled arterial hypertension and mild urinary retention as sole sequel of a previous injury to the lower back. baseline renal function was normal with a calculated creatinine clearance of 100 ml / min (cockroft formula). chemotherapy consisted of a 3-weekly intravenous administration of carboplatin (auc 5 mg / ml min) over 30 min and was well tolerated, grade 1 nausea, fatigue and grade 2 thrombocytopenia (53 10/l) being the only adverse effects. on day 8 of the 4th cycle, however, the patient was admitted because of nausea, constipation and acute urinary retention. post - renal acute renal failure (calculated creatinine clearance 40 ml / min) was completely reversible after catheterization. at this time, she first complained about visual impairment in her right eye (lack of focus and scattered blind spots). the optic nerve head was at that time more prominent in the right eye than in the left eye, and there were some hemorrhages within the nerve fiber layer. differential diagnosis of increased intracranial pressure due to meningeosis carcinomatosa or cerebral metastasis and anterior ischemic optic neuropathy (aion) due to anemia were discussed. an mri of the brain showed no signs of intracranial pressure, leptomeningeal carcinomatosis or pathology of the optic nerve. the serum tumor marker ca-125 had normalized (from 1,541 u / ml pre-, and 114 u / ml postoperatively) and complete remission was confirmed by abdominal ultrasound and chest x - ray. however, after the 5th cycle the visual acuity decreased further, and new visual field losses in the left eye appeared. at the same time an increase of the bilateral papilledema was observed, particularly in the left eye. in contrast, the hemorrhages in the right eye had almost disappeared, and some signs of ischemia were now noted. visual acuity had dropped to 20/100 in the left eye and there was a left relative afferent pupillary defect. diagnostic lumbar puncture was performed and intracranial pressure was measured at 10 cm h2o. based on the normal intracranial pressure, the differential diagnosis of pseudotumor cerebri, a condition characterized by ophthalmopathy, papilledema and elevated intracranial pressure, was dismissed. biochemical (protein, glucose, ldh) and cytological examination of the cerebral fluid was normal, without malignant cells. tests for specific antibodies were not diagnostic : anti - cns and anti - yo antibodies titers were (marginally) elevated in the serum, but not in the cerebral fluid. serum titers of anti - hu, anti - ri, anti - amphiphysin, anti - cv2, anti - tr, anti - ma - antibodies and antibodies against retinal rods, cones, mueller cells as well as plexiform structures were all negative. in view of these results, an autoimmune syndrome - unusual in the context of ovarian cancer - seemed very unlikely. based on the history, the clinical picture and the exclusion of other conditions via mri, cerebral fluid examination and serum antibody - titers, the diagnosis of bilateral carboplatin - induced papilledema was made. chemotherapy was stopped after the 5th cycle and prednisolone was initiated at a dose of 100 mg / d for 2 weeks, then tapered over 10 weeks. while the visual acuity in the right eye was stable, (2 years after cessation of chemotherapy), the optic nerve head showed some signs of optic atrophy with increased pallor and reduced visibility of the nerve fiber layer. the optic nerve head was at that time more prominent in the right eye than in the left eye, and there were some hemorrhages within the nerve fiber layer. differential diagnosis of increased intracranial pressure due to meningeosis carcinomatosa or cerebral metastasis and anterior ischemic optic neuropathy (aion) due to anemia were discussed. an mri of the brain showed no signs of intracranial pressure, leptomeningeal carcinomatosis or pathology of the optic nerve. the serum tumor marker ca-125 had normalized (from 1,541 u / ml pre-, and 114 u / ml postoperatively) and complete remission was confirmed by abdominal ultrasound and chest x - ray. however, after the 5th cycle the visual acuity decreased further, and new visual field losses in the left eye appeared. at the same time an increase of the bilateral papilledema was observed, particularly in the left eye. in contrast, the hemorrhages in the right eye had almost disappeared, and some signs of ischemia were now noted. visual acuity had dropped to 20/100 in the left eye and there was a left relative afferent pupillary defect. diagnostic lumbar puncture was performed and intracranial pressure was measured at 10 cm h2o. based on the normal intracranial pressure, the differential diagnosis of pseudotumor cerebri, a condition characterized by ophthalmopathy, papilledema and elevated intracranial pressure, was dismissed. biochemical (protein, glucose, ldh) and cytological examination of the cerebral fluid was normal, without malignant cells. tests for specific antibodies were not diagnostic : anti - cns and anti - yo antibodies titers were (marginally) elevated in the serum, but not in the cerebral fluid. serum titers of anti - hu, anti - ri, anti - amphiphysin, anti - cv2, anti - tr, anti - ma - antibodies and antibodies against retinal rods, cones, mueller cells as well as plexiform structures were all negative. in view of these results, an autoimmune syndrome - unusual in the context of ovarian cancer - seemed very unlikely. based on the history, the clinical picture and the exclusion of other conditions via mri, cerebral fluid examination and serum antibody - titers, the diagnosis of bilateral carboplatin - induced papilledema was made. chemotherapy was stopped after the 5th cycle and prednisolone was initiated at a dose of 100 mg / d for 2 weeks, then tapered over 10 weeks. while the visual acuity in the right eye was stable, (2 years after cessation of chemotherapy), the optic nerve head showed some signs of optic atrophy with increased pallor and reduced visibility of the nerve fiber layer. in our case report, we postulate a direct toxic effect of carboplatin, as it was described in the 1970s for cisplatin. in the case of cisplatin cortical blindness, clinically, a loss of visual accuracy up to full blindness, often loss of color vision and scotomas were seen. usually these symptoms are unilateral and initially mild, so that more chemotherapy is given before the diagnosis is made. a correlation with renal insufficiency and arterial hypertension was reported [1, 2, 3 ]. the symptoms are usually reversible within weeks to months after cessation of the platinum treatment [2, 3 ], and the restoration of color vision can take up to 1 year. it seems to be a cumulative toxicity, as all cases occurred after several cycles of chemotherapy. furthermore, pharmacokinetic studies of cisplatin show a continual rise of the drug concentration in the cerebrospinal fluid with each cycle. it is possible that the cerebrospinal fluid concentration is important for the occurrence of this toxicity : in one patient with blindness and a seizure equally high concentrations of cisplatin in the cerebrospinal fluid and the serum were measured. the early recognition of this potentially severe side effect is paramount, since stopping chemotherapy before irreversible damage occurs is crucial. it can occur at standard doses using creatinine - clearance dependent dose calculations (auc) and it can be partly irreversible. since carboplatin is extensively used in a variety of common tumors in the palliative as well as the curative setting, this potentially severe side effect has to be kept in mind. | we report on a patient with carboplatin - induced bilateral papilledema, as it was described in the 1970s for cisplatin. loss of visual accuracy up to full blindness, often loss of color vision and scotomas can be seen as a result of cortical blindness, macula degeneration, retrobulbar neuritis and papilledema. these symptoms are mostly unilateral and initially mild, so that more chemotherapy is given before the diagnosis is made. the symptoms are usually reversible within weeks to months after cessation of the platinum treatment. the therapeutic strategy is stopping the platinum treatment. in addition the empiric use of corticosteroids is suggested. |
eight - year - old male presented with 360 peripheral corneal endotheliopathy and edema, granulomatous keratic precipitates, and mild iritis od. twenty months later, os presented similarly and anterior chamber inflammatory cells, vitreous snowballs, and retinal vasculitis were observed ou. this is the first case of endotheliopathy as the first manifestation of pars planitis and as a cause of a secondary central cornea ectasia developed white eye, mild anterior chamber reaction, snowballs in the anterior vitreous, snowbanks in the peripheral retina and pars plana, retinal vasculitis, and cystoid macular edema. the association of a peripheral corneal endotheliopathy (pce) and pars planitis starting in childhood has been previously described. we report a cpp case in which pce was the first clinical manifestation, and which led to secondary corneal ectasia. on the first visit, an 8-year - old male presented because the patient s mother noticed a white reflex in the right eye for about 2 years that became more evident with time. his visual acuity od was 20/200 with improvement to 20/60 with pinhole occlusion, os 20/20. od showed mild ciliary injection, mild peripheral corneal edema, a 360 peripheral endothelial opacity delimitated by mashed potato keratic precipitates, and mild iritis (fig. 1). vitreous examination revealed few cells and a single snowball in the inferior, peripheral retina, near an area of apparent retinal vasculitis. granulomatous kps delineated the involved endothelial area pictures showing the right eye with a peripheral corneal stromal oedema and endothelial opacity. granulomatous kps delineated the involved endothelial area a diagnosis of pars planitis was suspected and fluorescein angiography (fa), ppd, vdrl, and fta - abs were ordered. prednisolone acetate (prednefrin, allergan) q2h and timolol 0.5% (imot, laboratorios sophia, guadalajara, mxico) bid were started ; the steroid was slowly tapered over a period of 1 month. all laboratory tests were either negative or normal and fluorescein angiography ou was also normal. examination showed va : od 20/400 with improvement 20/80 with pinhole occlusion, os 20/20 ; iop : 10/14 mmhg, with pronounced peripheral corneal stromal oedema and endothelial opacity od. granulomatous keratic precipitates (kps) delineated the involved endothelial area, and some deep stromal peripheral vessels were found. javal s keratometric readings were od 40.00/46.25 10 and os 44.00/45.00 5, he showed decreased corneal sensitivity, and there was no anterior chamber or vitreous inflammation. both the stromal and endothelial opacities diminished and so the corticosteroids were tapered during the next 2 months. six months later, a corneal topography (orbscan topography system ii, orbscan inc., 2 shows the findings) od shows a with the rule astigmatism with asymmetric bowtie pattern. central pachymetry was similar in both eyes (od 619 m, os 617 m) but the peripheral pachymetry in the od was 900 m thicker than the os in the superior area and 60 m in the inferior and temporal area. in the left eye (fig. specular microscopy (topcon sp-2000p noncontact specular microscope, topcon corp., tokyo, japan) of the right eye showed decreased endothelial cell density (1,636 cell / mm) with pleomorphism and polymegatism ; the left eye was normal (3,141 cell / mm). anterior elevation map within normal values ; posterior elevations map shows a central steepening of 96 mm ; and pachymetry map with increase of peripheral thickness. anterior elevation map within normal values ; posterior elevations map shows a central steepening of 96 mm ; and pachymetry map with increase of peripheral thickness. the keratometric map shows a asymmetrical bowtie pattern with an inferior steepest left eye orbscan topography map. the keratometric map shows a symmetrical bowtie three years after the initial visit, an examination showed a decreased va ou, being od 20/400 ; os 20/30. his iop was normal and his od showed corneal findings very similar to those previously described, in the left eye a peripheral endothelial opacity in the inferior sector of the cornea was observed. mild to moderate iritis and vitreous cells+ were found ou. snowballs in the inferior peripheral retina and some vascular sheathing were also seen. in both eyes, fa showed hyperfluorescence of the peripheral capillary vessels, parietal hyperfluorescence of the medium - sized veins, and optic disc and perifoveal hyperfluorescence ou. a complete blood cell count, urinalysis, angiotensin - converting enzyme, and chest x - ray were carried out. an autoimmune endotheliopathy (ae) associated with a corneal graft rejection was first described by khodadust and attarzadeh. this condition is characterized by a peripheral endothelial opacity and stromal edema, initially located in the lower quadrants, which may progress in a circumferential pattern. small and mutton fat kps arranged linearly at the junction of the oedematous and non - oedematous portions of the cornea may also be present. later, the same authors, as well as lucchese and tessler, described similar findings in cases of pars planitis and sarcoidosis patients. autoimmune responses to the corneal endothelium, and a fibrous metaplasia of the endothelium secondary to damage caused by kp, have been suggested to explain these findings. ae has been described in 18.8% to 25.4% of cpp patients and manifests bilaterally in 70% of cases. we have hypothesized that the peripheral swelling of the cornea may induce a modification of the central curvature. these changes can be explained using the conceptual model proposed by roberts, in which the lamellar collagen structure of the cornea is conceived of as a series of stacked fibrils with interlamellar spaces filled with an extracellular matrix between each layer. the collagen lamellas are in tension, due to intraocular pressure from below, and their ends are held tightly by the limbus. the amount of water at the extracellular matrix is related to the strain force at the lamellae. the biomechanical response of the cornea in the present case could be the consequence of differences in the amount of swelling throughout the cornea and alterations in the stress distribution. the peripheral corneal edema may function as a belt that exerts its force centripetally, allowing the central dome of the cornea to be displaced anteriorly, thus increasing its curvature. a change in the shape of the cornea has been demonstrated to occur in the swollen eye but not in the normal hydration state. using small - angle x - ray diffraction, boote. demonstrated that in physiologic conditions collagen fibrils pack around the peri - pupillary cornea (the interfibrillar space is 5% to 7% smaller than in the peripheral cornea). in the present case, this may have played a role in limiting swelling into the central cornea, explaining why the corneal edema was limited to the periphery. this report is interesting because, to the best of our knowledge, this is the first report of a case in which corneal endotheliopathy is the first manifestation of pars planitis and induced the development of a secondary central cornea ectasia. | purposeto report a case of corneal ectasia secondary to pars planitis corneal endotheliopathymethodsclinical case description and proposed hypothesis regarding development of corneal ectasiaresultseight - year - old male presented with 360 peripheral corneal endotheliopathy and edema, granulomatous keratic precipitates, and mild iritis od. a progressive corneal ectasia then developed. twenty months later, os presented similarly and anterior chamber inflammatory cells, vitreous snowballs, and retinal vasculitis were observed ou. classic pars planitis was diagnosedconclusionthis is the first case of endotheliopathy as the first manifestation of pars planitis and as a cause of a secondary central cornea ectasia developed |
although sarcoidosis has been identified as a specific disease entity for more than one century, the specific cause of sarcoidosis is unknown. in addition, the diagnostic criteria, treatment algorithms, method of follow - up and natural course of the disease have not been clearly elucidated. these concepts reflect our deeper understanding of this enigmatic disease. in terms of the diagnosis of sarcoidosis, presently, this is a clinical diagnosis as there is no reliable diagnostic test. in lieu of a diagnostic test these criteria will be outlined in this manuscript. although these diagnostic criteria are based on clinical data and have been vetted by world experts in the field, they are arbitrary and inexact. there is a general consensus that corticosteroids are the drug of choice when treatment is required. several alternative agents may be used, usually as corticosteroid sparing agents, because of the development of significant corticosteroid toxicity. the assessment of the efficacy of alternative anti - sarcoidosis medications is complex for many reasons, including distinguishing the effects of corticosteroids from the study drug and reliably assessing disease activity. the diagnosis of sarcoidosis is never secure but is arbitrarily made when the statistical likelihood of alternative diagnoses becomes too small to warrant further investigation. a patient may present with a constellation of clinical findings that is so specific for sarcoidosis that the diagnosis may be made empirically without the need for a confirmatory biopsy (left side of figure 1). if these rare presentations are not observed, the diagnosis requires a tissue biopsy revealing granulomatous inflammation (usually noncaseating), exclusion of alternative causes of granulomatous inflammation, and documentation of systemic disease (right side of figure 1). the diagnosis of sarcoidosis may be established without performing a tissue biopsy (left side of figure). this requires the presence of specific clinical findings that are highly specific for the diagnosis (one of the conditions in the box on the left side of the figure). if none of these specific clinical presentations occurs, then the diagnosis requires tissue biopsy confirmation of granulomatous inflammation, exclusion of alternative causes for the granulomatous inflammation, and documentation that the disease is systemic and is, therefore, not confined to a single organ (pathway on the right side of the figure). abbreviations : cxr, chest radiograph ; ncg, noncaseating granulomas ;, controversial criterion. first, granulomatous inflammation is, by itself, inadequate to establish a diagnosis of sarcoidosis. in one recent study of 131 lung biopsy specimens revealing necrotic granulomatous lesions without an obvious etiology, 52 (40%) remained unknown, and only 4/79 (5%) of those where the etiology was determined were found to have sarcoidosis. second, the diagnosis of sarcoidosis requires that alternative causes of granulomatous inflammation are excluded. although biopsy specimens are usually examined for the presence of mycobacteria and fungi, other alternative granulomatous diseases are more problematic to exclude. in one german sarcoidosis clinic, patients diagnosed with sarcoidosis completed a detailed questionnaire concerning occupations where exposure to beryllium was possible. over 7 years, 84 patients had positive responses to this questionnaire, and 34/84 (40%) of these patients were confirmed to have chronic beryllium disease, rather than sarcoidosis, on the basis of beryllium lymphocyte proliferation testing. atypical mycobacteriosis hypersensitivity pneumonitis pneumoconiosis : beryllium (chronic beryllium disease), titanium, aluminum aspiration of foreign materials wegener s granulomatosis (sarcoid - type granulomas are rare) necrotizing sarcoid granulomatosis (nsg) atypical mycobacteriosis granulomatous histiocytic necrotizing lymphademitis (kikuchi s disease) sarcoid reaction in regional lymph nodes to carcinoma non - hodgkin s lymphomas granulomatous lesions of unknown significance (the glus syndrome) atypical mycobacteriosis reaction to foreign bodies : beryllium, zirconium, tattooing, paraffin, etc. primary biliary cirrhosis non - hodgkin s lymphomas infectious mononucleosis non - hodgkin s lymphomas giant cell myocarditis adapted from reference and reprinted with permission of the american thoracic society. third, sarcoidosis is a systemic disease that is not confined to the lung or any organ in isolation. some features that show sarcoidosis is a systemic illness are that the disease is associated with anergy, non - granulomatous inflammatory conditions such as erythema nodosum, systemic symptoms such as fatigue and small fiber neuropathy, and that the disease may recur in transplanted allografts. usually, confirmation that the disease is systemic involves identifying granulomatous inflammation in at least two organs. indeed, idiopathic granulomatous diseases of some isolated organs are thought to be distinct from sarcoidosis, such as idiopathic granulomatous hepatitis. more than one decade ago, the national institute of health sponsored a case control etiologic study of sarcoidosis (access) that developed an organ assessment instrument to document a second organ being involved with sarcoidosis, short of performing a biopsy. the instrument classified the likelihood of several specific clinical scenarios being caused by sarcoidosis in an organ as definite, possible provided that a) granulomatous inflammation had already been identified in another organ, and b) alternative causes for the clinical scenario could reliably be excluded. for example, if histologic evidence of granulomatous inflammation was found on skin biopsy, then eye sarcoidosis was considered for the purposes of access, definite or probable involvement was considered as sarcoidosis involvement in that organ. therefore, the access instrument allowed for the diagnosis of sarcoidosis to be established by confirming the presence of sarcoidosis in a second organ, without the need to biopsy that second organ. more recently, the world association of sarcoidosis and other granulomatous disorders (wasog) has updated the access assessment instrument to take into account new advances in diagnostic technology. however, an issue that has recently surfaced is whether the requirement of documenting involvement of a second organ / systemic disease is universally required for the diagnosis of sarcoidosis. despite the aforementioned idiopathic granulomatous diseases in single organs that appear to be distinct from sarcoidosis, there may be specific presentations in single organs that are highly specific for sarcoidosis. two such examples would be lupus pernio (disfiguring facial sarcoidosis) and chest imaging, revealing mediastinal adenopathy plus parenchymal lung nodules in a perilymphatic distribution. if granulomatous inflammation was identified in such clinical situations and no other evidence of granulomatous inflammation was identified in other organs, most would consider the diagnosis of sarcoidosis secure. indeed, most large sarcoidosis series reported by sarcoidosis experts, including the multicenter access trial investigators, have reported a large proportion of patients as having sarcoidosis with only a single organ involved (366/736, 49.7%). as a counter - argument to this viewpoint, a recent retrospective analysis of patients with concomitant sarcoidosis and connective tissue diseases (ctds) suggested that a significant proportion of them may have had solely a ctd, and that the granulomatous inflammation may have been a result of the ctd. since ctds are not recognized as a cause of granulomatous inflammation, several of these patients may have been misdiagnosed as having sarcoidosis. summing up these data, we believe that sarcoidosis can be diagnosed when a single organ is involved, provided that alternative causes have been excluded and the presentation is highly specific for the diagnosis. we do believe that confirming granulomatous inflammation in a second organ does raise the specificity of the diagnosis, albeit with a potential drop in sensitivity. the entire diagnosis discussion to this point has focused on a set of criteria that are arbitrary. these criteria require that the patient has a clinical presentation suggestive of sarcoidosis and that alternative causes of granulomatous inflammation are reasonably excluded. these are not objective, rigorous conditions. obviously, sarcoidosis must have a specific cause or causes associated with specific immune mechanisms. recently, chen and colleagues demonstrated intense expression and wide distribution of serum amyloid a (saa) within the sarcoidosis granuloma that exceeded that found in all other granulomatous diseases that were examined. saa appeared to have been produced by macrophages and giant cells within the sarcoid granuloma. these authors postulated that saa could consolidate a poorly soluble protein aggregate to form a nidus for granuloma formation. saa may disrupt the clearance of an antigen within the granuloma that allows for its persistence. the granulomatous inflammation in sarcoidosis may result from a prolonged immunogenic response to a persistent antigen, resulting in immune system exhaustion. as the identification of saa may be a specific marker for sarcoidosis - related granulomatous inflammation hopefully, saa or another specific biomarker to be developed in the future will make the previous discussion of diagnosis obsolete. furthermore, such a biomarker may answer the question as to whether several other granulomatous diseases of unknown cause such as blau s syndrome, granulomatous lesions of unknown significance (glus syndrome), granulomatous - lymphocytic interstitial lung disease, and necrotizing sarcoid granulomatosis, are truly distinct entities from sarcoidosis or whether they fall under the sarcoidoses umbrella. sarcoidosis does not mandate treatment because the disease may never cause symptoms or organ dysfunction, may remit spontaneously, and therapy (particularly corticosteroids) is associated with a myriad of significant side effects. sarcoid granulomas are thought to result from the interaction of an antigen with the immune system. it has been conjectured that granulomatous inflammation in sarcoidosis is necessary to clear the antigen. if this theory is correct, it is plausible that effective anti - sarcoidosis therapy may resolve granulomatous inflammation that may result in failure of clearance of the putative sarcoidosis antigen(s). in this scenario, when treatment is withdrawn, the antigen may still be present and could lead to recurrent granulomatous inflammation and, therefore, relapse. this schema is supported by data that suggest that a relapse of sarcoidosis is more common in patients previously treated for sarcoidosis, or with higher doses of corticosteroids. in addition, this concept is consistent with the premise that anti - sarcoidosis treatment improves granulomatous inflammation but does not alter the natural course of the disease [21,3136 ]. therefore, the granulomatous inflammation of sarcoidosis may be beneficial and should probably be left unsuppressed, provided that it does not result in significant patient symptoms or organ dysfunction. when treatment is indicated for sarcoidosis, corticosteroids are considered the drug of choice because they appear to have the greatest likelihood of efficacy and work most rapidly. although a consensus statement of the american thoracic society, european respiratory society, and wasog have recommended 20 to 40 mg of daily prednisone equivalent for the initial treatment of pulmonary sarcoidosis, the lower end of that dosage range appears to be adequately effective. a recent review of acute pulmonary exacerbations of sarcoidosis found that 20 mg of daily prednisone for a median of 3 weeks returned spirometry and pulmonary symptoms back to baseline. indeed, a regimen of 15 mg / day of prednisone was shown to be effective for sarcoidosis more than 40 years ago. little is known concerning the optimal algorithm for tapering corticosteroids, although relapses with such tapers occur in a significant percent of cases. although corticosteroids are currently entrenched as the drug of choice for most forms of sarcoidosis, alternative agents are often used because of the frequency of the development of corticosteroid side effects. usually, these drugs are corticosteroid - sparing as often a small amount of corticosteroid must be continued for adequate efficacy. these corticosteroid - sparing drugs should be considered for patients deemed at risk of developing corticosteroid side effects and most patients who require long - term corticosteroid therapy. a recent delphi study of 36 sarcoidosis experts reached a consensus that methotrexate be considered the second - line agent for pulmonary sarcoidosis. in an unusual study design, two european medical centers were compared where one used methotrexate as a second - line anti - sarcoidosis agent and the other used azathioprine. the reduction in corticosteroid dose and improvement of pulmonary function was not statistically different between these two groups. however, the rate of drug side effects, particularly the rate of infection, was significantly greater in the patients receiving azathioprine as a second - line agent. leflunomide has also been shown to be an effective anti - sarcoidosis medication in a large number of sarcoidosis patients in the united states. we believe that the use of second - line agents should be individualized based on the patient s potential for toxicity and the organ that is being treated. in regards to this latter point, antimalarial agents are particularly effective for skin sarcoidosis as well as for sarcoidosis - induced derangement of vitamin d metabolism causing hypercalcemia, hypercalciuria, nephrolithiasis and renal insufficiency. as these drugs have been shown to be better tolerated than other treatments in rheumatoid arthritis, they may be preferred for the aforementioned forms of sarcoidosis. over the past decade, several studies have shown a benefit of tumor necrosis factor alpha (tnf-) antagonists for the treatment of sarcoidosis. these studies have included case series [4750 ] and a randomized double - blind placebo - controlled trial. there is a sound rationale for this therapy because tnf- is released by macrophages recovered from sarcoidosis patients and tnf- is thought to be integrally involved in the development of the granulomatous inflammation. infliximab appears to be a particularly effective for lupus pernio (disfiguring facial sarcoidosis), neurosarcoidosis, and pulmonary sarcoidosis. adalimumab may be effective for sarcoidosis although, extrapolating from clinical reports, the optimal dose may need to be higher than that routinely used in rheumatoid arthritis. a recently completed placebo - controlled trial of golimumab failed to show a benefit over placebo for the treatment of pulmonary and cutaneous sarcoidosis. furthermore, all tnf- antagonist agents have been occasionally associated with relapse, worsening, or development of sarcoidosis [5963 ]. these occurrences are most commonly reported with etanercept and are postulated to be the result of tnf- antagonist - induced stimulation of interferon - gamma production leading to granulomatous inflammation. personalized or precision medical care where therapy is individualized based on the patient s specific clinical features or genetics. one example of such care is the observation that the presence of the tnf- g-308a polymorphism in rheumatoid arthritis patients is associated with a more favorable response to tnf- antagonist therapy than those who do not possess this polymorphism. recently, these results have been duplicated in a sarcoidosis cohort who received infliximab or adalimumab. rituximab is a monoclonal antibody directed at the c20 cell surface antigen of b - lymphocytes. although the granulomatous inflammation of sarcoidosis is thought to primarily develop through an interaction with t - lymphocytes, there is often abnormal b - lymphocyte activity, as demonstrated by the frequent development of a polyclonal gammopathy in active sarcoidosis. several case reports and open - label case series have shown the potential efficacy of rituximab in the treatment of sarcoidosis [6972 ]. the results of recent sarcoidosis drugs trials have uncovered several issues that may be of critical importance in designing future studies. first, subjects with severe forms of sarcoidosis may have a greater potential to respond to therapeutic agents than those with mild disease. a post - hoc analysis of a randomized placebo - controlled trial of infliximab versus placebo for pulmonary sarcoidosis demonstrated the greatest improvement in percent predicted forced vital capacity (fvc) at 24 weeks in those with lower baseline fvc, st. although this finding could be explained by a ceiling effect where those with mild disease have less room to show significant improvement, it is also possible that those with more severe disease have more severe active granulomatous inflammation. although it has previously been thought that pulmonary sarcoidosis patients with fibrocystic disease may have burnt - out inactive disease, a recent study demonstrated that 85% of such patients have significant lung uptake on positron emission tomography (pet), suggesting that they have active disease. because sarcoidosis may result in both treatable granulomatous inflammation and irreversible fibrosis, it is imperative to identify subjects with active disease in clinical sarcoidosis trials. this will require the establishment of reliable biomarkers of active granulomatous inflammation that can be used to select appropriate patients. a second issue of importance in many sarcoidosis trials is distinguishing study drug effects from corticosteroid effects. alternative agents are still in great demand because of the numerous significant toxicities of corticosteroids. most clinical trials of potential sarcoidosis patients enroll subjects who are corticosteroid - dependent, as these are the patients who would be potential candidates for these drugs and it would be problematic to taper them off corticosteroids and risk disease exacerbation. because of the efficacy of corticosteroids, it is problematic to detect additional benefits of the study drug in such patients. as an example of this issue, in a randomized placebo - controlled trial of infliximab versus placebo for pulmonary sarcoidosis, although the trial was positive, those receiving more than 15 mg / day of daily prednisone equivalent did not demonstrate an appreciable additional benefit from infliximab. potential trial designs to circumvent this issue include a) non - inferiority comparison trials versus corticosteroids in de novo sarcoidosis patients with corticosteroid rescue for study drug failure ; b) including a corticosteroid withdrawal phase in the trial ; and c) initiating a corticosteroid taper prior to trial enrollment and only enrolling subjects who deteriorate. probably the single most challenging issue in the design of a clinical sarcoidosis trial is the establishment of an appropriate endpoint. the presence of disease activity would be an adequate endpoint for a uniformly progressive disease such as lung cancer or tuberculosis. as previously mentioned, the presence of disease activity should probably be an entry criterion for sarcoidosis clinical trials. in addition, measurement of disease activity may be an appropriate endpoint to detect anti - sarcoidosis activity of a drug. however, reduction of disease activity is an unreliable endpoint to determine if a drug is useful for the treatment of sarcoidosis. however, such impairment is usually mild in sarcoidosis and poorly correlates with impaired quality of life, which is what patients usually care about most, so this is an appropriate clinical endpoint for most sarcoidosis trials. however, pulmonary symptoms and impaired quality of life are often not directly related to sarcoidosis, so this endpoint is not uniformly reliable. because this issue is complex and unresolved, the granulomatous inflammation of sarcoidosis can be measured by various methods (left box).the endpoint of granulomatous inflammation would be appropriate to detect the anti - sarcoidosis activity of a drug. however, granulomatous inflammation from sarcoidosis may not result in physiologic impairment (middle box). when physiologic impairment occurs, it may be mild and not lead to the development of symptoms. therefore, the presence of active sarcoidosis (left box) or physiologic impairment (middle box) may be inadequate clinical endpoints to base decisions on whether sarcoidosis patients require therapy. if physiologic impairment leads to significant functional impairment and/or worsening quality of life (right box), then therapy for sarcoidosis is required. therefore, a rational clinical endpoint would need to incorporate the patients functional status and health - related quality of life. abbreviations : ace, angiotensin converting enzyme ; bal, bronchoalveolar lavage ; ef, ejection fraction ; hrqol, health - related quality of life ; mri, magnetic resonance imaging ; pet, positron emission tomography ; pft, pulmonary function test ; pro, patient reported outcome measure. adapted from. the diagnosis of sarcoidosis remains a challenging issue principally because there is no specific reliable diagnostic test available. various algorithms and criteria have been established as diagnostic guidelines that may be useful while we remain hopeful for the development of a gold standard diagnostic test. such trials involve overcoming several problematic issues, but this can be accomplished with prudent thought and planning. marc a. judson is a consultant for janssen, celgene, novartis, mitsubishi - tanabe, and questcor. | this manuscript outlines recent advances in the diagnosis and treatment of sarcoidosis. the diagnosis of sarcoidosis can occasionally be made on clinical grounds without a confirmatory biopsy when very specific clinical findings are present. otherwise, the diagnosis requires histologic evidence of granulomatous inflammation, exclusion of alternative causes, and evidence of systemic disease. because there is no available diagnostic test for sarcoidosis, the diagnosis is never completely secure. instruments have been developed to establish the presence of sarcoidosis in a second organ and hence establish the systemic nature of the disease. corticosteroids remain the drug of choice for the treatment of sarcoidosis. additional sarcoidosis medications are most commonly used as corticosteroid - sparing agents. recent clinical sarcoidosis drug trials have exposed important issues that may confound trial results, including selecting patients with active disease, identifying study drug effects in patients receiving concomitant corticosteroids, and establishing proper study endpoints. |
the surgical procedure used to create flaps involves tissue damage because some areas may exceed the territory supplied by a given blood vessel, leading to several degrees of ischemia.1 moreover, microvascular perfusion is seriously impaired with failing procedures.2,3 vasoactive drugs can be used to ameliorate the ischemia process and extend survival. buflomedil and pentoxifylline have often been used in the treatment of peripheral artery disease and microcirculatory deficit, resulting in improved flap viability.47 buflomedil is an alpha - receptor blocking agent and a non - specific calcium antagonist. it can also inhibit platelet aggregation, increase red blood cell deformability, and decrease peripheral vascular resistance, thus restoring microcirculatory perfusion.8 buflomedil appears to improve nutritional blood flow in ischemic tissue in peripheral or cerebral diseases. consequently, it increases walking distances in patients suffering from intermittent claudication.9 pentoxifylline is a xanthine - derived phosphodiesterase inhibitor that enhances camp levels, theoretically via the same actions as buflomedil,10 i.e., it has beneficial effects on white blood cell function and hemorheological conditions.11 the effects of buflomedil and pentoxifylline administration on functional capillary density, tissue viability based on blood flow presence / absence, and survival of skin flaps in hamsters have yet to be elucidated. this recently developed microscopy method, originally described by winkelman,12 can be used to non - invasively study the microcirculation with reflected light without any fluorescent dye.13 olivier and co - workers showed that ops technology is useful to directly observe microvascular blood flow in skin flaps and suggested post - operative monitoring of free - tissue transfers.14 the ops technique has been incorporated into a portable device, the cytoscan (cytoscan, cytometrics inc., this study tested the efficiency of ops imaging for the prognosis of flap viability, and compared the effects of acutely administered buflomedil and pentoxifylline on the microcirculation using a random - pattern skin flap survival test. experiments were performed according to the protocols approved by the ethical committee of the state university of rio de janeiro, rj, brazil (cea/215/2007) using 24 male 10-week - old syrian golden hamsters (mesocricetus auratus, botucatu, so paulo, sp, brazil) weighing 110130 g. hamsters were divided into three groups. the control group received 0.1 ml of vehicle (c, 0.9% nacl), the buflomedil (b) group received 3 mg / kg / day of buflomedil (abbott, so paulo, brazil), and the pentoxifylline (p) group received 14.5 mg / kg / day of pentoxifylline (aventis pharma, so paulo, brazil). all injections were administered intraperitoneally 1 hour before skin flap preparation and for 7 days post - operatively at 12-hour intervals. animals received an appropriate laboratory diet of nuvital (nuvilab, curitiba, pr, brazil) and water provided ad libitum. animals were anesthetized with 30 mg / kg of sodium pentobarbital (pentobarbital sodique, sanofi, paris, france, 60 mg / ml) intraperitoneally. throughout the surgery and subsequent experiment, the temperature of the animals was maintained at 36.5 c with a heating pad controlled by a rectal thermistor (ltb 750 thermostat system, uppsala, sweden). the dorsal flap was dissected, preserving the sacral arteries, and detached from its panniculus carnosus. the area was subsequently sutured to the dorsal region of the animal using simple stitches. the surgical protocol was based on the mcfarlane skin flap model (olivier.)14,15 adapted to hamsters. each dorsal randomized skin flap measured 2 x 6 cm and was divided into 12 fields of 1 cm in diameter. each of the two fields formed one band to be analyzed (figure 1). an ops probe, with a 10x objective and a final magnification of 450x, was applied to these fields from the base of the flap to the apex (figure 1). images of the microcirculation were video recorded, stored in vhs format, and subsequently processed by capimage.16 sterile mineral oil was applied between the probe and skin to improve the coupling and reliability of the readings. functional capillary density (fcd) was defined as the total length of capillaries with flowing red blood cells in the microscopic area (mm / mm) under observation. the distance from the skin flap base to the blood flow cessation (distwith flow) field was measured at 1 h, 24 h, and 7 days after surgery and compared to viable skin flaps. a transparent sheet was placed on the dorsal skin flap and drawings of the areas with blood flow and necrosis were performed. the percentage of viable area (% va) was calculated by dividing the healthy area by the total area on the seventh post - operative day using autocad 2000 (autodesk, san rafael, usa) (figure 2). the presence or absence of blood flow in the studied skin flaps was quantified in all 12 fields, grouped in bands (figure 1) after 1 and 24 h. the mean values of the fields from each band are reported. the data obtained were compared to the blood flow in the viable area observed on the seventh postoperative day. for clarity, the results are presented as the mean standard error of the mean (s.e.m.). for statistical analysis, mann - whitney u tests, anovas, and wilcoxon matched pair tests were used to compare groups, and p - values < 0.05 were considered as statistically significant. each dorsal randomized skin flap measured 2 x 6 cm and was divided into 12 fields of 1 cm in diameter. each of the two fields formed one band to be analyzed (figure 1). an ops probe, with a 10x objective and a final magnification of 450x, was applied to these fields from the base of the flap to the apex (figure 1). images of the microcirculation were video recorded, stored in vhs format, and subsequently processed by capimage.16 sterile mineral oil was applied between the probe and skin to improve the coupling and reliability of the readings. functional capillary density (fcd) was defined as the total length of capillaries with flowing red blood cells in the microscopic area (mm / mm) under observation. the distance from the skin flap base to the blood flow cessation (distwith flow) field was measured at 1 h, 24 h, and 7 days after surgery and compared to viable skin flaps. a transparent sheet was placed on the dorsal skin flap and drawings of the areas with blood flow and necrosis were performed. the percentage of viable area (% va) was calculated by dividing the healthy area by the total area on the seventh post - operative day using autocad 2000 (autodesk, san rafael, usa) (figure 2). the presence or absence of blood flow in the studied skin flaps was quantified in all 12 fields, grouped in bands (figure 1) after 1 and 24 h. the mean values of the fields from each band are reported. the data obtained were compared to the blood flow in the viable area observed on the seventh postoperative day. functional capillary density (fcd) was defined as the total length of capillaries with flowing red blood cells in the microscopic area (mm / mm) under observation. the distance from the skin flap base to the blood flow cessation (distwith flow) field was measured at 1 h, 24 h, and 7 days after surgery and compared to viable skin flaps. a transparent sheet was placed on the dorsal skin flap and drawings of the areas with blood flow and necrosis were performed. the percentage of viable area (% va) was calculated by dividing the healthy area by the total area on the seventh post - operative day using autocad 2000 (autodesk, san rafael, usa) (figure 2). the presence or absence of blood flow in the studied skin flaps was quantified in all 12 fields, grouped in bands (figure 1) after 1 and 24 h. the mean values of the fields from each band are reported. the data obtained were compared to the blood flow in the viable area observed on the seventh postoperative day. for clarity, the results are presented as the mean standard error of the mean (s.e.m.). for statistical analysis, mann - whitney u tests, anovas, and wilcoxon matched pair tests were used to compare groups, and p - values < 0.05 were considered as statistically significant. all animals investigated did not demonstrate blood flow after the third band of the skin flap, independent of the postoperative time. figure 3 shows the fcd for the control, buflomedil, and pentoxifylline groups on bands i, ii, and iii 1 and 24 hours after the surgical procedure. a decrease in fcd was observed from band i to band iii in all groups analyzed after 24 hours. the buflomedil group showed higher fcd values compared to the control or pentoxifylline group for each band at 1 and 24 h. conversely, no statistical differences were observed for distwith flow between the control, buflomedil, and pentoxifylline groups. although all groups displayed increased distances between 24 h and the seventh day, they reached similar final distances (control = 2.73 cm, buflomedil = 2.78 cm, and pentoxifylline = 2.70 cm) (figure 4). the perfusion / non - perfusion interface was easily identified, and there was no difference in the percentage of viable areas among the control, buflomedil, and pentoxifylline groups (50.7, 52.9, and 47.0, respectively). finally, the mean percentage of the preserved area was approximately 50% for all groups. on the seventh post - operative day, we were able to confirm the presence of blood flow, which was detected after 1 h using ops imaging of the first three bands for all groups. using this technique, however, we were unable to correlate all areas that lacked blood flow with a given time line. for band ii, an area of 100% in the control, buflomedil, and pentoxifylline groups without blood flow 1 h after surgery developed into perfused zones 7 days after surgery. the prediction of blood flow absence (in %) after 1 h and on postoperative day 7 for the control, buflomedil, and pentoxifylline groups for band iii is shown in figure 6. note that the percentage of fields without blood flow detected by cytoscan in controls decreased from 75% after 1 h to 60% after 7 days. when a skin flap is raised, injury to the blood vessels and sympathetic nerves causes hemodynamic instability, leading to blood flow abnormality and a disturbance in tissue oxygenation.17 in this work, two approaches were used to evaluate skin flap success. we used buflomedil and pentoxifylline and determined their effects on the microcirculation and skin flap survival (fcd, distwith flow, % va), and the ops imaging technique to predict flap viability over time following surgery. since significant microvascular damage is present during skin flap preparation, the development of early necrotic areas is expected. indeed, all fields on bands iv, v, and vi did not present any blood flow, resulting in tissue death. the fcd decreased in all groups from band i to band iii and was significantly lower on the distal part of the flap compared to its base after 24 h. the buflomedil group demonstrated higher values of fcd compared to the control and pentoxifylline groups. these results are consistent with the results reported by langer and colleagues, who validated the use of ops imaging against intravital microscopy and measured fcd on the skin flaps of hairless mice.18 harder. also showed a decrement in fcd in several segments from the base to the apex of the skin flaps of mice.19 buflomedil has been shown to have positive effects on microvascular reperfusion injury by decreasing leukocyte adhesion.20 however, szczesny. showed that buflomedil is ineffective in avoiding a decrease in the fcd of local skin blood perfusion during general anesthesia using intravital fluorescence microscopy in hairless mice.21 in addition, resting skin flux motion activity at the hallux in patients with intermittent claudication evaluated by laser doppler fluxometry was not increased after buflomedil treatment.22 it is possible that the results presented herein could be explained by better buflomedil effects on the redistribution of blood flow after ischemia compared to pentoxifylline. in fact, briguglio. demonstrated an attenuation of ischemia - induced histological loss and damage to pyramidal cells in rats treated with buflomedil.23 on the other hand, fdc showed favorable results and distwith flow did not change among the control, buflomedil, and pentoxifylline groups at any time point analyzed. for each group, increasing values of distwith flow were detected from 1 hour to 7 days. conflicting results have been reported concerning the actions of buflomedil and pentoxifylline on surface length necrosis on flaps or microcirculation. quirinia and colleagues did not find any differences in wound healing for buflomedil compared to the control (no treatment) in rats24 ; however, kamler and. showed an acceleration of wound surface area reduction after chronic ischemia.25 on the other hand, pentoxifylline elicited improved cardiovascular autonomic function, but failed to reduce microalbuminuria in type 2 diabetic patients, revealing a microcirculatory defect.26 the percentage of viable area on the seventh day after the surgical procedure did not differ among the groups studied and remained at approximately 50%, consistent with the buflomedil results described by biondo - simes.27 this result could be explained by the following scenarios. the treatment duration was not sufficient to produce favorable blood flow redistribution (treatment was initiated 1 h before surgery and lasted 7 days), allowing only acute effects to be detected. bayat and colleagues chronically pretreated rats for 30 days with pentoxifylline before skin flap preparation and observed an increase in flap survival.28 emrecan. showed that pentoxifylline could have an acute effect on the skeletal muscle of rabbits in an ischemia - reperfusion model.29 our group selected buflomedil (3 mg / kg / day) and pentoxifylline (14.5 mg / kg / day) administration for 7 days post - surgery to elucidate the acute drug effects. another possibility could be that buflomedil and pentoxifylline are not capable of improving the microcirculation without any previous impairment of this structure. a lower percentage of viable skin flap areas in pretreated rats has been demonstrated with nicotine, which decreases blood flow to peripheral vessels by 40%. the percentage of viable areas was larger for the buflomedil and pentoxifylline groups than for rats administered nicotine.30 finally, severe impairment of the microcirculatory network during the surgical procedure might interfere with drug action. it is well known that thin skin flaps may jeopardize the circulation.31 in the present study, the dorsal skin flap and sacral arteries containing the panniculus carnosus were preserved. it is likely, however, that some degree of arteriovenous shunting occurred over 24 h in the early and/or late post - operative periods, thereby limiting the skin survival in areas with marginal blood flow.32 using a pig skin flap model, kerrigan. observed that the dermal vessels of an ischemic island flap were dramatically affected.3 we also observed several distal random ischemic regions, which may have determined the final survival of the flap. all fields in which blood flow was present after 1 and 24 h (detected by cytoscan) remained viable 7 days after surgery. all fields without blood flow on band ii and 20% of the fields on band iii in all groups recovered after 7 days. thus, the cytoscan was more efficient in predicting fields without blood flow on band iii, which had direct contact with the necrotic area and was subjected to unstable hemodynamics. it must be noted that some fields with unpredictable blood flow recovery will normally be present,33,34 and it should be emphasized that the ops technique appeared to be more accurate than the clinical evaluation of skin flap necrosis.14 other evaluations of microcirculation techniques also exist, such as intravital fluorescence microscopy,35 but the portability of this technique is limited. laser doppler flowmetry36 also appears to be a promising technique for use with ops imaging. buflomedil presented higher values of fcd compared to the control and pentoxifylline groups in the skin flap hamster model. however, no differences were observed for the distance from the skin flap base to blood flow cessation or the percentage of viable skin flaps among groups. orthogonal polarization spectral imaging is a very useful tool to assess skin flap viability by counting fields with and without blood flow, showing the ability to confirm 100% of the areas with and 80% of the areas without flow over time. | objectivethis study investigated the effects of buflomedil and pentoxifylline, both of which are used in reconstructive surgery of hamster skin flap microcirculation, and evaluated the skin flap survival rate by orthogonal polarization spectral imaging.methodtwenty-four adult male syrian golden hamsters were divided into three groups : a control (c, 0.1 ml 0.9% saline), buflomedil (b, 3 mg / kg / day), and pentoxifylline group (p, 14.5 mg / kg / day). treatments administered intraperitoneally were initiated 1 hour before skin flap preparation and continued for 7 days post - operatively at 12-hour intervals. preparations (skin flaps) were divided into 12 fields, which were organized into six bands. functional capillary density (fcd, in mm / mm2), distance from the skin flap base to blood flow cessation (distwith flow, in cm), percentage of viable skin (va, in%), and qualitative analysis of blood flow by orthogonal polarization spectral imaging were performed at 1 and 24 hours and on the seventh post - operative day.resultbands iv, v, and vi presented no flow independent of time. the functional capillary density group b was higher than that of groups c and p, primarily after 24 hours. all groups showed an increase in d with time but reached similar final distances (c = 2.73, b = 2.78 and p = 2.70 cm). moreover, the percentage of viable areas remained at approximately 50%. the orthogonal polarization spectral imaging was useful to assess viability by counting fields with and without blood flow.conclusionsfunctional capillary density values were higher in the buflomedil group compared to the control and pentoxifylline groups in this model. functional capillary density did not influence d or the percentage of va, and the technique showed favorable potential to assess / predict the viability of skin flaps within 1 h after surgery. |
in 2010, there were 1,490,000 hiv - infected pregnant women and 390,000 children became infected with hiv. mother - to - child transmission of hiv can occur during pregnancy, during birth, or during breastfeeding. the risk of transmission is 1530% in nonbreastfeeding populations and breastfeeding adds an additional 520% risk for an overall transmission rate of 2045%. in 2010, the world health organization (who) released the guidelines on antiretroviral drugs for treating pregnant women and preventing hiv infection in infants with the goal of reducing mother - to - child transmission to less than 5% and virtually eliminating hiv infection in children by 2015 [3, 4 ]. infant feeding by hiv - infected women remains a public health dilemma for developing countries. although breastfeeding involves a considerable risk of hiv transmission, nonbreastfed infants are exposed to higher risk of death in resource - limited setting [5, 6 ]. according to 2010 who guidelines, national health authorities should decide whether health services will principally counsel and support hiv - infected women to either breastfeed and receive antiretroviral interventions or avoid breastfeeding, as the strategy that will most likely give infants the greatest chance of hiv - free survival. developed countries have decided to recommend formula feeding for hiv - exposed children due to the low infant mortality and the risk of hiv transmission through breastfeeding, whereas in developing countries the health and survival benefits of breastfeeding exceed the risk of hiv transmission. however, most of the evidence for recommending breastfeeding in developing countries comes from clinical trials performed in sub - saharan africa. data of formula feeding on child growth and mortality from pmtct programmes in other continents are scarce, and some developing countries are supporting the use of formula feeding in their national pmtct programmes [711 ]. india is the third country in the world in terms of hiv infected people, and it is estimated that 43,000 pregnant women were living with hiv in india in 2009. in this study, we investigated the effect of formula feeding and breastfeeding on mortality, growth, and hiv transmission of children enrolled in a pmtct programme in a rural district of india. in india, the neonatal mortality rate was 34 deaths/1000 live births and the infant mortality rate was 50 deaths/1000 live births in 2009. andhra pradesh is the state with the highest burden of hiv - infected people in india. rural development trust (rdt) is a nongovernmental organization that has three hospitals in anantapur. in these hospitals, medical care of hiv - infected people is provided free of cost, including medicines and consultation or admission charges. the vicente ferrer hiv cohort study (vfhcs) is an open cohort study of all hiv - infected patients who have visited the rdt hospitals since june 2006. taking into account data from the andhra pradesh state aids control society that estimates that 15,721 hiv - infected people are living in the district of anantapur, the cohort is fairly representative for the district as it covers approximately 70% of all hiv - infected patients. for this study, hiv - infected women from the vfhcs database who became pregnant between january 1st 2008 and december 5th 2010 were included in the analysis. the selection of patients from the database was executed in january 17th 2012. in order to reduce the number of children infected by hiv in close collaboration with governmental integrated counselling and testing centres, which provided free hiv testing to all pregnant women in the district, hiv - positive pregnant women were followed by a group of 30 community health workers spread across the district. hiv - infected pregnant women initiated triple antiretroviral therapy (art) regardless of the cd4 lymphocyte count. delivery by caesarean section was offered to all women for reducing the risk of hiv transmission at birth, unless they had an hiv viral load below 1000 copies / ml in the third trimester of pregnancy. in women who were diagnosed with hiv during labour, a single fixed - dose combination tablet of zidovudine 300 mg, lamivudine 150 mg, and nevirapine 200 mg was given, and caesarean section was performed only if there was no membrane rupture. however, women who initiated art less than one month before delivery were counselled about the risk of hiv transmission through breastfeeding and formula feeding was offered for six months free of cost. women who decided to give formula feeding to their babies were admitted to hospital for training on safe methods of replacement feeding such as boiling water for milk preparation and using cup feeding instead of bottle feeding [2, 16 ]. art to the mother was continued after delivery if the cd4 lymphocyte count was below 350 cells/l at the moment of initiating art. in women who initiated art with high cd4 lymphocyte count, art was stopped either one month after stopping breastfeeding or immediately after delivery in those women who decided to give formula feeding to their children. if the art regiment included a nonnucleoside reverse transcriptase inhibitor (nevirapine or efavirenz), two nucleoside reverse transcriptase inhibitors were continued for two extra weeks in order to reduce the risk of developing nevirapine or efavirenz resistance due to their longer life span. newborn prophylaxis was also given. before the publication of the who 2010 guidelines, single (zidovudine or nevirapine) or triple (zidovudine, lamivudine, and nevirapine) drug prophylaxis in syrup form was given to newborns for 7 to 42 days according to the risk of hiv infection during delivery. after the publication of who 2010 guidelines, all newborns received 28 days of zidovudine syrup according to their weight. for identifying the moment of hiv transmission, we aimed to perform hiv viral load at birth, after six weeks of delivery and after six weeks of stopping breastfeeding. children were considered to be infected intrauterus, during delivery, or during breastfeeding if the hiv viral load was positive at birth, after six weeks of delivery, or six weeks after stopping breastfeeding, respectively. children who did not have any hiv viral load determination were considered as hiv negative if their hiv serology was negative at any time after six months of age and were considered as hiv positive if they had a positive hiv serology after 18 months of age. measurement of hiv viral load was performed utilizing two different commercial assays, a real - time hiv-1 rna polymerase chain reaction assay (cobas taqman hiv-1 v1.0, roche diagnostics, mannheim, germany) until march 2011 and a reverse transcriptase activity assay (exavir load version 3, cavidi, uppsala, sweden) since march 2011. during the visits of children to hospitals, growth parameters such as weight, length / height, head circumference, mid - upper arm circumference, triceps skinfold thickness and subscapular skinfold thickness were collected. hiv - exposed children were followed up until 18 months of age with regular visits to the hospitals and with home visits by outreach workers. statistical analysis was performed using stata statistical software (stata corporation, release 11, college station, texas, usa). we used kaplan - meier curves and log - rank test for comparing mortality in children who received breastfeeding or formula feeding. time was measured from birth to death or the last visit date. for calculating z - scores of anthropometric indicators according to who child growth standards for age and sex, we utilized the who macro for stata (igrowup_standard.ado). who child growth standards were calculated from a group of selected children from different parts of the world who received optimal nutrition according to who standards : no known health or environmental constraints to growth, mothers willing to follow specific feeding recommendations (i.e., exclusive or predominant breastfeeding for at least four months, introduction of complementary foods by the age of six months and continued partial breastfeeding up to at least 12 months), no maternal smoking before and after delivery, single term birth, and absence of significant morbidity. among all anthropometric markers, we selected severe underweight (weight - for - age < 3 z - scores) because it has demonstrated to be a good indicator for predicting mortality in infants from india, especially in infants aged less than six months. during the study period, 391 hiv - infected pregnant women were identified and 336 (85.9%) entered in the pmtct programme before delivery. of the 55 pregnancies that did not enter in the programme before delivery, five children were lost to follow up and 12 out of 50 (24%) were hiv infected although 29 (58%) of them received formula feeding. characteristics of the 336 women who entered in the programme before delivery are described in table 1. the median age was 23 years, and the median height was 152 cm. the proportion of underweight in mothers was 39%, and approximately half of them were primipara. the proportion of pregnant women with cd4 lymphocyte count below 200 and 350 cells/l were 9% and 29%, respectively. zidovudine was used in 76% of women, and nevirapine was used more frequently than efavirenz. viral load in the third trimester was available for 199 women and 175 (88%) of them had a viral load below 1000 copies / ml. seven percent of women delivered at home, and caesarean section was performed in 39% of the cases. zidovudine syrup was used more commonly than other newborn prophylaxis regimens, and 51% of children received formula feeding. in figure 1, we present a flow diagram of the outcomes of all hiv - positive pregnancies that entered in the pmtct programme before delivery. of the three pregnant women who had spontaneous abortion, two started art (zidovudine, lamivudine, and nevirapine) before having the abortion. all voluntary terminations of pregnancy occurred between the third and the fifth month of pregnancy. of the 11 children who died during the first week of life, weight was recorded in five and in four of them the birthweight was less than 1.5 kg. the perinatal mortality rate was 22/329 (67 deaths/1000 total births, 95% confidence interval (ci) 45 to 99), the neonatal mortality rate was 12/318 (38 deaths/1000 live births, 95% ci 22 to 65), and the infant mortality rate was 27/318 (85 deaths/1000 live births, 95% ci 59 to 121). the overall transmission of hiv was 3.7% (95% ci, 2 to 6.7), although 12 (4%) were lost to follow up and 18 who had a negative viral load after six weeks of life were still on breastfeeding at the end of the study period. antenatal and perinatal preventive measures were able to reduce the rate of hiv transmission to 1.8% (95% ci, 0.7 to 4.1) in those children who had a viral load determination after the age of six weeks. of those breastfed children who had a negative viral load determination after aged six weeks, 3.1% (95% ci, 1.2 to 7.8) acquired hiv during breastfeeding. in this group, art was initiated at least one month before delivery in all but one of the cases (99.2%). the characteristics of the ten children who became hiv infected are presented in table 2. to study the effect of the type of feeding on infant mortality we excluded children who died during the first week of life to avoid causes of the death related to perinatal complications [22, 23 ]. kaplan - meier survival curves of infants by type of feeding are presented in figure 2. three quarters of deaths (12 out of 16) occurred during the first trimester of life. compared to breastfed children, formula - fed children had a crude hazard ratio for death of 15.2 (95% ci, 2 to 114.8) (p = 0.008). in a multivariable analysis, the hazard ratio for death in formula - fed children was 13.5 (95% ci, 1.7 to 108.2) (p = 0.014) adjusted by low birth weight (< 2.5 kg), low cd4 lymphocyte count of the mother (< 200 cells/l), home delivery, illiteracy of the mother and less than one month on art before delivery. the cumulative mortality at three months was 7.3% (95% ci, 4.1 to 12.7) for formula - fed infants and 0.66% (95% ci, 0.1 to 4.6) for breastfed infants. the cumulative mortality at 12 months was 9.6% (95% ci, 6.7 to 18.5) for formula fed infants and 0.68% (95% ci, 0.1 to 4.7) for breastfed infants. free - of - hiv survival was also significantly higher in breastfed children (126/131, 96.2%) than in formula - fed children (130/151, 86.1%) (p = 0.003). when questioning mothers about the symptoms of the child before death, eight reported gastrointestinal symptoms, three reported fever, three reported seizures, one reported respiratory symptoms, and one reported low consciousness. the proportion of children with severe underweight by type of feeding during the first 18 months of life is presented in figure 3. the proportion of infants with severe underweight who received formula feeding was higher in the first two months of life, reaching a pick of 40% at two months, but it was reduced notably afterwards. the proportion of infants with severe underweight who received breastfeeding experienced progressive reduction over time. the mean and 95% ci of anthropometric markers (weight, length / height, body mass index, weight for length / height, head circumference, mid - upper arm circumference, triceps skinfold thickness, and subscapular skinfold thickness) for age and sex by type of feeding during the first 18 months of life are presented in figure 4. whereas biomarkers of breastfed infants remained more or less stable over time, formula - fed infants experienced a rapid decline during the first two months of life and a rapid improvement after the third month of life, achieving similar or superior values to breastfed infants after the ninth month of life. in this study, the use of formula feeding was associated with increased risk of mortality and lower hiv - free survival compared to breastfeeding. in india, 98% of children are breastfed and it is estimated that 54% of all deaths before age of five years are related to malnutrition. the introduction of formula feeding for averting the transmission of hiv imposes an additional handicap to an already malnourished population. as it is shown in figures 3 and 4, formula - fed children experience an acute decline in anthropometric markers during the first two months of life, when most of deaths occurred. our findings are in accordance with the results of other studies performed in resource - limited settings that have shown an increased morbidity and mortality in hiv - exposed children who received formula feeding [7, 26, 27 ]. also in india, hiv - exposed children from a large government hospital who were formula fed had an increased risk of hospitalization. although the overall proportion of hiv - infected children was 3.7%, prenatal and perinatal interventions achieved a hiv transmission rate of 1.8%, which is similar to the rate of hiv transmission in developed countries where the majority of children receive formula feeding. the rate of hiv transmission due to breastfeeding was 3.1%, which is slightly higher than the rate reported in clinical trials in africa [30, 31 ]. the breastfed infants were heavier and taller during the first two months, but lighter and shorter thereafter. looking at the graph of weight for length / height (a marker of acute malnutrition) in figure 4, we can observe a decline after stopping breastfeeding at six months of age compared to who growth standards, which were created from a population of infants who were breastfed at least until the age of 12 months. however, we did not observe an increased mortality in the breastfed group after weaning at age 5 - 6 months as it was seen in clinical trials in africa with abrupt weaning at the age of four months [33, 34 ]. this observation is important because the 2010 who guidelines recommend breastfeeding until the age of 12 months, but the initiation of mixed feeding can increase the risk of hiv transmission. new studies comparing morbidity, mortality, and hiv transmission in children who receive breastfeeding for six or twelve months may clarify this matter. the proportion of women with underweight was similar to the proportion of women from rural areas with underweight described in the national family health survey, a large - scale survey conducted in a representative sample of households throughout india. in the same study, it was observed that 48% of children under five years had stunting (length / height for age < 2 z - scores), 20% had wasting (weight for length / height < 2 z - scores) and 43% had underweight (weight for age < 2 z - scores). these data can explain the low values in length / height, weight for length / height and weight observed in figure 4. however, anthropometric markers related to fat deposits such as triceps and subscapular skinfold thickness had similar or even higher values than who growth standards. these differences may be explained by the low quantity of proteins in the diet in poor and rural areas of india [15, 25 ]. the mother 's art was initiated at least one month before delivery in almost all children who received breastfeeding. it is possible that the rate of hiv transmission due to breastfeeding will rise if breastfeeding is encouraged to mothers who initiate art late in pregnancy or during labour. new studies are needed to assess hiv - free survival if breastfeeding is universally recommended regardless of the time on art before delivery. even in rural areas of india, universal art to hiv - infected pregnant women is able to reduce mother - to - child transmission to less than 5%. although breastfeeding can increase the rate of hiv transmission, formula feeding produces malnutrition during the first months of life, increases mortality and, therefore, has lower hiv - free survival compared to breastfeeding. the results of this study confirm the results of clinical trials performed in sub - saharan africa and support the implementation of universal art regardless of cd4 count to all hiv - infected pregnant women and breastfeeding for all hiv - exposed children in order to achieve who goals of elimination of paediatric hiv without increasing infant mortality in developing countries with high levels of malnutrition. | we describe a programme for the prevention of mother - to - child transmission (pmtct) of hiv that provided universal antiretroviral therapy (art) to all pregnant women regardless of the cd4 lymphocyte count and formula feeding for children with high risk of hiv transmission through breastfeeding in a district of india. the overall rate of hiv transmission was 3.7%. although breastfeeding added a 3.1% additional risk of hiv acquisition, formula - fed infants had significantly higher risk of death compared to breastfed infants. the cumulative 12-month mortality was 9.6% for formula - fed infants versus 0.68% for breastfed infants. anthropometric markers (weight, length / height, weight for length / height, body mass index, head circumference, mid - upper arm circumference, triceps skinfold, and subscapular skinfold) showed that formula - fed infants experience severe malnutrition during the first two months of life. we did not observe any death after rapid weaning at 5 - 6 months in breastfed infants. the higher - free - of hiv survival in breastfed infants and the low rate of hiv transmission found in this study support the implementation of pmtct programmes with universal art to all hiv - infected pregnant women and breastfeeding in order to reduce hiv transmission without increasing infant mortality in developing countries. |
optimal cutoff value of alt level to precisely estimate the presence of fatty liver was as low as 28.0 u / l. we should consider the possibility of fatty liver even when alt level is within normal range in subjects with poorly controlled type 2 diabetes. |
|
atopic dermatitis (ad) is a common skin disease manifested clinically by chronic inflammation and characterized histologically by skin infiltration of inflammatory cells including predominantly lymphocytes, eosinophils, and mast cells. it affects 1020% of children and 13% of adults in developed countries, and the prevalence is increasing. many of these patients also suffer from asthma and allergic rhinitis along with intense itching and skin infection. although the pathogenesis and etiology of ad remain to be completely understood, this multifactorial disease likely results from a complex crosstalk between genetic and environmental factors. exaggerated th2 response, disruption of the epidermis barrier functions, high levels of serum ige, and decreased production of antimicrobial peptides (amps) are the key findings in ad. data from human and animal studies indicate that ad is a th2 dominant inflammatory skin disease at the acute stage followed by th1 involvement at the chronic stage of the disease. specifically, this notion of th2-dominance in ad is validated by a mouse model we have successfully generated through overexpressing il-4, a key th2 cytokine, in the basal epidermis using a epidermis - specific keratin 14 promoter. the il-4 transgenic (tg) mice spontaneously develop skin lesions, serological abnormalities, and skin infection, which fulfil the clinical and histological diagnostic criteria for human ad. consistent with an inflammatory disease process, upregulation of chemokines, proinflammatory cytokines, b cell activation molecules, angiogenic factors, and several critical adhesion molecules has been found in these tg mice. the jak - stat pathway is a classical signal transduction pathway for numerous cytokines and growth factors. the binding of ligands to their receptors leads to jak activation, which in turn phosphorylates and activates stats. the jak family includes jak1, jak2, jak3, and tyk2, and the stat family includes stat1, stat2, stat3, stat5a / b, and stat6. as a negative regulator of the jak - stat pathway, the socs family consists of cytokine - inducible sh2 domain - containing protein (cis) and socs17. socss may act on the activation loop of jaks, or they may interact with phosphotyrosines in the cytoplasmic domain of cytokine receptors, suppressing the jak - stat pathway with a negative feedback mechanism. in this review, we will discuss the roles that the jak - stat pathway plays in the pathophysiology of ad. since ad is a th2-dominant disease, examination of how the jak - stat pathway regulates th2 differentiation would help us to understand the possible roles that jak - stat play in ad. it is well established that il-4 promotes the differentiation of th2 cells, which in turn produce il-4, il-5, il-10, and il-13. the study of il-4 and the il-4 receptor -null mice demonstrated clearly that il-4 is required for a th2 response and the production of th2 cytokines. in addition, studies from the il-4 tg mice demonstrated upregulation of the th2 cytokines. consistently, the il-4 tg mice generated on a th2-dominant strain, balb / c mice, develop earlier onset and worse ad - like skin lesions than the il-4 tg mice generated on a th1-dominant strain, c57bl/6 mice, suggesting that the th2 systemic immune milieu, in addition to cutaneous th2 immune milieu, plays an essential role in the pathogenesis of ad. additionally, knocking out bcl-6, a transcription factor functioning to regulate il-4 signal transduction, leads to th2-mediated hyperimmune response in many organs, further supporting the regulatory function of il-4 on th2 immunity. importantly, il-4 signals through the jaks - stat6 pathway to regulate th2-related target genes in lymphocytes, firmly supporting a role of jak - stat in th2 regulation. thymic stromal lymphopoietin (tslp), known to promote th2 differentiation, to activate natural killer t cells and basophils, and to affect b cell maturation, has been reported to be associated with ad. while the murine tslp receptor signals via stat3, stat5/tec, a src type kinase, the human tslp receptor activates stat1, stat3, stat5/jak1, and jak2. it has been reported that histamine enhances the secretion of th2 cytokines such as il-4, il-5, il-10, and il-13 and inhibits the production of th1 cytokines. histamine was shown to stimulate il-13 production through the jak - stat pathway in a murine th2 cell line. in addition, tyrphostin, a jak - stat pathway inhibitor, reversed the effects of histamine on ifn, il-5, and il-10 production. horr. have shown that histamine, by acting through the h4 receptors on t cells, can suppress stat1 activation and help to drive the th2 response, leading to the development of ad. it is generally accepted that tyk2, jak2, and stat4, which are il-12 signaling pathway components, are essential for th1 cell differentiation, while jak1, jak3, and stat6, which are il-4 signaling components, are critical for th2 differentiation. in addition, stat5a / b, which are involved in the upregulation of gata3 and il-4r, and stat3, which helps stat6 bind to its target genes, also play some roles in th2 cell differentiation. stat6 regulates genes involved in th2 and b cell differentiation, ige class switching, and mhc class ii production. while stat4 null mice fail to generate th1 cells and conditional stat3 knockout mice have an exaggerated th1 response, stat6 null mice have impaired th2 cell development and ige class switching. on the contrary, mice expressing constitutively active stat6 (stat6vt) develop an ad - like skin lesion. in addition, several stat6 polymorphisms are also associated with a predisposition to allergic diseases and high levels of ige. a jak3 mutation in severe combined immunodeficiency (scid) patients leads to the absence of t cells and natural killer cells and the production of dysfunctional b cells. a loss - of - function mutation in tyk2 leads to defects in multiple cytokine signaling pathways including type i ifn, il-6, and il-12. these patients showed impaired th1 differentiation and accelerated th2 differentiation with clinical manifestations including unusual susceptibility to various microorganism infections, ad - like skin lesions, and high serum ige levels, suggesting a regulatory role of the jak family in th2 differentiation. another tyk2 null mutation and hypomorphic mutations in stat3 are associated with the hyper - ige syndrome (hies), a primary immunodeficiency characterized by ad like - skin lesions, susceptibility to infections, and high serum ige levels. in this syndrome, il-12 and ifn/ signal pathways are defective, thus blocking the th1 differentiation. as a result, the th2 response is exaggerated, leading to similar clinical findings as ad. because stat3 is important for b cell proliferation and differentiation, hies patients have decreased number of memory b cells as compared with ad patients. as a negative regulator of the jak - stat pathway, suppressor of cytokine signaling 2 (socs2) deletion leads to increased susceptibility to ad as compared with wild - type mice. these mice show an exaggerated th2 response along with significantly elevated serum ige levels, eosinophilia and low il-17 levels. socs2-knockout cd4 t cells display constitutively high levels of stat6 phosphorylation as compared with wild type t cells. in addition, il-2-induced stat5 activation, also involved in the early th2 differentiation, is exaggerated in the socs2-knockout cd4 t cells. on the other hand, il-12-stimulated stat4 phosphorylation is unaffected, and il-6 mediated stat3 phosphorylation is inhibited. since it is generally accepted that the development of the th1, th2, and th17 immune responses are mediated by stat4/stat1, stat6/stat5, and stat3 respectively, the increased stat6/stat5 activation and suppressed stat3 activation naturally lead to an exaggerated th2 response at the expense of th17. another example of the jak family involvement in th2 immunity is shown in nc / nga mice that develop ad - like skin inflammation. nakagawa. reported that pyridone 6 (p6), a pan - jak inhibitor, delayeded the onset and reduced the severity of ad - like skin lesions in nc / nga mice. p6 suppressed the ifn-/stat1, il-2/stat5, and il-4/stat6 signaling pathways strongly while it suppressed il-6/stat3 pathway only modestly, resulting in reduction of the th1 and th2 responses but the enhancement of the t17 response. eosinophil skin infiltration is frequently observed in the skin lesions of ad patients and ad mouse models. although stat6 null mice have defects in th2 differentiation and ige class switching, crossing these mice with nc / nga mice can not prevent the development of ad - like skin lesions. even though these mice do not produce ige and th2 cytokines, the histological features of their skin lesions are similar to that of ad. these authors suggested that a th2 response is not absolutely necessary for the development of ad - like skin lesions ; instead, ifn- and eosinophil skin infiltration may play an essential role. eosinophils express the il-31 receptor a (il-31ra), and il-31 has the ability to delay the apoptosis of eosinophils and to stimulate the secretion of pro - inflammatory cytokines and chemokines through jak - stat. in addition, il-5, mainly involved in b cell proliferation and differentiation, was shown to induce eosinophilia when upregulated. conversely il-5 or il-5 receptor chain (il-5r) il-5 signals through the jak2-stat1/stat5 and map kinases pathways to regulate genes involved in cell proliferation, survival and effector functions. in the skin, regulatory t cells (tregs) help to regulate the immune response, and in ad, treg function is suppressed. depleting treg cells significantly increased the severity of the skin inflammation in ova - sensitized mice. in t cells, il-4-activated stat6 upregulates gata3, the master regulator of th2 cells. in addition, stat5a / b and stat3 may also play important roles in the regulation of foxp3. on the other hand it was reported that the ifn- gene transfer stimulates treg related cytokines and improves ad - like symptoms of nc / nga mice. this finding, however, seems to contradict the notion that ifn- is a promoting factor for ad. on the clinical level, treatment with a low - dose cyclosporine a is effective in reducing skin inflammation with simultaneous increase of treg population in ad patients, further supporting a role of treg in ad. stat3, mediating the il-23 and il-6 pathways, is critical for the differentiation of th17 cell. in the early stage of human ad, the number of th17 cells is increased, and il-17 has been reported to upregulate adhesion molecules on keratinocytes, enhancing t cell - keratinocyte adhesion and t cell - mediated cytotoxicity. however, in chronic ad, the th17 pathway is suppressed, which could account for the amp deficiency in ad. in the skin lesions of ad patients as well as ad mouse models, the number of mast cells is increased. il-9 stimulates vegf production from human mast cells through the activation of stat3 in an in vitro experiment. interestingly, il-9 serum levels in ad patients are not different from those of non - ad controls even though il-9 and its receptors are upregulated in the lesional skin of the ad patients, suggesting that the upregulation of vegf in mast cells via il-9 may be a localized cutaneous phenomenon. the prominent increase of mast cell vegf production and angiogenesis in an ad animal model also supports the role of jak - stat in this immune pathway being a major cell type of the skin and possessing the ability to participate in various immune responses, epidermal keratinocytes are likely an active player in the skin inflammation of ad. we and others have shown that the dysregulation of the keratinocyte genes by il-4 play an important role in ad. the type i receptor consisting of il-4r and il-2rc chains is expressed in hematopoietic cells, while the type ii receptor consisting of il-4r and il-13r1 is expressed in other cell types including keratinocytes. in keratinocytes, both il-4 and il-13 binds to il-4r and il-13r1. il-13r2, which was reported as a dominant negative inhibitor for both il-4 and il-13, binds to il-13 with high affinity, but lacks a significant cytoplasmic domain. we have previously reported that il-13r2 is upregulated by il-4 in keratinocytes. in ad patients this may be due to a negative feedback mechanism available to keratinocytes to maintain homeostasis. we have also shown that il-4 upregulates chemokines ccl8, ccl24, ccl25, ccl26, ccl3l1, cxcl6, and cxcl16, all known for their roles in the pathophyisology of ad. the eotaxin subfamily that consists of ccl11/eotaxin-1, ccl24/eotaxin-2, and ccl26/eotaxin-3 plays an important role in ad. cysteine receptor 3 (ccr3), which are expressed predominantly on eosinophils, recruiting these cells to the inflammatory site. skin infiltrating eosinophils release a variety of cytotoxic granule proteins to cause tissue damage. in the regulation of ccl26, though both stat3 and stat6 are phosphorylated by il-4, only stat6 is functionally activated. detailed analysis of the promoter of the ccl26 gene has shown that the stat6 response element consists of two palindromic half sites ttc and gaa spaced by four nucleotides. others also reported that stat6, different from other stat members, prefers the stat sites with four spacing nucleotides. in addition to tyrosine phosphorylation, stat3 activation may also involve serine phosphorylation and lysine acetylation. knockout mice studies indirectly support our results in that while stat6 null mice demonstrated defects in eosinophil tissue infiltration, the stat3 cre - loxp knockout mice did not show similar defects. furthermore, il-4 and il-13 double knockout mice, when sensitized with ovalbumin, develop a skin lesion characterized by intact cd4 dermal infiltration, severely diminished eosinophil infiltration, and undetectable ova - specific ige levels, suggesting that stat6 activation is required for eosinophil skin infiltration in ad. in addition to chemokines, il-4 also upregulates pro - inflammatory factors, such as il-1, il-19, il-20, il-25, il-27, il-12r2, il31ra, and nitric oxide synthase 2 (nos2). moreover, we demonstrated that il-4 signals through the jak - stat pathway to regulate il-19 expression in keratinocytes. using pcr array, we have found that that il-4 upregulates angiogenic or lymphangiogenic - related genes, such as vegf, ang-1, angl-4, igf1, notch4, pgf, and mcp-1. using the il-4 tg mice and cell culture, we showed that cd11b macrophages attracted to the skin lesion by mcp-1 may account for lymphangiogenesis observed in ad by secreting vegf - c. while il-4 upregulates chemokines, pro - inflammatory factors, angiogenic factors in keratinocytes, it downregulates antimicrobial peptides (amps) or factors involved in apm production. other groups have also demonstrated that the dysregulation of amps affects the development of ad. human -defensin-3 (hbd-3), which is significantly decreased in ad, is downregulated by il-4, il-10, and il-13. in addition, hbd-2 and human cathelicidin (ll-37) were also found to be reduced in ad skin lesions as compared with psoriasis. suggested that low levels of ll-37 suppressed by il-4 and il-13 through stat6 may be the mechanism responsible for increased vaccinia virus replication as occurred in eczema vaccinatum of ad patients. skin barrier function defects are critical for the development of ad. in addition to the filaggrin mutations in some ad patients, cytokines they are downregulated by il-4 and il-13 in keratinocytes, and in stat6 transgenic mice, their expression is significantly decreased as well. in addition, filaggrin, which is reduced in acute ad skin lesion, is also suppressed by il-4 and il-13 in keratinocytes. interestingly, while il-4 markedly reduces ceramide levels in the epidermis by downregulating the expression of serine - palmitoyl transferase-2, acid sphingomyelinase, and -glucocerebrosidase, th1 cytokines (gm - csf / ifn-/tnf-) increase the ceramide levels. taken together, the role that il-4 plays in the dysregulation of keratincoyte functions in ad involves the upregulation of chemokines, pro - inflammatory factors, and pro - angiogenic factors and the downregulation of amps and factors responsible for skin barrier function. pruritis in ad is induced by complicated crosstalks among keratinocytes, immune cells, and nerve fibers. the data from the nc / nga mice studies indicate that although il-4 is a key mediator of the inflammatory process in ad, il-31 might be the key causative factor for pruitus. in addition, il-31 transgenic mice developed a th2 immune response with severe pruritic skin lesions similar to ad. il-31 is upregulated in the skin of ad patients, and it induces the itching sensation either by direct stimulation of the dorsal root ganglion fibers that express il-31ra or by indirect stimulation of pruritic factors from keratinocytes. il-31 receptor activation in keratinocytes induces calcium influx and stat3 mediated -endorphin production, which may contribute to the peripheral itching in ad. thus far, data from our laboratory and others seem to indicate an influential role of jak - stat in the pathogenesis of ad, with il-4 being a key mediator (figs. 1 and 2). specifically, the jak - stat pathway is instrumental for the th2 cell differentiation. in the immune milieu of ad, the enhancement of th2 cell proliferation and their release of various cytokines via the jak - stat pathway is likely the critical factor for the inflammatory responses in ad. this th2 immune upregulation could then lead to b cell maturation and plasma cell differentiation, resulting in the hyper - secretion of ige. similarly, this hyper th2 immune milieu triggers epidermal cells to release various chemokines, pro - inflammatory cytokines, and angiogenic factors that participate in ad pathophysiology. moreover, il-5 released from this hyper th2 milieu could activate eosinophils that are attracted to the skin by the eotaxin subfamily, further worsening the ad condition. in addition, by way of il-31, an inducer of pruritus, ad becomes increasingly intensified. figure 1. proposed mechanism of jak - stat involvement in atopic dermatitis (ad) development, part in addition to the filaggrin gene mutation defects in some ad patients, il-4 is also able to downregulate barrier proteins filaggrin, loricrin and involucrin through the jak - stat pathway making the epidermis more penetrable by allergens and pathogens. once penetrated through the epidermis, allergens / pathogens are detected by dendritic cells, which become subsequently activated to present these antigens to nave th0 cells. the th0 cell can then differentiate into the th2 cell through the jak1,3-stat6 pathway under the influence of il-4. in the th0 cells, the stat6 pathway can also upregulate gata3, a master regulator of th2 cells. gata3 in turn suppresses foxp3, the master regulator in treg cells, thus allowing more t cells to be activated. proposed mechanism of jak - stat involvement in atopic dermatitis (ad) development, part ii. here, th2 cells play a significant role. by their abilities to provide il-4 and il-5 stimulation via the jak - stat pathway, immature b cells could be differentiated into mature b cell and plasma cells would undergo antibody heavy chain switching to ige class. the subsequent binding of ige to skin mast cells could lead to release of histamine, which is known to exacerbate ad. similarly, this hyper th2 immune milieu, particularly il-4, could trigger epidermal cells to produce and release various chemokines (such as ccl26), pro - inflammatory cytokines, and angiogenic factors, leading to ad pathophysiology. moreover, il-5 released from this hyper th2 milieu could, through jak - stat pathway, activate eosinophils, and attract them to the skin via ccl26, further worsening the ad condition. in addition, by way of il-31, an inducer of pruritus, ad symptoms could become increasingly intensified. our understanding of the jak - stat pathway and its relationship to the dysregulated immune response and keratinocyte function is still in its infancy. what exact roles do the different jaks and stats play in the pathogenesis of ad ? answering these questions will enable us to target more specifically the key components of the complex pathophysiology of ad, thus providing the best treatment for this disease. | atopic dermatitis (ad), a common chronic inflammatory skin disease, is characterized by inflammatory cell skin infiltration. the jak - stat pathway has been shown to play an essential role in the dysregulation of immune responses in ad, including the exaggeration of th2 cell response, the activation of eosinophils, the maturation of b cells, and the suppression of regulatory t cells (tregs). in addition, the jak - stat pathway, activated by il-4, also plays a critical role in the pathogenesis of ad by upregulating epidermal chemokines, pro - inflammatroy cytokines, and pro - angiogenic factors as well as by downregulating antimicrobial peptides (amps) and factors responsible for skin barrier function. in this review, we will highlight the recent advances in our understanding of the jak - stat pathway in the pathogenesis of ad. |
we thank leigh conroy for her help with the matlab algorithms and dr iris kulbatski for her help with editing the manuscript. this work was supported by funding provided to r. dacosta by the canadian institutes for health research. | the dorsal skinfold window chamber (dswc) model is a unique tool that enables analysis of various aspects of tumor biology and therapeutic response. although the protocol for the murine dswc model is standardized, certain tumors fail to grow or require a particular environment to promote growth. given such limitations, we optimized the dswc model for a slow - growing tumor that regresses spontaneously in the standard protocol. we further characterized the vascular network in the tumor model compared with that of non - tumor - bearing mice and observed significant differences in multiple parameters related to vascular structure and function. |
pharmacovigilance is the science and activities relating to the detection, assessment, understanding, and prevention of adverse effects of drugs or any other possible drug - related problems. recently, its concerns have been widened to include herbals, traditional and complementary medicines, blood products, and biological. the purpose of pharmacovigilance is to detect, assess, and understand, and to prevent the adverse effects or any other possible drug - related problems, which is not only confined to chemical drugs, but extended to herbal, traditional, and complementary medicines, biologicals, vaccines, blood products, and medical devices. the history of the use of herbs as medication is as old as history itself. some authors state that the first recorded use of herbs for medical treatment began over 4000 years ago. traditional chinese medicine centers on interactions between the body and the environment. a mixture of treatments, including herbs, acupuncture, and massage, is then prescribed. an example of the types of herbal products can be seen in table 1. in the united states, a combination of european, chinese, ayurvedic, an other unconventional treatments influenced the use of herbs to the present day. in almost all the traditional systems of medicine, adverse events may also arise from the misuse of the wrong species of medicinal plants, incorrect dosing, errors in the use of herbal medicines by healthcare providers and consumers, interactions with other medicines, and use of products contaminated with potentially hazardous substances, such as toxic metals, pathogenic microorganisms, and agrochemical residues. herbal products from different cultures the following examples demonstrate the range of problems encountered with the use of herbal medicines and products. some herbal products were found to contain 0.1 to 0.3 mg of betamethasone per capsule after some patients developed corticosteroid - like side effects.owing to misidentification of the medicinal plant species, plant materials containing aristolochic acid were used for manufacturing herbal products, which caused severe kidney failure in patients in several countries.reports have been received by drug safety monitoring agencies of prolonged prothrombin times, increased coagulation time, subcutaneous hematomas, and intracranial hemorrhage associated with the use of ginkgo biloba.one of the most well - known traditionally used herbal medicines caused severe, sometimes fatal cases of interstitial pneumonia, when used in conjunction with interferon. some herbal products were found to contain 0.1 to 0.3 mg of betamethasone per capsule after some patients developed corticosteroid - like side effects. owing to misidentification of the medicinal plant species, plant materials containing aristolochic acid were used for manufacturing herbal products, which caused severe kidney failure in patients in several countries. reports have been received by drug safety monitoring agencies of prolonged prothrombin times, increased coagulation time, subcutaneous hematomas, and intracranial hemorrhage associated with the use of ginkgo biloba. one of the most well - known traditionally used herbal medicines caused severe, sometimes fatal cases of interstitial pneumonia, when used in conjunction with interferon. list of the unapproved ayurvedic medicinal products found on the canadian market thus far, which have been analyzed by health canada and found to contain high levels of lead, mercury, and arsenic, are as follows : karela tablets, produced by shriji herbal products, indiakarela capsules, produced by himalaya drug, indiakarela capsules, produced by charantia, uk (specifically batch # 12011)maha sudarshan churna powder, produced by zandu pharmaceuticals, mumbai, indiamaha sudarshan churna powder, d and k pharmacy, bhavnagar, indiamaha sudarshan churna powder, produced by chhatrisha, lalpur, indiamaha sudarshan churna powder, produced by dabur india, new delhi, indiasafi liquid, produced by hamdard - wakf - pakistansafi liquid, produced by hamdard - wakf - indiayograj guggul tablets, produced by zandu pharmaceuticals, mumbai, indiasudarshan tablets, produced by zandu pharmaceuticals, mumbai, indiashilajit capsules, produced by dabur india, new delhi, india karela tablets, produced by shriji herbal products, india karela capsules, produced by himalaya drug, india karela capsules, produced by charantia, uk (specifically batch # 12011) maha sudarshan churna powder, produced by zandu pharmaceuticals, mumbai, india maha sudarshan churna powder, d and k pharmacy, bhavnagar, india maha sudarshan churna powder, produced by chhatrisha, lalpur, india maha sudarshan churna powder, produced by dabur india, new delhi, india safi liquid, produced by hamdard - wakf - pakistan safi liquid, produced by hamdard - wakf - india yograj guggul tablets, produced by zandu pharmaceuticals, mumbai, india sudarshan tablets, produced by zandu pharmaceuticals, mumbai, india shilajit capsules, produced by dabur india, new delhi, india in order to provide consistency in the naming of herbs in adverse reaction (ar) reports, the who collaborating centre for international drug monitoring has recommended the use of proper scientific binomial names for herbs used in medicine, including the use of such names (where this information is available) in the coding of ar reports. it is equally important for the authors of published ar case reports to identify the specific product(s) involved, including label and manufacturer information, specific ingredients, and dose employed. published case reports would also benefit from analysis of the suspect product used, for contamination and adulteration, or species identification, where possible. an ar is defined as a noxious and unintended response to a marketed health product, which occurs at doses normally used or tested for the diagnosis, treatment, or prevention of a disease or the modification of an organic function. it is undeniable that plants have an important role in the development of modern medicines. more than 60 to 70% of modern medicines in the world market are directly or indirectly derived from plant products. high - profile issues such as ars associated with ephedra and aristolochia have shown that hmps can produce toxicity in human beings. however, it is difficult to identify the causative agent associated with the ars encountered because traditional herbal preparations often contain multiple ingredients. the who database has over sixteen thousand suspected herbal case reports. due to the lack of clinical trials for most hmps, postmarket pharmacovigilance is a critical source of safety information ; however, the assessment of ars associated with hmps offers unique challenges in the quantity and quality of available information. the quality of ars depends in part on educating potential reporters of what types of data are valuable for assessment. such educational efforts can take the form of online educational modules, such as those directed at naturopathic doctors in canada. in order to provide consistency in the naming of herbs in ar reports, the who collaborating centre for international drug monitoring has recommended the use of proper scientific binomial names for herbs used in medicine, including the use of such names (where this information is available) in the coding of ar reports. it is equally important for the authors of published ar case reports to identify the specific product(s) involved, including label and manufacturer information, specific ingredients, and dose employed. published case reports would also benefit from analysis of the suspect product used, for contamination and adulteration, or species identification, where possible. a lack of, or incorrect, herbal identification has been noted as a potential source of confusion in published case reports of hmp toxicity. in order to provide consistency in the naming of herbs in adverse reaction (ar) reports, the who collaborating centre for international drug monitoring has recommended the use of proper scientific binomial names for herbs used in medicine, including the use of such names (where this information is available) in the coding of ar reports. it is equally important for the authors of published ar case reports to identify the specific product(s) involved, including label and manufacturer information, specific ingredients, and dose employed. published case reports would also benefit from analysis of the suspect product used, for contamination and adulteration, or species identification, where possible. an ar is defined as a noxious and unintended response to a marketed health product, which occurs at doses normally used or tested for the diagnosis, treatment, or prevention of a disease or the modification of an organic function. it is undeniable that plants have an important role in the development of modern medicines. more than 60 to 70% of modern medicines in the world market are directly or indirectly derived from plant products. high - profile issues such as ars associated with ephedra and aristolochia have shown that hmps can produce toxicity in human beings. the most common adverse effects reported are hepatic and renal problems. however, it is difficult to identify the causative agent associated with the ars encountered because traditional herbal preparations often contain multiple ingredients. due to the lack of clinical trials for most hmps, postmarket pharmacovigilance is a critical source of safety information ; however, the assessment of ars associated with hmps offers unique challenges in the quantity and quality of available information. the quality of ars depends in part on educating potential reporters of what types of data are valuable for assessment. such educational efforts can take the form of online educational modules, such as those directed at naturopathic doctors in canada. in order to provide consistency in the naming of herbs in ar reports, the who collaborating centre for international drug monitoring has recommended the use of proper scientific binomial names for herbs used in medicine, including the use of such names (where this information is available) in the coding of ar reports. it is equally important for the authors of published ar case reports to identify the specific product(s) involved, including label and manufacturer information, specific ingredients, and dose employed. published case reports would also benefit from analysis of the suspect product used, for contamination and adulteration, or species identification, where possible. a lack of, or incorrect, herbal identification has been noted as a potential source of confusion in published case reports of hmp toxicity. there are several ways in which we can move forward in attempting to provoke pharmacovigilance in herbals. introduce pharmacovigilance concepts into the curriculum of herbals at the undergraduate and postgraduate level.make reporting of adverse reactions to regulatory mandatory for herbal formulations.human resource development is a key feature for the success of this enterprise. it will be necessary to train herbal experts in the science of pharmacovigilance and include them not only in reporting but also in assessment of the adverse reactions.healthcare professionals should remain vigilant for potential interactions between herbals and prescription medications, especially when it involves medications with narrow therapeutic indices. due to the wide use and easy availability of herbal medicines, the safety and quality of herbal medicine should be ensured through greater research, pharmacovigilance, greater regulatory control, and better communication between patients and health professionals. the recommended approach is to include herbal medicines in existing national pharmacovigilance systems or, where such systems have not yet been developed, to establish comprehensive national pharmacovigilance systems which incorporate coverage of herbal medicines. pharmacovigilance in herbal medicine in india is perhaps an unthought of concept as yet ; we do not need herbal thalidomide to wake the pharmacovigilance community to the need of the hour. it will be necessary to train herbal experts in the science of pharmacovigilance and include them not only in reporting but also in assessment of the adverse reactions. healthcare professionals should remain vigilant for potential interactions between herbals and prescription medications, especially when it involves medications with narrow therapeutic indices. due to the wide use and easy availability of herbal medicines, the safety and quality of herbal medicine should be ensured through greater research, pharmacovigilance, greater regulatory control, and better communication between patients and health professionals. the recommended approach is to include herbal medicines in existing national pharmacovigilance systems or, where such systems have not yet been developed, to establish comprehensive national pharmacovigilance systems which incorporate coverage of herbal medicines. pharmacovigilance in herbal medicine in india is perhaps an unthought of concept as yet ; we do not need herbal thalidomide to wake the pharmacovigilance community to the need of the hour. | herbal formulations being widely accepted therapeutic agents as antidiabetics, antiarthritics, hepatoprotectives, cough remedies, memory enhancers, and adaptogens. the commonest myth regarding herbal medicines is that these medicines are completely safe, and can therefore be safely consumed by the patient on his / her own, without a physician 's prescription. this belief has led to large - scale self - medication by people all over the world, often leading to disappointing end - results, side - effects, or unwanted after - effects. there is an increasing awareness at several levels of the need to develop pharmacovigilance practices for herbal medicines. the current model of pharmacovigilance and its associated tools have been developed in relation to synthetic drugs, and applying these methods to monitoring the safety of herbal medicines presents unique challenges in addition to those described for conventional medicines. several problems relate to the ways in which herbal medicines are named, perceived, sourced, and utilized. this may be because of differences in the use of nonorthodox drugs (e.g., herbal remedies) which may pose special toxicological problems, when used alone or in combination with other drugs. the purpose of pharmacovigilance is to detect, assess, and understand, and to prevent the adverse effects or any other possible drug - related problems, related to herbal, traditional, and complementary medicines. |
this study was approved by the local ethics committee, and each patient gave informed consent for participation. the clinical diagnosis of pd was made according to the uk brain bank criteria.11 at the time of the study, none of the patients had ever taken medications for pd. all patients underwent a detailed clinical evaluation, including laboratory tests for lipid profiles and serum homocysteine. at the time of study enrollment, all included patients were evaluated using the unified parkinson s disease rating scale (updrs) and the modified hoehn and yahr staging score. patients fasted the night before the test, except for a small amount of water, if they were thirsty and abstained from drinking alcohol or coffee the day before the study. continuous electrocardiographic and non - invasive blood pressure monitoring was used in each patient (ym6000, mediana tech., san antonio, tx, usa). after 30 min of supine rest, the test (20 min at 60) was performed using the manumed special tilt1-section (enraf nonius, rotterdam, the netherlands). by consensus, orthostatic hypotension was defined as a fall in blood pressure of at least 20 mm hg systolic pressure and/or 10 mm hg diastolic pressure between 2 and 5 minutes after tilt.12 all patients enrolled in the study underwent conventional mri with a 3.0-t system (magnetom verio, 3 t, siemens). axial t2 weighted images, complemented with coronal fluid attenuated recovery images, were used to rate the wmh. the mr images were rated using the semiquantitative visual rating system of scheltens.13 this rating scale provides four sum scores in a semiquantitative way ; periventricular hyperintensities (06), deep wmh (024), basal ganglia hyperintensities (030), and infratentorial hyperintensities (030). periventricular hyperintensities were identified as continuous, confluent areas of high signal intensity adjacent to the anterior or posterior horns of the lateral ventricles (cap) and along the lateral ventricles (bands). absence of lesions was a score of 0, 5 mm lesions was a score of 1, and a score of 2 was given for lesions > 5 mm. wmh, located in the deep and subcortical white matter, were separately rated in the frontal, temporal, parietal, and occipital regions according to the previously described criteria.13 these rating criteria were also applied to regions of the basal ganglia (caudate, putamen, globus pallidus, thalamus, and internal capsule) and infratentorial regions (cerebellum, midbrain, pons, and medulla). the independent sample t - test or pearson s chi - square test was used to compare the differences between the groups categorized by the blood pressure response during orthostasis (non - oh and oh). given the potential influence of several variables on the results of wmh, we also conducted group comparisons with an analysis of covariance (ancova) including age, the presence of hypertension and diabetes, low density lipoprotein (ldl)-cholesterol, and serum homocysteine levels as confounding covariates. patients were also divided into no to mild and severe wmhs groups based on two extremes by tertile distribution. the relationship between blood pressure profiles monitored during tilt table testing tilt and wmh were tested using spearman s rank correlation coefficients. the statistical analysis was performed with spss software version 15.0 (spss, inc., this study was approved by the local ethics committee, and each patient gave informed consent for participation. the clinical diagnosis of pd was made according to the uk brain bank criteria.11 at the time of the study, none of the patients had ever taken medications for pd. all patients underwent a detailed clinical evaluation, including laboratory tests for lipid profiles and serum homocysteine. at the time of study enrollment, all included patients were evaluated using the unified parkinson s disease rating scale (updrs) and the modified hoehn and yahr staging score. patients fasted the night before the test, except for a small amount of water, if they were thirsty and abstained from drinking alcohol or coffee the day before the study. continuous electrocardiographic and non - invasive blood pressure monitoring was used in each patient (ym6000, mediana tech., san antonio, tx, usa). after 30 min of supine rest, the test (20 min at 60) was performed using the manumed special tilt1-section (enraf nonius, rotterdam, the netherlands). by consensus, orthostatic hypotension was defined as a fall in blood pressure of at least 20 mm hg systolic pressure and/or 10 mm hg diastolic pressure between 2 and 5 minutes after tilt.12 all patients enrolled in the study underwent conventional mri with a 3.0-t system (magnetom verio, 3 t, siemens). axial t2 weighted images, complemented with coronal fluid attenuated recovery images, were used to rate the wmh. the mr images were rated using the semiquantitative visual rating system of scheltens.13 this rating scale provides four sum scores in a semiquantitative way ; periventricular hyperintensities (06), deep wmh (024), basal ganglia hyperintensities (030), and infratentorial hyperintensities (030). periventricular hyperintensities were identified as continuous, confluent areas of high signal intensity adjacent to the anterior or posterior horns of the lateral ventricles (cap) and along the lateral ventricles (bands). absence of lesions was a score of 0, 5 mm lesions was a score of 1, and a score of 2 was given for lesions > 5 mm. wmh, located in the deep and subcortical white matter, were separately rated in the frontal, temporal, parietal, and occipital regions according to the previously described criteria.13 these rating criteria were also applied to regions of the basal ganglia (caudate, putamen, globus pallidus, thalamus, and internal capsule) and infratentorial regions (cerebellum, midbrain, pons, and medulla). the independent sample t - test or pearson s chi - square test was used to compare the differences between the groups categorized by the blood pressure response during orthostasis (non - oh and oh). given the potential influence of several variables on the results of wmh, we also conducted group comparisons with an analysis of covariance (ancova) including age, the presence of hypertension and diabetes, low density lipoprotein (ldl)-cholesterol, and serum homocysteine levels as confounding covariates. patients were also divided into no to mild and severe wmhs groups based on two extremes by tertile distribution. the relationship between blood pressure profiles monitored during tilt table testing tilt and wmh were tested using spearman s rank correlation coefficients. the statistical analysis was performed with spss software version 15.0 (spss, inc., among the 117 patients enrolled, 46 were men and 71 were women. the mean age at examination was 68.5 10.1 years, and the mean duration of motor symptoms was 2.1 1.0 years. the severities of parkinsonian motor symptoms were as follows : mean total updrs score was 24.7 17.9 (part 1, 2.2 2.3 ; part 2, 7.2 5.8 ; part 3, 14.8 10.7). twenty - nine patients had hypertension (hbp), 24 had diabetes mellitus (dm), and 106 were non - smokers. the presence or absence of hypertension and diabetes, and current / past smoking habits did not affect cerebral wmh (hbp vs. no hbp group : 14.9 8.6 vs. 11.9 9.0, f = 1.295, p = 0.258 ; dm vs. no dm group : 15.8 6.7 vs. 11.8 9.3, f = 2.118, p = 0.148 ; non - smoker vs. ex - smoker vs. current - smoker 12.9 8.9 vs. 9.8 10.1 vs. 11.7 8.4, f = 0.259, p = 0.772, by ancova controlling for age, ldl - cholesterol and serum homocysteine). higher ldl - cholesterol and serum homocysteine levels also did not correspond to increasing wmh scores (r = 0.004, p = 0.965 in ldl - cholesterol ; r = 0.071, p = 0.494 in homocysteine by spearman s rank correlation). clinical and laboratory profiles of the groups with and without orthostatic hypotension are presented in table 1. data from patients with and without orthostatic hypotension were similar for age, gender, duration of illness, severity of parkinsonian motor symptoms, and proportion of patients with hypertension and diabetes. among the 42 patients with oh, 29 were clinically symptomatic (69.1%). the wmh scores were higher in the orthostatic hypotension group compared to those in the non - oh group after controlling for age, the presence of hypertension and diabetes, ldl - cholesterol and serum homocysteine. in addition, a trend of more wmh lesions in the supratentorial region of the oh group was also found (table 2). on correlation analysis, orthostatic diastolic blood pressure change was positively correlated with white matter changes (figure 1a and b). on the other hand, supine blood pressures were closely correlated with the wmh scores of the whole brain (figure 1c and d). when comparing groups with extremes of wmhs, the severe wmh group had higher supine systolic bp and larger fall in bp during orthostasis (figure 2). epidemiological investigations in healthy populations have shown that wmh predict the risk of cerebrovascular disease, cognitive dysfunction, and mortality.1 advanced age, arterial hypertension, diabetes, and elevated serum homocysteine levels are risk factors for increasing wmh.24,14,15 however, these associations remain unclear in patients with pd.5,6 in this study, the presence or absence of cerebrovascular risk factors was not associated with wmh in patients with pd. this finding is consistent with previous studies and suggests that other nonvascular factors contribute to the vascular brain damage in patients with pd. orthostatic hypotension is common in patients with pd and appears more frequently as disease progresses and influence subjective symptoms, quality of life, and disease treatment.16 recent investigations in patients with multiple system atrophy indicate that supine systolic blood pressure and/or blood pressure changes during orthostasis are significantly and independently correlated with wmh.17,18 however, a similar phenomenon was not confirmed in patients with pd.18 prior studies were not performed in patients with early stage and the enrolled patients were not free from drug effects. in this study, we enrolled only newly diagnosed patients with early stage pd who had not taken any dopaminergic medication. because dopaminergic drugs can aggravate orthostatic hypotension, this result suggests that this association was independent from anti - parkinsonian treatment. we found that the orthostatic hypotension group had more severe wmh changes and that the differences in wmh were more significant in the deep white matter region. although the hemodynamic consequences during orthostasis have not been clarified, it was reported that systemic blood flow is inversely associated with wmhs in the elderly.19 because orthostatic hypotension leads to recurrent blood pressure drops, this could result in cerebral hypoperfusion and impact neurocirculatory failures, perhaps via cerebral small - vessel disease.20 supine blood pressures can also contribute myriad episodes of cerebral hypo- and hyper - perfusion to white matter injuries. in addition to neurocirculatory insufficiency, degenerative processes associated with aging and disease itself may contribute to the generation of wmh in pd. in summary, our results indicate that hemodynamic changes associated with orthostatic hypotension may be associated with white matter changes in patients with pd. | background and purpose : several reports on the elderly population have suggested that orthostatic hypotension is associated with white matter hyperintensities (wmh) ; however, little information is available on patients with parkinson s disease (pd).methods : we analyzed the association blood pressure profiles during tilt table testing with wmh scores in 117 patients with pd. wmh were rated using the semiquantitative visual rating system proposed by scheltens.results:the presence of orthostatic hypotension was associated with increasing tendency of wmh score and the blood pressure changes during tilting and supine blood pressure were positively correlated with increasing wmh score.conclusions:this finding indicates that hemodynamic changes associated with orthostatic hypotension may be associated with white matter changes in patients with pd. |
male sprague - dawley rats (n=18 : exposed n=9 ; sham exposed n=9) were individually housed before and during experimental protocols. all protocols were approved by the saint louis university animal care and use committee. during the experiment, animals were between the ages of 4 and 7 months, age matched between groups. the rats were exposed at a stable temperature (2324c) and were maintained in a 12 h light / dark cycle ; lights on at 7 a.m. food and water were available ad lib. two identical exposure structures were used (one charged, the other not) located in a 104 m room with masonry walls and 4 m high ceiling. the large room allowed for the exposure and sham exposure group to be in the same room. the exposure apparatus was designed and constructed with the assistance from the parks college of engineering at saint louis university. two sonotube reinforced cardboard tubes 8 ft long and 2 ft in diameter served as framework for the exposure structures. one tube was wrapped with 18 gauge insulated copper wire ; the other was not wound and served as the sham exposure environment. the exposure apparatus was energized by a standard 110 v, 60 hz outlet and a step - down transformer (a variac) providing the necessary voltage of 8 v that excited the wire from a three - phase, four - wire supply to create a horizontal, linearly polarized 1000 milligauss (or 1 gauss or 1.010 t) magnetic field within the exposure volume. the magnetic field in the sham exposure sonotube was less than 1 mg and was 7 m away from the exposure. the animals were singly housed with the cages being constructed of translucent polypropylene 1298, fitted with 2 high pvc filters as covers., five electrodes for sleep recording were implanted according to standard procedures (38). these five placements, in a pin - plug assembly, permitted the fastening of stainless steel jeweler screws (080 by 1/8 in.) that allowed for ecog recordings from the parietal - frontal and the parietal - occipital regions of the brain. the pin plug was then permanently attached to the skull using dental cement, forming a mound that permanently seals the skull, electrodes, and plug as a single unit. post operatively, the animal received post - operative injections of 0.1 mg / kg buprenorphine twice daily for 3 days. after surgery, the animals were allowed to recover for 1 week at which time two animals were placed in the sleep lab for another week for acclimation. the animals were continually maintained on a 1212 hours schedule throughout the experiment and were staggered in twos as they entered the exposure room. at the end of acclimation, the animals were placed into the exposure apparatus, which involved being placed into the plastic cages described above with one rat in the energized tube, and the other in the sham non - energized tube. exposure was 24 hours a day with the exception of approximately 10 min each day for handling and care of the animals when the coils were deactivated. animals were staggered into the acclimation room and exposure facilities two at a time each week until no more than four animals were in each tube at once. field measurements ensured a uniform exposure for all rats in the energized tube. on the final 3 days of exposure (day 28, 29, 30), urine was collected every 6 hours from the basin of the cage at the end and middle of the dark period and at the end and middle of the light period. standard metallic metabolic cages could not be used due to the configuration of the apparatus and due to possible eddy currents being generated by the materials of typical metabolic cages. to collect the urine during the 3 days of collection, the cages were fitted with a porous plastic grid affixed approximately one inch from the basin of the cage, this allowed for urine to pass freely to the basin while the grid contained any expelled feces. the urine, after collection, was centrifuged and the supernatant stored at -80c until all animals urine had been collected. separately, the dark and light samples for each rat were then pooled and volume measured. analysis of the urine involved an elisa kit (alpco, dunham, nh) for 6-sulphatoxymelatonin (amt6), the main urine metabolite of plasma melatonin. this metabolite is considered to be a reliable measure of circulating melatonin (39). urine creatinine was also measured using the jaffe method (40). while the measurement of amt6 is an adequate alternative to plasma melatonin measurements, and less invasive, the only disadvantage to the procedure is the uncertainty of whether the entire urine fraction has been collected (41). when animals drink greater than average volume, the amount of fluid filtered increases in the kidneys and the amount of urine concentration decreases. in this study, the interest is the amount of circulating melatonin in the blood, as measured indirectly by a urine metabolite. in the case of an animal that drinks more than average, the amt6 per ml of urine will appear low due to the large dilution. creatinine is diluted the same way but is known to be excreted at a constant rate, filtered and excreted in the urine with no diurnal rhythm (41). therefore, it is possible to relate the concentration of amt6 to the concentration of a known, evenly excreted substance and report the melatonin in units of creatinine, as done here. after urine was collected from each pair (one each from exposed and sham conditions), the animals were placed into the sleep facility for subsequent analysis of sleep architecture. the physiological psychology laboratory, which houses the sleep research center, has facilities to analyze sleep on two animals at a time. the group of animals exposed (n=9) was similar to that of the sham exposed (n=8). unequal numbers resulted from a sham exposed animal that could not be recorded due to the head - piece coming dislodged from the skull. the rats were placed in the animal sleep room immediately following removal from exposure where total sleep time during night and day, waking time, low - voltage sleep, high - voltage sleep, and paradoxical sleep (rem sleep) were analyzed over a 24-hours period. sleep tracings were amplified by a grass polysomnograph model 78d and interfaced with a power mac g4. electroencephalogical tracings were scored by the first author according to the method described by rechtschaffen (38). the design of the experiment is outlined below : surgically implant cortical electrodes in 18 animals.one week recovery time.one week adaptation time in sleep chamber.one month continuous exposure at 1000 mg in exposure facility ; half the animals sham exposed.urine collection for last 3 days of exposure.sleep analysis in sleep chamber upon removal from exposure apparatus. surgically implant cortical electrodes in 18 animals. one month continuous exposure at 1000 mg in exposure facility ; half the animals sham exposed. urine collection for last 3 days of exposure. male sprague - dawley rats (n=18 : exposed n=9 ; sham exposed n=9) were individually housed before and during experimental protocols. all protocols were approved by the saint louis university animal care and use committee. during the experiment, animals were between the ages of 4 and 7 months, age matched between groups. the rats were exposed at a stable temperature (2324c) and were maintained in a 12 h light / dark cycle ; lights on at 7 a.m. food and water were available ad lib. two identical exposure structures were used (one charged, the other not) located in a 104 m room with masonry walls and 4 m high ceiling. the large room allowed for the exposure and sham exposure group to be in the same room. the exposure apparatus was designed and constructed with the assistance from the parks college of engineering at saint louis university. two sonotube reinforced cardboard tubes 8 ft long and 2 ft in diameter served as framework for the exposure structures. one tube was wrapped with 18 gauge insulated copper wire ; the other was not wound and served as the sham exposure environment. the exposure apparatus was energized by a standard 110 v, 60 hz outlet and a step - down transformer (a variac) providing the necessary voltage of 8 v that excited the wire from a three - phase, four - wire supply to create a horizontal, linearly polarized 1000 milligauss (or 1 gauss or 1.010 t) magnetic field within the exposure volume. the magnetic field in the sham exposure sonotube was less than 1 mg and was 7 m away from the exposure. the animals were singly housed with the cages being constructed of translucent polypropylene 1298, fitted with 2 high pvc filters as covers. before exposure, five electrodes for sleep recording were implanted according to standard procedures (38). these five placements, in a pin - plug assembly, permitted the fastening of stainless steel jeweler screws (080 by 1/8 in.) that allowed for ecog recordings from the parietal - frontal and the parietal - occipital regions of the brain. the pin plug was then permanently attached to the skull using dental cement, forming a mound that permanently seals the skull, electrodes, and plug as a single unit. post operatively, the animal received post - operative injections of 0.1 mg / kg buprenorphine twice daily for 3 days. after surgery, the animals were allowed to recover for 1 week at which time two animals were placed in the sleep lab for another week for acclimation. the animals were continually maintained on a 1212 hours schedule throughout the experiment and were staggered in twos as they entered the exposure room. at the end of acclimation, the animals were placed into the exposure apparatus, which involved being placed into the plastic cages described above with one rat in the energized tube, and the other in the sham non - energized tube. exposure was 24 hours a day with the exception of approximately 10 min each day for handling and care of the animals when the coils were deactivated. animals were staggered into the acclimation room and exposure facilities two at a time each week until no more than four animals were in each tube at once. field measurements ensured a uniform exposure for all rats in the energized tube. on the final 3 days of exposure (day 28, 29, 30), urine was collected every 6 hours from the basin of the cage at the end and middle of the dark period and at the end and middle of the light period. standard metallic metabolic cages could not be used due to the configuration of the apparatus and due to possible eddy currents being generated by the materials of typical metabolic cages. to collect the urine during the 3 days of collection, the cages were fitted with a porous plastic grid affixed approximately one inch from the basin of the cage, this allowed for urine to pass freely to the basin while the grid contained any expelled feces. the collection period for 3 days the urine, after collection, was centrifuged and the supernatant stored at -80c until all animals urine had been collected. separately, the dark and light samples for each rat were then pooled and volume measured. analysis of the urine involved an elisa kit (alpco, dunham, nh) for 6-sulphatoxymelatonin (amt6), the main urine metabolite of plasma melatonin. this metabolite is considered to be a reliable measure of circulating melatonin (39). urine creatinine was also measured using the jaffe method (40). while the measurement of amt6 is an adequate alternative to plasma melatonin measurements, and less invasive, the only disadvantage to the procedure is the uncertainty of whether the entire urine fraction has been collected (41). when animals drink greater than average volume, the amount of fluid filtered increases in the kidneys and the amount of urine concentration decreases. in this study, the interest is the amount of circulating melatonin in the blood, as measured indirectly by a urine metabolite. in the case of an animal that drinks more than average, the amt6 per ml of urine will appear low due to the large dilution creatinine is diluted the same way but is known to be excreted at a constant rate, filtered and excreted in the urine with no diurnal rhythm (41). therefore, it is possible to relate the concentration of amt6 to the concentration of a known, evenly excreted substance and report the melatonin in units of creatinine, as done here. after urine was collected from each pair (one each from exposed and sham conditions), the animals were placed into the sleep facility for subsequent analysis of sleep architecture. the physiological psychology laboratory, which houses the sleep research center, has facilities to analyze sleep on two animals at a time. the group of animals exposed (n=9) was similar to that of the sham exposed (n=8). unequal numbers resulted from a sham exposed animal that could not be recorded due to the head - piece coming dislodged from the skull. the rats were placed in the animal sleep room immediately following removal from exposure where total sleep time during night and day, waking time, low - voltage sleep, high - voltage sleep, and paradoxical sleep (rem sleep) were analyzed over a 24-hours period. sleep tracings were amplified by a grass polysomnograph model 78d and interfaced with a power mac g4. electroencephalogical tracings were scored by the first author according to the method described by rechtschaffen (38). the design of the experiment is outlined below : surgically implant cortical electrodes in 18 animals.one week recovery time.one week adaptation time in sleep chamber.one month continuous exposure at 1000 mg in exposure facility ; half the animals sham exposed.urine collection for last 3 days of exposure.sleep analysis in sleep chamber upon removal from exposure apparatus. one month continuous exposure at 1000 mg in exposure facility ; half the animals sham exposed. independent t - tests were performed to analyze differences among the two groups of animals on the dependent variable of 6-sulphatoxymelatonin per unit of creatinine. all creatinine levels were converted to mg / ml and divided by the concentration of amt6s that was expressed in ng / ml. the end results were expressed as 6-sulphatoxymelatonin per mg of creatinine. total sleep time during dark, total sleep time during light, low - voltage sleep, high - voltage sleep, and paradoxical sleep were all analyzed by mann - whitney u non - parametric test since the levine 's test for homogeneity of variance demonstrated a non - normal distribution of percentages within each variable. the amt6s production of rats during the dark period was marginally significantly increased (t=1.75, p=0.05 [one - tailed ]) in the exposed animals as compared to the sham exposed. light - time levels of both groups did not demonstrate a significant difference as shown in fig. 2. sleep architecture during the dark was not significantly altered from control animals in all stages examined (fig. paradoxical sleep was slightly decreased in the exposed animal but was not statistically significant (u=24.5, p=0.18). sleep during light - time hours showed similar features as dark - time (fig. 4). there were continued slight paradoxical sleep differences during sleep in the dark. the primary goal of this project was to examine the effects of continuous 60 hz magnetic fields on the production of melatonin and on the sleep architecture of rats. the results here do not support the notion that magnetic fields reduce melatonin production as suggested by the melatonin hypothesis. further, this study demonstrated a magnetic field that induced a marginally statistically significant increase in dark 6-sulphatoxymelatonin (amt6s) levels. subsequent sleep analysis indicates there is little effect of a 1000 mg magnetic field on the sleep architecture of rats, at least not within the first day after 1 month 's continuous exposure. the animal literature is inconsistent with respect to the effects of magnetic fields on melatonin levels. inhibitory effects of magnetic fields on melatonin production covering a variety of exposure paradigms have been found (19, 4247). there is also a preponderance of negative results (29, 31, 32, 35, 44, 48, 49). this report demonstrated a mild stimulatory effect, which has also been reported (36, 50). (36) administered a 300 mg field as an independent variable to test extinction of a conditioned reaction in rats. they found that in the presence of the field, the animals had an increased retention of conditioned reactions and, more relevant to this study, an increase in serum melatonin. the findings were significant only during illumination as the differences at night, while still in the direction of increased melatonin in exposed rats, were not significant. the experimenters did not speculate as to why the melatonin results were different from the melatonin hypothesis. another study that reported an increase in melatonin from magnetic fields was after a 24 hours exposure to a 1000 mg field (50). this experiment demonstrated no differences in 6-sulphatoxymelatonin during the period of exposure but did show a statistically significant increase in the melatonin metabolite the day following exposure as compared to a baseline measurement. similarly, an epidemiological study found railway workers had no differences in 6-hydroxymelatonin sulfate (another metabolite found in urine) during exposure, but levels increased briefly after the work ceased (51). a study performed on a non - mammal also found increases in melatonin after exposure to magnetic fields (52). using a teleost fish (brook trout), results demonstrated a more than two - fold increase in serum melatonin in the exposed fish. first, the pineal glands may have been directly affected by an increase in calcium efflux into pineal photoreceptors, thereby increasing pinealocyte production of melatonin. or, results may be due to the well documented sensitivity of fish to electric fields that may have caused a stress response in the animals leading to the melatonin increase. a well - controlled laboratory study using a longitudinal design and using very high field strengths (52 g) also provides evidence for a possible compensatory response. using adult male rats, animals were exposed for 1, 3, 7, 15, or 21 days for 30 min each day. using blood melatonin as an end point, a depressed blood melatonin was found after 15 days, but the difference disappeared after 21 days (53). in another study that lasted 60 days examined electric blanket use of men and women, melatonin levels were measured indirectly using 6-hydroxymelatonin sulfate. about one - quarter of subjects in this experiment demonstrated lowered melatonin levels when blanket use first began and again at the completion of the study, but not when urine was sampled during the middle (30 days into experiment) of the study (18). this may again provide further evidence to an adaptive response in at least some individuals after continuous exposure protocols but possible dysfunction in pineal gland activity in an unpredictably altered magnetic field environment, either during onset or offset of exposure. interestingly, upon further inspection of the numerous articles where no statistically significant effect was reported on melatonin production, at least one study found melatonin levels that varied not only as a function of how long exposure lasted but also as a function of time of collection (31). female sprague - dawley rats were exposed to 1000 mg for 2, 4, 8, and 13 weeks of exposure. after 2 weeks exposure, animals had an increase by 20% in melatonin 5 hours after lights out but a 40% decrease 6 hours after lights out, a significant difference. subsequent measurements at 4, 8, and 13 weeks of continuous exposure found no significant differences between sham exposed and exposed animals blood melatonin levels at either 5 or 6 hours after darkness. however, as time passed, there seemed to be a trend toward an increase in melatonin in the exposed group, albeit a statistically non - significant difference. 31) determined that a 1000 mg field had no suppressive effect on the production of melatonin. this was stated even though at 2 weeks a significant decrease in melatonin was found 6 hours into the dark cycle. the authors suggest the effect did not last into the latter portions of the experiment due to an adaptation to the presence of the field. when attempting to replicate the findings at 2 weeks and compare to the effects of 1 week exposure and a 1 day exposure, loscher. in fact, they found moderate increases 6 hours into the dark cycle in melatonin in the exposed animals after 1 and 2 weeks. in addition to a possible adaptation or compensation phenomenon, loscher 's lab indicated a possible phase shift of melatonin determined by assaying samples collected at different times during the nocturnal cycle. in our laboratory, no hourly measures were made of the amt6s but urine samples were instead pooled for a single night and day measure. the urine of our rats was collected four times a day (in the middle and end of light and dark periods) because we supposed that from a pathophysiological view, the total nocturnal melatonin production is more significant than the phase shift or peak amplitude of the nocturnal melatonin profile. an hourly collection may have provided additional insights as to whether our results reflect an adaptation phenomenon or a phase - shift in the normal melatonin rhythm. our melatonin data seems to indicate that at our field strength, 30 days of exposure may slightly increase the pineal production of melatonin as measured by urine collected over the entire dark period in nine rats as compared to nine sham exposed animals. there are studies examining intracellular free calcium (ca) as a function of magnetic field exposure. this discrepancy may explain why some studies find an increase in serum melatonin and some a decrease. if ca efflux is reduced at the level of the pineal, it seems reasonable that the subsequent increase in intracellular calcium could increase melatonin production. using the same logic, if there is less free calcium in the pinealocyte, less melatonin would be released. no study has been found that examined the sleep in the rat after continuous exposure to a magnetic field. (15) examined human sleep after an acute exposure of a relatively weak field (10 mg). in their experiment, human subjects were exposed for one night and sleep was analyzed during the exposure period. they found significant decreases in slow - wave sleep, paradoxical sleep, and subjective measures of sleep quality as compared to non - exposed controls. in our study, animals were not exposed per se during the sleep analysis. we removed them after 30 days of exposure to a much higher field strength (1000 mg) and immediately placed them in the sleep room for analysis of sleep for 24 hours. notwithstanding a measurable increase in melatonin in exposed rats, no significant differences were found in any portion of the animal sleep structure as compared to sham exposed. melatonin has been established to have soporific actions in humans (62), but the evidence in the nocturnal rodent is less clear. in the rat, both an increase in sleep (56) and a decrease (57) have been reported. in one study, rats were injected with a moderate dose (3 mg / kg) of melatonin at dark onset and found that melatonin briefly inhibited paradoxical sleep, but found no overall hypnotic effects of the melatonin group (58). it should be noted that in our study, we also found slight decreases in paradoxical sleep in the exposed animals with increased melatonin (p=0.18). overall, in a nocturnal animal, melatonin levels are likely to induce changes that are typical for the dark period of the species. in the nocturnal rat, it should not be surprising that we found a slight decrease in paradoxical sleep in the exposed animals when melatonin levels were higher. we did find mild, non - significant changes in slow wave and in paradoxical sleep in the exposed animals. but, overall, we can conclude that a 1-month exposure to a field strength of 1000 mg at 60 hz does not seem to significantly affect the sleep of rats upon removal from the exposure. while we intended to focus on the power frequency spectrum (e.g., 60 hz), it is worth noting that there have been experiments utilizing frequencies such as in the cellular phone range that have also impacted serum melatonin levels in rats. (59) found that a 900 mhz microwave level field induced oxidative changes in the hippocampus which was interestingly modulated by melatonin administration. meanwhile, koyu. (60) demonstrated no effects on serum melatonin levels from either a 900 mhz or 1800 mhz field. concerns about magnetic fields across many frequencies are a confusing biomedical and public health controversy. the research is far from being unequivocal in the search for a true melatonin theory of emf exposure. many studies performed in this area are of sound scientific methodology, but when all literature is examined, there is considerable inconsistency and even abject contradiction. the problem is that we need to determine what biologically significant exposure really is, and the identification of a true possible mechanism of action would be useful. in the laboratory, more replication of interesting results and confirmations of possible associations that have been found in field studies even if the epidemiological findings in this area that spawn laboratory hypotheses are deemed spurious, further low - cost exposure studies may have an important impact on our society as additional magnetic fields are likely to be encountered in the environment. this project was funded by an internal grant from the graduate school of saint louis university. | backgroundstudies investigating the effect of power frequency (5060 hz) electromagnetic fields (emf) on melatonin synthesis in rats have been inconsistent with several showing suppression of melatonin synthesis, others showing no effect and a few actually demonstrating small increases. scant research has focused on the ensuing sleep patterns of emf exposed rats. the present study was designed to examine the effects of extremely low power frequency electromagnetic fields (emf) on the production of melatonin and the subsequent sleep structure in rats.methodseighteen male sprague - dawley rats were exposed to a 1000 milligauss (mg) magnetic field for 1 month. urine was collected for the final 3 days of the exposure period for analysis of 6-sulphatoxymelatonin, the major catabolic product of melatonin found in urine. subsequent sleep was analyzed over a 24-hour period.resultsmelatonin production was mildly increased in exposed animals. although there were no statistically significant changes in sleep structure, exposed animals showed slight decreases in rem (rapid eye movement) sleep as compared to sham (non - exposed) animals.conclusionspower frequency magnetic fields induced a marginally statistically significant increase in melatonin levels in exposed rats compared to control. subsequent sleep analysis indicated little effect on the sleep architecture of rats, at least not within the first day after 1 month 's continuous exposure. varying results in the literature are discussed and future research suggested. |
knowledge of the anatomical variations of the axillary region has become more relevant with increasing surgeries of this region for breast cancer, reconstruction procedures and axillary by - pass. lager 's arch or the axillary arch (aa) is the best - known variant structure in the axilla. it is a muscular or fibromuscular slip of varying dimensions, extending from the latissimus dorsi (ld) muscle about the middle of the posterior axillary fold, and crosses over the neurovascular structures, to join the under surface of the tendon of the pectoralis major, the coracobrachialis, or the fascia over the bicepsbrachii [1, 2 ]. a 60-year - old female presented to our breast clinic with a left breast lump sized 3 2.5 cm incidentally felt since 3 months. it was gradually increasing in size and was not associated with any other breast complaints. she was evaluated with mammography that suggested a birads 3 lesion at 12 oclock position. subsequently, her metastatic work - up included abdominal pelvic ultrasound, chest computed tomography and bone scan. intra - operatively, during the axillary lymph node dissection, we encountered an unusual muscle slip crossing the axilla from the ld muscle to the posterior surface of the pectoralis major muscle anterior to the axillary vein. all neurovascular structures and lymphoid tissue were lying posterior to this abnormal muscle (fig. 1). figure 1:aa muscle extending from ld muscle to under surface of pectoralis major tendon in the left axilla crossing over axillary neurovascular structures (in view). aa, axillary arch ; pm, pectoralis major ; ld, latissimus dorsi muscle ; av, axillary vein ; tnb, thoracodorsal neurovascular bundle ; ltn, long thoracic nerve. aa muscle extending from ld muscle to under surface of pectoralis major tendon in the left axilla crossing over axillary neurovascular structures (in view). aa, axillary arch ; pm, pectoralis major ; ld, latissimus dorsi muscle ; av, axillary vein ; tnb, thoracodorsal neurovascular bundle ; ltn, long thoracic nerve. the lymph nodes lateral and beneath the arch were successfully dissected, and the arch itself was left undisturbed. the procedure was uneventful, and the patient had a good post - operative recovery. on follow - up, 21 months after surgery, the patient was alive and free of known disease. however, it was karl langer in 1846 who gave a more accurate description of this variant, so thereafter, it was named after him [24 ]. throughout the literature, several terms have been used to describe the muscular variant running from the ld muscle towards the pectoralis major muscle : aa muscle, axillopectoral muscle and also their translations in different languages. in the following text, the anatomical description of the aa is variable among authors. according to testu 's classification (1884), the complete aa extended between the ld and the tendon of the pectoralis major near its insertion on the humerus ; the incomplete one extended from the ld to the axillary fascia, biceps brachii muscle, coracobrachialis muscle, the distal end of the bicipital groove and the inferior edge of pectoralis minor muscle or coracoid process. the case we encountered was of unilateral complete aa. according to many anatomic texts, however, it has been recognized in only 0.25% during axillary surgical procedures [1, 2, 4, 5 ]. the difference in surgical and anatomical incidence reflects a failure of reporting or identification during surgery. also, this discrepancy may be attributed to the specific aim of cadaveric studies to identify such anatomic anomalies. this should be kept in mind as it can be confused with axillary lymphadenopathy or soft tissue tumour [1, 3 ]. aa can act as entrapment site for the neurovascular bundle during some arm movements causing circulatory deficiency, chronic pain and paraesthesia. simple division of the arch is curative in such cases [2, 5 ]. jelev, through his extensive work, introduced a new definition of clinical aa as a variant muscular structure in the axilla that is a possible entrapment site for the nerves and vessels., the existence of a superficial or deep aa could be suspected according to the vessels or nerves mostly affected. it can positively correlate its presence with neurovascular entrapment symptoms. also, it can assess its anatomic relations [1, 5 ]. the surgical significance of the aa is 2-fold : (i) it may hide some axillary nodes, and (ii) it may mislead the dissection into a supra axillary plane. a group of lateral axillary nodes may be concealed under the aa while crossing over the axillary vein. missing these nodes during axillary node dissection imposes a risk for local recurrence in patients with breast cancer and melanoma. this also can lead to inaccurate staging, which in turn could negatively affect adjuvant and systemic therapy decisions for breast cancer. this may lead the surgeon to dissect in a plane above the axillary vein increasing the risk of injury to the axillary artery and brachial plexus. during sentinel node biopsy, the aa can pose difficulty as it stretches in the hyper abducted position shifting the nodes higher. in order to clearly identify, the anatomic landmarks the arch can be divided at the level of the axillary vein. furthermore, some authors suggest division of the arch in all cases to prevent possible post - operative axillary vein compression and associated lymphoedema [3, 4 ]. in our case, there was no added morbidity related to dissection of nodes beneath and lateral to the arch. the presence of the aa may precipitate lymphoedema in cases where ld myocutaneous flap is used for breast reconstruction. for this reason, the aa should be divided if there is a possibility of a ld flap being required in the future. as in our case, most reported clinical cases describing the aa have been identified during axillary surgical procedures [2, 4 ]. it can cause confusion during routine axillary surgery for breast cancer, which can both affect procedure safety and misguide further treatment decisions [3, 4 ]. | langer 's arch is the best - known anatomic variant of definite surgical implication in the region of the axilla. this rare anomaly is a muscular slip extending from the latissimus dorsi (ld) muscle to the tendons, muscles or fasciae around the superior part of the humerus. in this report, we present a rare case of left axillary arch. during modified radical mastectomy for breast cancer, we encountered an abnormal muscle slip crossing the axilla from the ld muscle to the posterior surface of the pectoralis major muscle anterior to the neurovascular structures. preoperative knowledge is essential to identify such unusual anomaly and avoid potential complications both intra- and postoperatively. |
a substantial body of research reports of illness related distress and its impact on quality of life for the person living with illness as well as for their significant others (i.e., spouses and family caregivers) is available. there are, however, few studies that focus on the family system and describe illness experiences from the perspective of the family as a unit. these studies show that living with illness is experienced as family vulnerability, helplessness, strain, and suffering, as a struggle to make sense and maintain normality in family living and as bringing about difficulties in family relationships. the results from the above reported system focused studies point to a need to expand the focus for care in order to support families living with illness and also to broaden the concept of health to embrace the family as a unit, that is, family health. family health has been described as an interactional, holistic, and dynamic phenomenon positing circular causality and comprising biological, psychological, spiritual, sociological, and cultural aspects of well - being both on an individual and family level. the theoretical ground for this understanding is system theory which focuses on interactions among the various parts of a system and the system as a whole. one example of this systemic view is newman 's theory of health as expanding consciousness in which individuals are seen as open systems that constantly interact with their environment. one family member 's illness is reflected in the pattern of family interaction and when these patterns are made conscious, health will be gained. although research findings and system theories suggest that chronic illness has an impact on the family as a unit, the health care system is typically patient focused and family systems needs are notably unmet. recently published reviews of family interventions [814 ] show that interventions provided by health care often are characterized as psychoeducational versus relationship focused. additionally, interventions are mostly directed towards the individual family member or towards the partner dyad. outcomes targeting individual outcomes are domineering. responding to families ' experiences of distress and the lack of systems oriented support from health care, the majority of family systems nursing interventions are grounded in the so - called calgary models, calgary family assessment model and calgary family intervention model, and in the illness belief model. the aim of these interventions is to support family health by creating a context for change. this context is facilitated in the interactions between the participants by assessing the families ' situations and intervening by posing reflective questions targeting the problems experienced by the families. changes that are hoped for concern the families ' ways of thinking and responding in relation to the illness situation and to those problems experienced, having impact on their well - being. this may embrace change or modification of constraining beliefs and strengthening of facilitating beliefs, of finding alternative ways of talking about the family situation and the discovery of new meanings [15, 16 ]. a small but growing body of qualitatively designed outcome studies focusing on families ' experiences points at these interventions to be a healing experience, improving family relationships [18, 19 ], alleviating experiences of suffering [20, 21 ], and psychologically empowering [22, 23 ]. for the development of family systems support with the purpose to improve and promote family health when living with illness, knowledge about the nature of the intervention processes and understanding of the potent working mechanisms are central. most of the interventions are theoretically grounded but have not been evaluated from how they are practiced. evaluations that focus on the processes that actually take place can contribute with knowledge of the working mechanisms in the intervention. this type of knowledge is a prerequisite for successful refinement and implementation in health care. therefore, the aim of this study was to describe the dialogue process and possible working mechanisms of the family health conversation (famhc). what dialogue events may be identified and how are they interrelated ? what characterizes the interactions between the participants ? an evaluative approach aiming at a description of the intervention process and the working mechanisms of this process was chosen. data consisting of transcripts from the interventions were analyzed within a hermeneutic tradition that emphasize the necessity to use ones pre - understanding in the interpretation process but still remain open to the phenomenon at hand. openness is supported by not using theories in this phase of the analysis and also by getting into a dialogue with the text in order to reach a first understanding. this understanding should be critically examined in relation to the text throughout the analysis [2527 ]. by going into the hermeneutical spiral, where the parts are considered in relation to the whole and vice versa, the interpretations can be validated so that the best possible interpretation is reached [26, 27 ], an understanding that is grounded in a fusion of horizons. to gain a deeper understanding of the phenomenon, the findings could be further reflected on in relation to theories or philosophical reasoning [26, 27 ]. the family health conversation model was developed by the last author and her colleagues at the linnaeus university in sweden during the late 1990s. the model is inspired by the calgary family assessment and intervention models and particularly the illness beliefs model. the conversation model builds on system oriented theories and models [6, 15, 28 ], change theory, and narration and reflection theory [26, 30, 31 ]. the model is used by the research team in clinical practice and in research [17, 18 ]. the structure of the model is three conversations, a closing letter, and an evaluative follow - up interview. the three conversations have somewhat different intentions. the first conversation with each family started with a discussion of the aims and of the family members ' expectations of how the conversations possibly could support them. then, each family member was invited to tell their story about how they experienced the family 's situation. based on these stories, the family and the two conversational leaders together agreed what to talk about and what changes might be desirable and possible for the families. the intention in the second conversation was to progress further towards minimizing the family 's suffering by illuminating family members ' beliefs and by strengthening facilitating beliefs and modifying constraining beliefs. the intention of the third conversation was much the same as the second but also included a termination of the series of meetings and a reflection of the process of change that had occurred. data for this study consisted of 15 transcribed famhcs with five families that had participated in a series of three conversations. a purposive sampling strategy was used to get a sample of families that varied according to chronic illness, time since onset, sex, and age of patients and family members. the five families that were included had all participated in famhc either at our campus - based center for research on families ' health (n = 3) or in their own homes (n = 2). they were either self - referred or recruited from a rehabilitation clinic at the local hospital (table 1). four nurses from the research team, of whom one is the second author of this paper, participated in three different pair constellations of conversational leaders. all nurses had education at advanced level and several years of experience with this type of conversations. this study was approved by the research ethics committee at linkping university, sweden (reference number 2010/51 - 31) and was conducted in accordance with the declaration of helsinki (1964). participants gave their written consent for the tape - recorded conversations to be used for research purposes. the analysis comprised two phases in relation to the use of theories : one inductive phase and one deductive phase. in the inductive phase, dialogue events were described and explored by the first author (carina persson) who was not yet familiar with the intervention model or its central assumptions and theories and thereby data could speak for itself. the transcribed conversations were read, one conversation at a time, and text segments focusing on dialogue events were identified. the identified text segments were reread focusing on the question : what are the characteristics of these dialogue events ? one further reading was done to identify sequence patterns within and between the dialogue events guided by the question : what is preceding and what follows ? the author strived to become involved in a hermeneutical dialogue [25, 26 ], that is, asking new questions to the text, gaining new understanding, and asking new questions, is it really so ? (see table 2 for one example of the analysis ' process). in the deductive phase, we wanted to further understand how the events and sequence patterns may support family health from a theoretical perspective. the results of the inductive analysis were reflected on using literature embracing health theory, systems intervention models, and philosophic literature highlighting phenomenon / concepts with meaning for health. overall, we found that family members narrated and explored the families ' concerns in interaction with the conversational leaders. the participants ' verbal actions were also analyzed separately, but it was obvious that a reciprocal pattern of actions - responses took place that generated the movement between the two dialogue events (see table 3). narrating episodes were characterized by their descriptive constituent and comprised the families ' experiences of living with illness. family members ' reactions, behaviors, and feelings in various daily situations and consequences of these were in focus. one example of this is illustrated in table 3 where the couple narrates their experiences of the woman 's difficulties getting ready in time due to her illness. the partner supports the woman 's narration by adding his perspective and his explanation of why she does not manage : she finds out other things to do and is not focused on what has to be done. at the end of this event the woman acknowledges the husband 's belief that she should focus on one thing at a time and accept her situation. earlier and also later in the conversation it is clear that the couple are distressed and that the issue has grown into a conflict between them. not being able to do things together has also contributed to having less in common and a sense of living their lives separate from each other. families constructed a collective narrative where family members took turns and acted as the primary or the primary narrator initiated and took the main responsibility for the narration while the secondary narrator spontaneously commented, added to, confirmed, or questioned the other 's story. these functions were evident and changed between all of the participating family members and were related to the issues brought up during the conversations. table 3 illustrates how the woman acts as a primary narrator brings up the problematic issue and takes responsibility to move on. her partner acts as the second narrator, adding his viewpoint and confirming her difficulties. in the conversations the primary narrator also took the opportunity to invite other family members to contribute to the narrative and sometimes narrate on behalf of the primary narrator. the latter occurred when the primary narrator found it too emotionally upsetting to narrate, or when he / she had memory or communication problems. one example of this is the following citation from one of the families where the woman finds it too upsetting to tell herself.woman with illness : i 've become rather tired the last couple of months. it 's been like a trauma between me and my son 's wife. she has forbidden me to meet my granddaughter who has turned one and a half. (turns to her partner), i ca n't go through it, i ca n't tell, i wo n't tell, no (starts to cry). woman with illness : i 've become rather tired the last couple of months. it 's been like a trauma between me and my son 's wife. she has forbidden me to meet my granddaughter who has turned one and a half. (turns to her partner), i ca n't go through it, i ca n't tell, i wo n't tell, no (starts to cry). the narrating episodes were initiated by an invitation from conversational leaders and only rarely by another family member (see table 3 for one example). in the initiating phase, mainly open questions were asked (how do you think then ? in what way ?). statement questions if the family member 's response was brief, the conversational leaders asked follow - up questions, made short one - syllable comments, or asked clarifying questions. if a narrator had contradictory experiences or opinions, a discussion took place before the narration was resumed or preceded into an exploring episode. we found that the exploring episodes comprised the family members ' reconsiderations and reflections on the situations in the narrating episodes. the participants examined the nature of the situations and discussed possible explanations. from the illustration in table 3, it is evident that the woman reconsiders the exploring question from the conversational leader that connects the previously narrated problematic issue to the belief that despite the illness, she expects herself to act as before the onset. the family members ' various reactions, behaviors, and beliefs were made visible and were also reconsidered in relation to those of other family members and to their impact on family relations. family members cocreated alternative explanations to why problematic situations occurred and reconsidered their own options to influence and take control over certain situations. family members ' understandings of various situations were reconsidered and sometimes altered, resulting in the development of a new communal understanding of the families ' experiences. in table 3 this is illustrated when the woman expresses a different interpretation of how the problematic issue may be understood. she concludes that you can not expect to be as before and that she should accept that her illness limits her ability. her partner can also see that this belief contributes to distress and brings about physical symptoms. the couple expresses a renewed and shared understanding of how the problematic issue may be understood., the conversational leaders took the initiative and facilitated exploration and family members responded by means of reconsideration and reflection. the conversational leaders asked questions, for example, when a problematic situation had been narrated or when different experiences or beliefs between family members were discerned., analyzing questions, or connecting questions (see table 4). we also found that follow - up questions were frequently asked in the exploring episodes. the conversational leader initiated the exploring episode by posing a comparative question has it always been like this ? and goes on by posing other comparative questions related to what has previously been narrated by the family members. finally the conversational leader a poses an interpretive question yes, do you demand too much of yourself ? the conversational leaders also offered the family the opportunity to listen to their reflections. this was most prominent when a family member had difficulties expressing him-/herself, for example, in cases of memory or expressive dysfunctions and when younger children participated in the conversations. to reach a possible understanding of how the conversations may support family health, we found it useful to consider newman 's theory of health as expanding consciousness, where health is seen as a synthesized phenomenon constituted by disease and nondisease. further, health is seen as the larger whole and disease and nondisease as reflections of this larger whole. from this viewpoint, it appeared to us that the working mechanisms of the famhcs may be understood as facilitating a spiral movement towards placing family nondisease (i.e., family health) in the foreground. this movement was driven by the verbal interactions between family members and the conversational leaders. it facilitated families ' narration and exploration of their experiences, a process in which family members developed an increased understanding of themselves and others and of their interactional patterns. newman 's health theory may also help us understand the importance of this increased understanding in relation to health. she defines nondisease and disease as explicit manifestations of the individual 's underlying interactional pattern embracing the individual 's interactions with the environment and recognizes a movement toward nondisease as a movement towards an expanded consciousness of this underlying pattern. to further understand this interpretation of the conversations as supporting health, we focused on possible meanings of what could be considered as essential factors in the process, interactional narrating, listening, and reconsidering (see figure 1). the starting point of the conversations was the invitation to the family members to narrate their experiences of the family 's situation living with illness. narration has previously been linked to well - being from an individual perspective and may be understood as essential for self - identity and for the understanding of one 's experiences. according to ricoeur, narration contributes to the constitution of the self and mediates self - understanding. this is made possible through the connection between the plot (constitution of action) in the narrative and the identity of the character (constitution of the self), which is constructed in the narrative. in the famhcs, family members also cooperated in the construction of a collective narrative. from a dialogic perspective, participating in a reflective dialogue with others facilitates the constitution of an identity with the community. this opportunity is opened up in a context where participants share their feelings and are joined in a shared language. the narrating episodes and the construction of a collective narrative within the famhcs may thus be seen as a way to increase individual as well as family well - being by facilitating the constitution of a self and support identity - building within the family. the major difference of family system interventions compared to interventions directed towards one individual is the possibility to bring forth family members ' different perspectives. in the famhcs, this offered an opportunity to become aware of multiple ways of being in the situation and of alternative ways of interacting. from a systemic standpoint, information about differences makes a difference to the system and from a therapeutic point of view this is regarded as a ground for change and plausible solutions to problematic situations. according to the illness beliefs model family beliefs are assumed to be connected to suffering and healing, where some beliefs may be facilitating and others constraining in relation to family health. beliefs are challenged and refined in interaction with others and a dialogue context is seen as a powerful way for a change in beliefs to take place. the famhcs may have offered the families a context for improving family health by making various beliefs visible and by linking beliefs to family members ' different experiences, a process that may have facilitated changes in constraining beliefs. narrating and listening seemed essential for reconsidering experiences in the following process. reconsidering in a dialogic form may have offered an opportunity for families to find new options and develop meaning and hope. meaning - making has been regarded as a relational activity, where meaning is generated and transformed in the response and reresponse from different voices in a dialogue. we found that a new communal understanding of the family 's experiences was developed in the process of reconsidering. the meaning of this shared understanding could possibly be understood through marcel 's philosophy of hope [33, 34 ]. the development and experience of hope is closely linked to intersubjectivity and the establishment of a we, which in turn is grounded in the sharing of concrete, lived experiences [34, s.234 ]. in the famhcs, family members ' sharing of their experiences and reconsidering may have improved family health by facilitating meaning - making and development of hope. the families ' narrating, listening, and reconsidering were undertaken in interaction between the participants in the conversations. the conversational leaders ' interactions with the families were found to differ in the two episodes identified. this difference in interactional patterns may be understood in relation to therapeutic interviewing, where various types of questions are linked to their different intentions : explanation of problem and revealing of current patterns, or development of insight. building on the narrating episodes, conversational leaders and families interacted so that explanation and insight were developed in the exploring episodes. the findings in our analyses indicate that the famhcs have a theoretically grounded potential to facilitate a movement towards family health. this theoretically driven argument is supported in qualitative studies of family systems nursing interventions when evaluated by families living with chronic illness in various phases [17, 20, 23 ]. in an integrative review of family responses from participating in systems nursing interventions only a few studies were found indicating that families did not benefit compared to standard care. in another study, six families living with different cancer illnesses in palliative phase described moments during the conversations as being emotionally demanding, although the overall experience was that of a healing and comforting experience. the invitation and facilitation of families ' narrating were found to be a starting point for the conversations analyzed in this study. the invitation to tell the family illness story has previously been related to unburdening oneself and as a way for making sense of suffering and finding hope from a family perspective when living with cancer illness in palliative phase. in an interpretative research synthesis study with the aim to develop an understanding of how narratives may be a path to health, the analyses resulted in a model where narrative understanding in a caring conversation was seen as consisting of three phases. the first phase involved the patient telling their story, the second was about narration of the suffering experience, and, in the third phase, the narrative was reconnected to the patient 's life story. going through this process meant going from understanding to interpretation and finally to creation of meaning in and of suffering in connection with illness. the analyses showed that families coconstructed a collective narrative grounded in the individuals ' various experiences. in addition family members ' understandings of various situations were reconsidered and sometimes altered so that a new communal understanding of the families ' experiences was developed. cybernetic theories imply the importance of viewing individual family members ' behaviors as interactional in their nature since feedback is continually received from others. one family member 's actions will inevitably influence the behavior of the others and vice versa. this could also be expressed as circular causality where forces in the family move in a circular fashion, implying that it is meaningless to search for the cause of an interpersonal event. one qualitative study including 16 families living with hiv / aids showed that one response to family nursing systems interventions is an increased understanding of family dynamics which opens up for change and contributes to the families ' health experiences and individual well - being. the results of the analyses in this study have to be considered in relation to the characteristics of the participating families, the methods chosen, and the preunderstanding of the researchers. the included families varied according to family structure, age, type of chronic illness, time living with illness since onset, and reason for participating (i.e., self - referred or participating in a research project). these variables could have had an impact on the conversations ; however, no such differences could be distinguished in relation to what was brought up or to the participants ' interactions. in some of the conversations a pattern was more easily distinguished or more or less dominating although it could be found in all of the conversations. this could be seen as strengthening the findings but you have to bear in mind that focus for the study was not to detect differences across families but to see common patterns in the conversations. one might argue that five families is a small sample size and thereby might jeopardize the credibility of the study. however, this choice must be considered in relation to the process of obtaining in - depth knowledge. data was comprehensive and consisted of 15 conversations, each approximately one hour long, allowing for an analysis that brought forth greater understanding in a circular and reflexive process moving towards a validated interpretation. the inductive analyses built on the first author 's (carina persson) distinctions of the included conversations. according to bateson, we see certain things as distinctions from the background. at the time of the inductive phase of the analyses, the first author was not familiar with conversational analyses or the theories and models that the intervention model is based upon. this opened up for an inductive and an outsider perspective which also limited what could be seen. to minimize the risk that essential dialogue events and interactional patterns were overlooked, the findings were discussed with colleagues at the family focused unit at the university and with the second author (eva benzein) who has expert knowledge and is skilled in practicing the model. in an attempt to find the most possible understanding of data, the first author strived to become involved in a critical dialogue with the text by making assumptions and actively searching for alternative ways of understanding. in this process, the emphasis was on reaching an understanding characterized by a coherent relationship between the parts and the whole. however, it should be emphasized that the choice of the literature in the deductive phase of the analyses is critical for the understanding of the conversational events as being supportive for family health. the choice was guided by the inductively generated findings and also by the authors ' preunderstandings. the findings should therefore be considered as one possible way of understanding the potent working mechanisms inherent in the conversations. the results from this study offer a description of one model for family systems nursing interventions and, additionally, a theoretically grounded interpretation of how this intervention may support family health. the interpretation showed that narrating, listening, and reconsidering in interaction may be crucial parts in the model. this type of knowledge can hopefully contribute to the successful implementation of family systems interventions in education and clinical practice, with the aim to meet the overlooked needs in care of families experiencing illness. | research shows that living with illness can be a distressing experience for the family and may result in suffering and reduced health. to meet families ' needs, family systems intervention models are developed and employed in clinical contexts. for successful refinement and implementation it is important to understand how these models work. the aim of this study was therefore to describe the dialogue process and possible working mechanisms of one systems nursing intervention model, the family health conversation model. a descriptive evaluation design was applied and 15 transcribed conversations with five families were analyzed within a hermeneutic tradition. two types of interrelated dialogue events were identified : narrating and exploring. there was a flow between these events, a movement that was generated by the interaction between the participants. our theoretically grounded interpretation showed that narrating, listening, and reconsidering in interaction may be understood as supporting family health by offering the families the opportunity to constitute self - identity and identity within the family, increasing the families ' understanding of multiple ways of being and acting, to see new possibilities and to develop meaning and hope. results from this study may hopefully contribute to the successful implementation of family systems interventions in education and clinical praxis. |
the nerve damage is more common after brachial plexus injury coming up at birth or it follows nerve trunks injury that occurred for different causes.15 primarily, options for the nerve repair are either direct neurorrhaphy or interposition of nerve grafting according to the appearance and the gap of nerve stumps. the neurotization is another technique of nerve repairing that offers an additional support for nerve recovery, especially in brachial plexus reconstruction.67 the insertion of nerve conduits (tubulization) for bridging a nerve defect has been recently proposed by many authors, who have obtained interesting results, mainly during adulthood.8 the tubulization can be carried out with two different kind of structure, namely biological or synthetic conduits. among the biological conduits, vein graft represents a biological structure that, filled up with muscle or nerve inductors, connects the nerve stumps while among the synthetic tubes different materials have been proposed. during the years, many both reabsorbable and not reabsorbable (polylactic acid, collagen, silicon etc.) synthetic nerve conduits have been used.9 although several reports about nerve conduit substitution of sensitive nerve in the adult have been published, few papers about mixed nerve repair have been written.1011 moreover, despite the fact that bridging nerve defect is not a new technique, up to now few reports about the use of tubulization in children have been published, especially since the repair is mostly directed to mixed or motor nerves rather than the repair of sensitive nerve trunks. although previous experiences in children have already been published, except for obstetrical brachial plexus reconstruction.1213 to the best of our knowledge there are no literature reports on upper and lower limb nerve repair by means of tubulization in pediatric age. in 2006, the st. louis group reported their experience in the use of re absorbable collagen nerve guides for primary repair of seven obstetrical brachial plexus lesions, performing a retrospective study on the first five cases. they concluded that their results were particularly encouraging considering that all patients had severe plexus lesions and hence they suggested that collagen nerve guides could be considered as an alternative to autologous nerve graft, especially in selected patients with limited nerve gap to repair. in 2007, the norfolk group focused on the use of biological tubulization with autologous vein graft as a possible alternative technique for brachial plexus repair. these authors reported on a case of primary nerve repair and a case of late nerve surgery, using vein grafts filled with minced nerve tissue. those authors reported a good recovery and concluded that tubulization techniques should be taken into account when autologous nerve grafts are insufficient for the reconstruction of extensive lesions or as an alternative to grafting in case of short nerve gap. they also added that the excellent results of reconstruction with veins filled with minced nerve tissue suggest the need for further studies of this technique in developmental age. taking into account the possibility of using more established techniques of nerve repair, aim of this study is to present the authors experience of using nerve tubes for nerve repair during developmental age, providing a contribution to define indications and limits of the method in children notwithstanding the fact of dealing with nonhomogeneous case series of different nerve trunks. nine cases were affected by obstetrical brachial plexus palsy (obpp) and six by peripheral nerve injuries (pni) of upper and lower limbs. the right side was involved in 8 cases (4 obp and 4 pni) and the left side in 7 (5 obpp and 2 pni). according to the type of lesion, the time elapsed from the injury before nerve repair was logically different. with regard to obpp patients, the damage was at birth and the time elapsed at the moment of surgery was 5.4 1.50 months standard deviation (sd) (range 48 months), whereas it was 8.7 5.07 years sd (range 2.813.2 years) for pni patients, in which nerve injury had occurred in a period ranging from 6 h to 6 months before surgery. the surgical consent was taken from parents of all patients, explaining the technique and pointing out that the results of nerve surgery even with nerve grafts are unpredictable. nerve conduits chosen for nerve repair consisted of reabsorbable collagen matrix tubes (neuragen). in the obpp group (nine cases), the surgical exploration of the brachial plexus was always carried out through a supraclavicular approach. five cases were suffering from complete brachial plexus palsy, three cases had a triradicular involvement while only one case presented as a partial involvement of the superior roots. four patients had an implant of a single conduit, three patients had two conduits while two had multiple insertions (three conduits). after a primary exploration of the lesion, we checked that whether a rupture or an avulsion had occurred or not, we proceeded to brachial plexus reconstruction. after excision of neuroma, the nerve reconstruction was performed using a single or multiple nerve tubes for bridging the nerve gap in all the nine patients. furthermore besides nerve conduits, five of them had nerve repairing by means of different techniques of nerve surgery. more precisely conduits were used for conjoining a direct nerve loss (i.e. c5c6 upper primary trunk), whereas nerve grafting was used for bridging and neurotizing different roots and trunks [table 1 ]. clinical details of obpp patients loss of nerve substance was observed in different proportions for each case. the mean nerve gap was 2.47 0.65 cm sd (range 0.52.8) for obstetrical palsy patients, whereas we found a gap of 1.75 0.33 cm sd (range 1.42.5) for peripheral nerve lesions. regarding pni patients, we explored the injured nerve through the classical surgical approach to the specific nerve trunk. in selected cases, with a loss of nerve substance, which did not allow direct repair was observed, the reconstruction was performed using nerve conduits. equally the scheme of surgery is presented together with causes of injury and time elapsed from damage to surgery [table 2 ]. as different nerves and sites of injury had to be tested, we assessed nerve injuries with different methods in order to measure the outcome and to define good, fair and bad results. the assessment of brachial plexus injuries was classified according to mallet score for the shoulder, gilbert score for the elbow and raimondi score for the hand.141516 pni group, the causes of nerve injury, characteristics of the conduit inserted for reconnecting nerve stumps and results the total brachial plexus injuries were evaluated as good for mallet iv / gilbert 45/raimondi 45 values, fair for mallet iii / gilbert 23/raimondi 3 and bad for mallet ii / gilbert 01/raimondi 12. similarly, upper or three - radicular brachial plexus injuries were assessed as good for mallet iv / gilbert 45, fair for mallet iii / gilbert 23 and bad for mallet ii / gilbert 01, without considering raimondi 's hand classification. the results of sensory nerves repairing were assessed according the nerve injuries committee scale of the british medical research council, modified by mackinnon and dellon, while motor - sensory mixed nerves were evaluated by means of the sakellarides scale.1718 the sensitive nerve results were assessed as good for values of s4/s3 +, fair for (s3/s2 +) and bad for m4/s3 +, fair for > m3/s3s2 + and bad for < m2/s2. patients assessment and evaluation scales have been inserted in order to provide separately the results of brachial plexus palsy and pni. furthermore for each group the preoperative assessment, the operative planning and postoperative evaluation are shown in tables. the mean followup was 5.2 1.68 years sd (range 2.87.7 years) for obpp and 4.08 1.76 years sd (range 1.36.5 years) for pni. in obpp patients, we observed 1 good (11%), 3 fair (33%), and 5 bad (56%) results. in the pni group, we observed four patients (67%) who had good results while only two (33%) had a bad outcome. five obpp patients, having presented a bad outcome, were in need of a second surgery, as usually happens after a failure of brachial plexus reconstruction [table 3 ]. according to the recent trends of nerve repair in obpp, a late nerve surgery was performed. the scheme of reconstruction for primary and late nerve surgery in some patients during the reoperation, we checked the previous effects of surgery. particularly, in the suprascapular nerve neurotization by means of accessory spinal nerve, we explored a site close to the one where conduits had been applied during the first surgical procedure. in three patients out of five, who underwent that specific neurotization, we constantly found a thick fibrosis and a pale yellowish thinned incontinuity structure as expression of the loosening of suture tension at the distal stump [figure 1 ]. the electrical stimulation of this nerve structure, coming out of the presumable conduit remnant, did not demonstrate any distal muscle contraction. the arrow is showing the residual of nerve conduit, while the forceps is indicating the presence of useless regenerated nerve in pni group, bad outcome occurred in patients affected by sciatic nerve injury. one of the two patients who had undergone primary nerve surgery was lost at long term followup while the second patient underwent reoperation for nerve reconstruction with autologous sural nerve grafts. among reconstructive techniques, the tubulization might have technical advantages in the presence of wide nerve loss when conventional nerve grafts are insufficient to provide the filling of the defect. therefore, the additional supply offered by nerve conduits can enlarge the reconstruction, overcoming the scarcity of nerve grafts, especially in young children. in fact in small children, when the autologous nerve supply is poor, that is, in brachial plexus reconstruction, it is sometimes difficult to find veins with appropriate diameter to create biological nerve guides. in these cases moreover, another advantage is the possibility of the sparing of autologous structures, which avoids the morbidity of the donor site and scars. commonly neuroma resection and nerve grafts are considered the gold standard for nerve repair both in brachial plexus palsy and in pnis.1920 the sural nerve is the most common donor nerve2122 but one must be aware that anatomical variations of the nerve are often present. additionally the incision at the calf for harvesting the nerve represents a further unexpected lesion that parents are dimly prepared to accept, especially because skin incisions produce very often excessive scarring. biological tubulization has been rarely proposed in childhood, because of the scarcity of vein supply, feasible for bridging nerve defects. indeed, harvesting vein grafts of adequate caliber useful for tubulization is challenging in young children. so as, although tubulization obtained both with biological and synthetic structures is not a new proposal referring to the adult, a few reports have reported about using nerve conduits in pediatrics. moreover, nerve lesions in pediatric age group are mostly related to mixed nerve injuries, on this subject there is no complete agreement of management, even for adult patients. particularly brachial plexus reconstruction is difficult because the large number of nerve graftings are commonly required. brachial plexus injuries in the children are mostly dependent on perinatal lesion rather than traumatic damage caused during infancy. conversely pni of upper or lower limbs are more common in pediatric trauma occurring during growth.23 peripheral nerve lesions differ on the type and the localization of trauma : open injuries are immediately treated while in closed lesions a waiting attitude is mandatory in order to evaluate a possible spontaneous recovery. considering the problems of nerve supply in pediatrics and taking into account that tubulization is a well - accepted technique in the adult, we decided to use nerve conduits in order to find out a solution to repair the nerve gap in the child. most of the papers were related to the sensory nerve, but some articles were concerning mixed nerve repair in the adult, particularly in the upper limb.242526 during the years, many authors have studied alternative methods for filling nerve gap,2728 so as nerve tubulization is nowadays widely used for digital nerve reconstruction in adulthood.29 in this sense tubulization has become a validated procedure that is now proposed as a good alternative for the repair of small nerve defects (< 30 mm). nerve repair by means of conduits during developmental age has been sparsely reported, even if two groups of researchers have obtained satisfactory results by using nerve conduits for obstetrical brachial plexus reconstruction. they proposed different conduits, namely collagen nerve guides and vein grafts, having drawn the same conclusions, recommending the conduits as an alternative to nerve grafting. however, none of the two groups has outlined contraindications in using nerve conduits in pediatric age. we have decided using collagen reabsorbable matrix conduit (neuragen) on the basis that this structure consists of a material commonly used in medical practice (i.e. suture, devices etc.,) and because it has been already experienced by one of the two groups of researchers. comparing our results with those of the two reports, we observed a high rate of failure (56%) in using conduits in brachial plexus repair. more precisely, in our case series of nine obpp patients, five cases required a new surgical approach. giving more details, three of those (60%), who had the surgical repair in a site close to the previous surgery, showed that the presumable remnant of the conduit appeared thinned as if there were a loosening of suture tension at the distal stump. this useless regenerated nerve did not show any distal response after electrical stimulation. during the second surgery in all cases, we performed selective neurotizations targeting specific muscles, according to the late nerve surgery nowadays preferred. the presence of thick fibrous tissue in the site of conduit implant has explained the regeneration failure, even though, in brachial plexus repair, the main problem is the particular anatomy of nerve axons and their continuous reorientation that turns out failure even with the conventional nerve graft. furthermore, another reason of failure possibly has been the length of the gap because, the longer the gap, the higher the risk of failure for the depletion of nerve regeneration. even if we did not need a nerve tube of extra length, the length of nerve conduits might represent a limit to the application of the method, particularly for the limited possibility of adapting the nerve gap. it is also important to mention that even brachial plexus reconstruction by means of sural nerve graft sometimes shows unsatisfactory results. in fact regardless of the technique of the repair many variable parameters influence the outcome of brachial plexus reconstruction (i.e., extension and severity of the lesions). the small group of patients treated with tubes does not allow to draw conclusion but, in our opinion, the use of tubulization should not be considered as a first choice in brachial plexus reconstruction in children, especially for long defects, as experimentally demonstrated.30 the situation has been different in pnis repair, where positive outcomes were generally observed. since the main nerve trunks of upper limb and sensory nerves of the hand have demonstrated satisfactory responses, the reliability of using nerve tubes can be affirmed, both in terms of the quality of and the time of the recovery.31 conversely bad results occurred only in the lower limb, particularly in sciatic nerve repair, both at the thigh and at fibular head level. the reasons of these negative results have been probably the same as those causing the bad results in brachial plexus repair, namely the vast anatomical complexity of the sciatic nerve, which presents a continuous axonal reorientation (particularly in the thigh) and the long distance between the site of lesion and the corresponding muscular groups. in addition, the failure of mixed nerve is higher than the sensory nerve. nerve tubulization for repairing nerve lesions in developmental age (children) can be considered as a valid alternative to nerve grafts in pni, especially of the upper limb. conversely, repair by means of nerve conduits in great trunks of the lower limb should be more cautious. equally in brachial plexus repair, the tubes can increase the possibility of nerve repair when conventional techniques are insufficient that is, in case of large nerve gap, especially in total palsy, but can not be considered as a first choice of treatment. finally in nerve reconstruction planning, the high cost of the tube should always be considered, although in harvesting nerve grafting, the possibility of avoiding the donor site morbidity is not an insignificant detail. | background : direct neurorrhaphy, nerve grafting interposition and neurotization are the options for nerve repair in children, whereas few reports about using nerve conduits (tubulization) are referred to pediatrics in the literature. the authors present their experience about nerve repairing by means of nerve tubes during the developmental age when the harvesting of nerve grafts and also vein grafts of adequate caliber for bridging nerve defects is difficult. a critical review of their case series offers indications for using nerve conduits in pediatrics.materials and methods : fifteen patients were treated using the nerve tubulization ; nine patients were affected by obstetrical brachial plexus palsy (obpp) while six were suffering from peripheral nerve injuries (pnis).results : in patients suffering from obpp, we observed 1 good, 3 fair and 5 bad results. in the pni group, we observed 4 patients who had good results while only 2 had a bad outcome. no fair results were observed.conclusions:in peripheral nerve repairing in children by using nerve conduits, the outcome has been widely effective even when dealing with mixed and motor nerve, thus nerve tubulization might be considered as an alternative to nerve grafting. conversely, considering the uncertain result obtained in brachial plexus repairing, the conduits can not be considered as a first choice of treatment in brachial plexus reconstruction. |
tumors arising on true vocal cords result in hoarseness at an early stage, and the incidence of positive lymph nodes for t1 - 2 glottic cancer is low due to minimal lymphatic drainage. based on clinical presentations and a favorable prognosis, the aim of therapeutic management is not only tumor control but also preservation of laryngeal function. radiotherapy (rt) and co2 laser excision are the first choice of treatments for early glottic cancer. although both modalities produce similar outcomes, rt is the preferred option for improved voice quality, especially in cases of bulky tumors, infiltrative growth pattern, and tumors with anterior commissural involvement. the historical data reported 5-year overall survival rates of 80%-85% and local control rate of 79%. in current retrospective studies, the 5-year local control rates were 85%-95% for t1 and 70%-80% for t2 with definitive rt, and cause - specific survival rates were usually higher than 90% [7 - 11 ]. however, the total rt dose, fractionation scheme, and target volume have been dependent on the institution s policy. the purpose of this study is to present our long - term results of definitive rt for early - stage squamous cell carcinoma of the glottis. we analyzed the prognostic factors that had a significant impact on local tumor control and evaluated patterns of failure. after receiving an approval from the institutional review board, we reviewed 438 patients with malignancy in the glottic larynx referred to our clinic between january 1981 and december 2010. two hundred twenty - two consecutive cases of t1n0 or t2n0 glottic cancer treated with definitive rt were identified. in our institution, patients with synchronous primary cancer and with medical history of prior surgery of the neck or chemotherapy were excluded. the american joint committee on cancer (ajcc) staging system used in this study was t1 for a tumor confined to a single (t1a) vocal cord or both (t1b) vocal cords, and t2 for a tumor with supraglottic or subglottic extension, with or without impaired vocal cord mobility. old cases of t2a and t2b before applying the sixth version of the ajcc s staging system were re - staged. magnetic resonance imaging, and positron emission tomography - computed tomography were not routinely performed. rt was delivered using two - dimensional (2d) conventional rt until 2005, and the three - dimensional (3d) conformal technique was used since 2006. the beam energy consisted of co and 4-mv or 6-mv photons, and co was used until 1992. right / left or right anterior oblique / left anterior oblique beams were used in 2d conventional rt, and the field size ranged from 20 to 42 cm based on the anatomy and extent of the disease. in the 3d conformal technique, the clinical tumor volume (ctv) was defined as the whole larynx, and the planning target volume was an expansion of ctv by 0.5 cm. the borders of the rt field were (1) superior : lower level of hyoid bone, (2) inferior : lower margin of cricoid cartilage, (3) anterior : beyond the skin surface, and (4) posterior : anterior border of the vertebral body of cervical spine. paired wedge filters of 15, 30, or 45 were used to improve dose homogeneity. the median total rt dose was 66 gy (range, 60 to 76 gy). t1 and t2 patients received a median of 65 gy (range, 60 to 76 gy) and 67 gy (range, 60 to 70.75 gy), respectively. the fraction size ranged from 1.75 to 2 gy until december 1998, and 2.5 gy was administered since january 1999. fifty - nine patients (38.1%) with t1 and 11 (16.4%) with t2 disease were treated with 2.5 gy / fx. the median overall treatment time was 48.0 days (range, 27.9 to 77.1 days) for t1 and 53.1 days (range, 32.1 to 69.0 days) for t2 disease. all patients were treated once daily with five fractions per week. as patients were treated with different doses and fractionation schemes, the biologically effective dose (bed) for tumors was calculated by considering tumor proliferation based on a linear quadratic model. the definition of bed is the total dose multiplied by relative effectiveness, which is the first part of the above equation. the second part consists of time - dependent factors that involve the compensatory effect of tumor repopulation during the treatment. 3) / : the dose at which the linear and quadratic portions of the cell killing are equal in radiation biology. 4) : the cell killing per gy on a log - linear plot of the linear quadratic model. the range of 0.3 - 0.35/gy has been accepted for most epithelial tumors, and 0.35/gy was used in the present study. 6) tk (day) : the kick - off time, denoting the time between the start of treatment and the initiation of accelerated proliferation during the treatment. 7) tp (day) : the potential doubling time, representing the average doubling time of cancer cells during the treatment. after the completion of rt, a radiation oncologist and/or otolaryngologist conducted regular follow - up evaluations. the pre- and post - rt change in voice quality were recorded as aggravated, ditto, and improved in a self - reported fashion. acute and late toxicities were assessed according to common terminology criteria for adverse events ver. overall survival (os) was defined as the interval from the start date of rt to the date of death. local recurrence - free survival (lrfs) was defined as the interval from the start date of rt to the date of the diagnosis of local tumor recurrence. the survival differences according to clinicopathological factors were evaluated by a two - sided log - rank test. the results of the analyses were declared statistically significant if the p - value was less than 0.05. statistical analyses were performed with spss ver. 20.0 software (ibm corp., armonk, ny). after receiving an approval from the institutional review board, we reviewed 438 patients with malignancy in the glottic larynx referred to our clinic between january 1981 and december 2010. two hundred twenty - two consecutive cases of t1n0 or t2n0 glottic cancer treated with definitive rt were identified. in our institution, patients with synchronous primary cancer and with medical history of prior surgery of the neck or chemotherapy were excluded. the american joint committee on cancer (ajcc) staging system used in this study was t1 for a tumor confined to a single (t1a) vocal cord or both (t1b) vocal cords, and t2 for a tumor with supraglottic or subglottic extension, with or without impaired vocal cord mobility. old cases of t2a and t2b before applying the sixth version of the ajcc s staging system were re - staged. magnetic resonance imaging, and positron emission tomography - computed tomography were not routinely performed. rt was delivered using two - dimensional (2d) conventional rt until 2005, and the three - dimensional (3d) conformal technique was used since 2006. the beam energy consisted of co and 4-mv or 6-mv photons, and co was used until 1992. right / left or right anterior oblique / left anterior oblique beams were used in 2d conventional rt, and the field size ranged from 20 to 42 cm based on the anatomy and extent of the disease. in the 3d conformal technique, the clinical tumor volume (ctv) was defined as the whole larynx, and the planning target volume was an expansion of ctv by 0.5 cm. the borders of the rt field were (1) superior : lower level of hyoid bone, (2) inferior : lower margin of cricoid cartilage, (3) anterior : beyond the skin surface, and (4) posterior : anterior border of the vertebral body of cervical spine. paired wedge filters of 15, 30, or 45 were used to improve dose homogeneity. the median total rt dose was 66 gy (range, 60 to 76 gy). t1 and t2 patients received a median of 65 gy (range, 60 to 76 gy) and 67 gy (range, 60 to 70.75 gy), respectively. the fraction size ranged from 1.75 to 2 gy until december 1998, and 2.5 gy was administered since january 1999. fifty - nine patients (38.1%) with t1 and 11 (16.4%) with t2 disease were treated with 2.5 gy / fx. the median overall treatment time was 48.0 days (range, 27.9 to 77.1 days) for t1 and 53.1 days (range, 32.1 to 69.0 days) for t2 disease. all patients were treated once daily with five fractions per week. as patients were treated with different doses and fractionation schemes, the biologically effective dose (bed) for tumors was calculated by considering tumor proliferation based on a linear quadratic model. the definition of bed is the total dose multiplied by relative effectiveness, which is the first part of the above equation. the second part consists of time - dependent factors that involve the compensatory effect of tumor repopulation during the treatment. 3) / : the dose at which the linear and quadratic portions of the cell killing are equal in radiation biology. 4) : the cell killing per gy on a log - linear plot of the linear quadratic model. the range of 0.3 - 0.35/gy has been accepted for most epithelial tumors, and 0.35/gy was used in the present study. 6) tk (day) : the kick - off time, denoting the time between the start of treatment and the initiation of accelerated proliferation during the treatment. 7) tp (day) : the potential doubling time, representing the average doubling time of cancer cells during the treatment. after the completion of rt, a radiation oncologist and/or otolaryngologist conducted regular follow - up evaluations. the pre- and post - rt change in voice quality were recorded as aggravated, ditto, and improved in a self - reported fashion. acute and late toxicities were assessed according to common terminology criteria for adverse events ver. overall survival (os) was defined as the interval from the start date of rt to the date of death. local recurrence - free survival (lrfs) was defined as the interval from the start date of rt to the date of the diagnosis of local tumor recurrence. the survival differences according to clinicopathological factors were evaluated by a two - sided log - rank test. the results of the analyses were declared statistically significant if the p - value was less than 0.05. statistical analyses were performed with spss ver. 20.0 software (ibm corp., armonk, ny). the median age was 60 years (range, 35 to 86 years), with male preponderance (male : female=43.4:1). t1a and t1b diseases were observed in 122 (55%) and 33 (15%) patients, respectively, and 56 (25%) patients had tumors with anterior commissural involvement. with the median bed15 of 74.67 gy15 (range, 67.88 to 85.96 gy15), a total bed15 74.67 gy15 was delivered to 107 patients (48%), and the fraction size was < 1.8 gy in 11 (5%), 1.8 gy in 115 (52%), 2 gy in 26 (12%), and 2.5 gy in 70 (31%) patients. the median overall treatment time was 50 days (range, 28 to 77 days). the photon energy of 4 mv or 6 mv was used in 129 (58%) patients. the median duration of follow - up was 85.2 months (range, 11.0 to 313.1 months). the 5-year rates of lrfs and ultimate lrfs with voice preservation were 87.8% and 90.3%, respectively (fig. 1a and b). in the univariate analysis for lrfs, t - stage (t1 vs. t2, p < 0.001), anterior commissural involvement (no vs. yes, p < 0.001), rt fraction size (2.5 gy vs. < 2.5 gy, p=0.042), and overall treatment time (< 50 days vs. 50 days, p=0.001) were statistically significant (table 2). in the multivariate analysis, t2 disease (hazard ratio [hr ], 2.30 ; 95% confidence interval [ci ], 1.08 to 4.94) and anterior commissural involvement (hr, 3.37 ; 95% ci, 1.62 to 7.02) were significantly poor prognostic factors. the 5-year lrfs rates were 94.7% and 72.0% for t1 and t2 disease, respectively (fig. 2a), and 91.9% and 75.7% for tumors without and with anterior commissural involvement, respectively (fig. the 5-year lrfs rates were 92.5% and 85.5% for an rt fraction size of 2.5 gy and < 2.5 gy, respectively (fig. 3a), and 92.4% and 83.2% for overall treatment time of < 50 days and 50 days, respectively (fig. the treatment failure involved local recurrences in 34 patients, and the median time to relapse was 1.4 years (range, 0.3 to 13.2 years). three patients had regional recurrences, and the median time to recurrence was 4.0 years (range, 2.5 to 4.3 years). the initial treatment failure did not include distant metastasis in any of the patients. in patients with local relapse four patients had recurred tumors in the area of the subglottis. in the 40 patients who underwent salvage treatment, salvage surgery (total laryngectomy, partial laryngectomy, laryngofissure and cordectomy, laser microsurgery, or neck node dissection) was performed in 37. four patients complained of aggravation of voice quality, and 16 reported an improved status or no change. at the time of analysis, death events due to aggravation of glottic cancer and other malignancies were observed in 16 and 15 patients, respectively. forty - seven patients had died of old age or other medical comorbidities. among the patients who received salvage treatment, 16 had died. there were seven and three events of cause - specific death and death from other malignancies, respectively. one hundred eighty - one (82%) patients reported an improvement of voice quality after the completion of rt. two and nine patients reported acute toxicities of grade 3 dysphagia and hoarseness, respectively, with symptoms subsiding within several months. grade 3 or higher chronic toxicities were not reported, and no patient required permanent tracheostomy due to persistent laryngeal edema. the median age was 60 years (range, 35 to 86 years), with male preponderance (male : female=43.4:1). t1a and t1b diseases were observed in 122 (55%) and 33 (15%) patients, respectively, and 56 (25%) patients had tumors with anterior commissural involvement. with the median bed15 of 74.67 gy15 (range, 67.88 to 85.96 gy15), a total bed15 74.67 gy15 was delivered to 107 patients (48%), and the fraction size was < 1.8 gy in 11 (5%), 1.8 gy in 115 (52%), 2 gy in 26 (12%), and 2.5 gy in 70 (31%) patients. the median overall treatment time was 50 days (range, 28 to 77 days). the photon energy of 4 mv or 6 mv was used in 129 (58%) patients. the median duration of follow - up was 85.2 months (range, 11.0 to 313.1 months). the 5-year rates of lrfs and ultimate lrfs with voice preservation were 87.8% and 90.3%, respectively (fig. 1a and b). in the univariate analysis for lrfs, t - stage (t1 vs. t2, p < 0.001), anterior commissural involvement (no vs. yes, p < 0.001), rt fraction size (2.5 gy vs. < 2.5 gy, p=0.042), and overall treatment time (< 50 days vs. 50 days, p=0.001) were statistically significant (table 2). in the multivariate analysis, t2 disease (hazard ratio [hr ], 2.30 ; 95% confidence interval [ci ], 1.08 to 4.94) and anterior commissural involvement (hr, 3.37 ; 95% ci, 1.62 to 7.02) were significantly poor prognostic factors. the 5-year lrfs rates were 94.7% and 72.0% for t1 and t2 disease, respectively (fig. 2a), and 91.9% and 75.7% for tumors without and with anterior commissural involvement, respectively (fig. the 5-year lrfs rates were 92.5% and 85.5% for an rt fraction size of 2.5 gy and < 2.5 gy, respectively (fig. 3a), and 92.4% and 83.2% for overall treatment time of < 50 days and 50 days, respectively (fig. at the time of analysis, tumor relapse was reported in 37 patients (17%). the treatment failure involved local recurrences in 34 patients, and the median time to relapse was 1.4 years (range, 0.3 to 13.2 years). three patients had regional recurrences, and the median time to recurrence was 4.0 years (range, 2.5 to 4.3 years). the initial treatment failure did not include distant metastasis in any of the patients. in patients with local relapse, 28 had ipsilateral vocal cord recurrences. in the 40 patients who underwent salvage treatment, salvage surgery (total laryngectomy, partial laryngectomy, laryngofissure and cordectomy, laser microsurgery, or neck node dissection) was performed in 37. four patients complained of aggravation of voice quality, and 16 reported an improved status or no change. at the time of analysis, death events due to aggravation of glottic cancer and other malignancies were observed in 16 and 15 patients, respectively. forty - seven patients had died of old age or other medical comorbidities. among the patients who received salvage treatment, 16 had died. there were seven and three events of cause - specific death and death from other malignancies, respectively. one hundred eighty - one (82%) patients reported an improvement of voice quality after the completion of rt. two and nine patients reported acute toxicities of grade 3 dysphagia and hoarseness, respectively, with symptoms subsiding within several months. grade 3 or higher chronic toxicities were not reported, and no patient required permanent tracheostomy due to persistent laryngeal edema. the primary aim of early glottic cancer treatment is to eradicate the tumor with preservation of the larynx. the maintenance of voice quality and swallowing function after the treatment is also very important. as there have been no randomized trials to compare conservative surgery with rt, the evaluation of treatment outcomes has been mainly based on retrospective studies. the present study is also a retrospective analysis of definitive rt for t1 - 2n0 glottic cancer. however, we analyzed the long - term results of large - scale data from a single institution. as shown in table 4, the 5-year rates of lrfs and os in the present study are comparable to the results of other series [7 - 11 ]. in the multivariate analysis, this result is consistent with that of other retrospective studies [14 - 17 ]. tumors at the anterior commissure tend to spread into the thyroid cartilage, resulting in early progression with partial cartilaginous or extra - laryngeal invasion. reported that the 5-year local control rates of tumors, with and without anterior commissural involvement, were 74% and 89% (p=0.0027), respectively. demonstrated that underestimation of the extent of the tumor due to insufficient radiological imaging and underdosage at the air - tissue interface could result in a negative impact of anterior commissural involvement on tumor control. although there is still some controversy, we suggest that anterior commissural involvement should be considered for routine staging systems. in previous studies, t2 stage was independently associated with a lower cure rate. a retrospective study which analyzed tis - t2 tumors identified that t2 disease was the only independently significant factor. burke. demonstrated that t - stage was significant in local control (p=0.0009) and underlined the treatment - related implications in t2 tumors with inferior tumor control. although both rt and co2 laser excision are the standard management options, conservative surgery can not guarantee voice quality in t2 or bulky tumors. to enhance the cure rate of early glottic cancer, accurate staging and discussion on optimal therapeutic modality are needed. decreasing the accelerated repopulation of cancer cells and shortening the overall treatment time are the main objectives. in a prospective randomized study, yamazaki. reported superior local control with 2.25 gy / fx compared with 2 gy / fx. more recently, moon. designed a randomized trial and demonstrated that local progression - free survival of a group that received 2.25 gy / fx was not inferior to that of a conventional arm. in our institution, the dose per fraction for early glottic cancer was 1.8 to 2.0 gy / fx until december 1998, and treatment with 2.5 gy / fx was started from january 1999. the absence of significant differences in the multivariate analysis according to fraction size (2.5 gy vs. < 2.5 gy) and overall treatment time (< 50 days vs. 50 days) may be attributed to an insufficient follow - up duration of hypofractionation in comparison with the conventional treatment. therefore, a longer follow - up is needed to assess the treatment efficacy of the hypofractionation strategy. this study calculated the tumor bed (gy15) considering both total rt dose and time - related factors. the local tumor control was not significantly different according to the value of bed 74.67 gy15 or < 74.67 gy15, and altering the cutoff values also did not result in statistically significant differences. although the level of tumor bed that is adequate to kill laryngeal tumor cells has not been definitively established, tong. demonstrated that total bed15 less than 65 gy15was related to poorer tumor control. considering that the lowest value of bed was 67.88 gy15 in the present study, we suggest that total bed15 was sufficiently high and a differential tumoricidal effect of definitive rt could not be verified. with regard to patterns of local failure, most recurrent tumors occurred in the ipsilateral vocal cord, with no isolated contralateral recurrence observed. therefore, we suggest that the prevention of ipsilateral vocal cord recurrence is important in early glottic cancer. as we treated the whole larynx in each case, the lack of contralateral failure could be due to the fact that the contralateral vocal cord was treated. more recently, dosimetric studies have reported the feasibility of the ipsilateral vocal cord intensity - modulated rt (imrt), mainly in t1 disease [21 - 23 ]. although the sparing of the organs - at - risk (contralateral vocal cord, arytenoids, swallowing muscles, carotid arteries, thyroid gland, and spinal cord) is the main advantage of the technique, the problems of tumor motion, hot spots, and contouring errors have limited the use of imrt in early glottic cancer. in a comparative study regarding imrt and 2d rt planning, imrt using image - guided technique resulted in comparable target coverage with significant reduction of radiation dose in normal tissues. the authors suggested the potential for better functional preservation with the equivalent tumor control in imrt. as mentioned in the present study, the poorer treatment outcomes of t2 disease and tumors with anterior commissural involvement remain unresolved. an earlier study demonstrated the importance of shorter overall treatment time and a larger fraction size to improve local control in t2 tumors. in a recent study on early glottic tumors with anterior commissural involvement, higher tumor bed and a larger fraction size were significantly positively associated with better local control, and the authors suggested a negative impact of underdosage at the air - tissue interface on the tumor control in such patients. since the increase in tumor dose is limited due to the normal tissue toxicities, the highly conformal rt technique can be beneficial in such high - risk subgroups. further analyses are necessary to investigate the feasibility of the new rt method in t2 disease or tumors with anterior commissural involvement. researchers at the university of texas southwestern performed a phase i study of unilateral vocal cord rt for t1a glottic cancer (choy h, personal communication). the sequential dose - fractionation scheme was 50 gy/15 fx, 45 gy/10 fx, and 42.5 gy/5 fx, and the results have not been reported yet. in our institution, patients with t1 or t2 single vocal cord tumors, without anterior commissural involvement, have undergone boost irradiation with the 3d conformal technique since january 2011. the dose - fractionation scheme involves initial treatment of the larynx (50 gy/20 fx), followed by boost irradiation of the unilateral vocal cord site (20 gy/8 fx), with a total of 70 gy administered to the primary lesion. between january 2011 and december 2012, a total of 20 patients underwent definitive rt with the boost technique. with short - term follow - up, the local tumor control of the new method was not found to be inferior to the conventional method. most inflammatory changes of the glottis were mild and asymptomatic, and no further aggravation of the gross findings was observed. fifteen patients had residual inflammatory signs on the laryngoscopic examinations at 6 months to 1 year after the completion of rt, of whom eight patients showed the ipsilateral hyperemia and wall thickening with the contralateral vocal cord relatively clear and well - healed. although the boost irradiation technique also treated the whole larynx, the dose reduction in the contralateral side with conformal rt technique showed favorable toxicity profiles. further studies are required to evaluate the clinical benefit of the single vocal cord irradiation in early glottic cancer. from the present long - term analyses of definitive rt in early glottic cancer, a high cure rate, tolerable toxicity, and favorable voice quality were obtained. t2 stage and anterior commissural involvement were the most important prognostic factors related to worse local control, and further investigations of the optimal treatment strategies in the high - risk tumors are needed. although a larger fraction size and shorter overall treatment time showed a potential relationship with better tumor control, longer follow - up is needed to confirm this finding. the higher incidence of ipsilateral local failure points out the importance of reducing recurrences at the site of the primary tumor. in the local field rt of t1 - 2n0 squamous cell carcinoma of the glottis, optimization of the rt method would be a remaining subject to improve the therapeutic index. | purposethis study evaluates the long - term results of definitive radiotherapy (rt) for early glottic cancer. clinical and treatment factors related to local control and patterns of failure are analyzed.materials and methodswe retrospectively reviewed 222 patients with t1 - 2n0 squamous cell carcinoma of the glottic larynx treated with definitive rt from 1981 to 2010. none of the patients received elective nodal rt or combined chemotherapy. the median total rt dose was 66 gy. the daily fraction size was < 2.5 gy in 69% and 2.5 gy in 31% of patients. the rt field extended from the hyoid bone to the cricoid cartilage.resultsthe median age was 60 years, and 155 patients (70%) had t1 disease. the 5-year rates of local recurrence - free survival (lrfs) and ultimate lrfs with voice preservation were 87.8% and 90.3%, respectively. t2 (hazard ratio [hr ], 2.30 ; 95% confidence interval [ci ], 1.08 to 4.94) and anterior commissural involvement (hr, 3.37 ; 95% ci, 1.62 to 7.02) were significant prognostic factors for lrfs. in 34 patients with local recurrence, tumors recurred in the ipsilateral vocal cord in 28 patients. there were no contralateral vocal cord recurrences. most acute complications included grade 1 - 2 dysphagia and/or hoarseness. there was no grade 3 or greater chronic toxicity.conclusiondefinitive rt achieved a high cure rate, voice preservation, and tolerable toxicity in early glottic cancer. t2 stage and anterior commissural involvement were prognostic factors for local control. further optimization of the rt method is needed to reduce the risk of ipsilateral tumor recurrence. |
the control of the disease in nigeria is coordinated by the national tuberculosis and leprosy control programme (ntblcp) in line with the stop tb partnership initiatives whose ultimate target is to eliminate tb as a public health problem (less than 1 case per million population) by the year 2050.1 however, nigeria is a setting where several healthcare options (medical pluralism),2 including orthodox medicine (public, private, or drug stores), traditional medicine, spiritual healers etc., operate freely ; the public health facilities within which the tb control programme operates is distanced from the people and are often not the first choice during health seeking decisions. therefore, for the above target to be achievable in nigeria using the current passive detection strategy, the people at the community level should be empowered with adequate knowledge of the growing burden of the disease and accessible potentials for cure. furthermore, nigeria is a very populous nation that is divided into several administrative units (states) with varying ethnicity, socio - economic and health indices. unfortunately, the public rarely knows the tb burdens from the states of nigeria, and this may be contributing to the prevailing inappropriate care seeking behaviour and poor awareness of the disease in nigeria.3 it thus seems that the tb data of various states ministries of health are meant only for generating national estimates and reports such that the general public as well as most health workers are not aware of the disease burden and accessibility of effective treatment. it is our believe that if information on the magnitude of tb burden generated from directly observed treatment, short - course (dots) centres in nigeria are fed back to the people through the existing community structures such as women meetings etc. this belief is supported by a study which showed that the average delay by patients in southern nigeria (including enugu state) before presenting to dots centres was 3 months, and the main reason for the poor use of dots facilities was ignorance.3 hence, it became important to articulate the reported burden and treatment outcomes of tb in enugu state in relation to the national burden so as to equip health workers in the state and beyond with the information that will help them to inform their clients and the general public. furthermore, to protect the vulnerable population of the state from tuberculosis, as stipulated in the stop tb strategy,1 it is hoped that enugu state 's ministry of health will be motivated to adapt this study results for use in community partnership towards tb control. the study was a retrospective and quantitative study of all available secondary data on tb cases registered annually (cohorts) by the tb control programme of the ministry of health, enugu state, nigeria for the period 2000 to 2009. the data source was the annual registration for tb from all the dots centres in the state. information on the number of tb cases of all categories, the treatment outcome for the cohorts of new sputum smear positive (ss+) cases, as well as the age and sex distribution of incident ss+ cohorts were included in the study. supplementary data for the national tb prevalence and new ss+ tb for 2004 - 2008 were retrieved from the world health organisation (who) online global tb database,4 and the 2008 annual report of the ntblcp.5 because dots became nationwide in 2004,5 retrieval of national data was restricted to 2004. however, epi info software version 3.5.1 was used where applicable for inferential statistics at 95% confidence level ; trends of tb cases were analysed using chi - square for trends. the definitions for categories of registered sputum smear positive tb patients in nigeria as well as the structure of the ntblcp in nigeria are described in a related publication.6 enugu state is one of the five igbo speaking states that constitute the southeast zone of the nigeria. tuberculosis control in the state is coordinated by the state 's tuberculosis and leprosy control programme. dots services were introduced in the state in 1994 and scaled - up in 1995. the state has dots services in all the 17 local government areas (lgas) and currently, there are 116 dots centres. information on the human immunodeficiency virus (hiv) prevalence per cohort of tb cases in enugu state, and further description of the study area are shown in a recent study.7 the modal age group for new ss + tb cases in enugu state was 25 - 34 years (29.8%), followed by 35 - 44 years (20.0%) while 0 - 14 years contributed the least (1.4%). with exception of the 0 - 14 age group, the total number of male cases registered per age group for the 5-year period was higher than that of females. details of the age and sex distributions of the new ss + ptb in enugu state, nigeria are shown in table 1. sex and age group distribution of new ss+tb cases in enugu state, 2005 - 2009 the reported annual number of all tb cases increased progressively from 914 cases in the year 2000 to 1684 in 2009 except in 2001 and 2004 when for each year, it was less than that of the preceding year [table 2 ]. from 2004 to 2009, all cases of tb increased at a mean rate of 7.4% per annum (median 5.2%) ; the least annual increase of 1.2% occurred from 2004 to 2005 while the highest (16.1%) was reported from 2007 to 2008. trends of tb case findings in enugu state, 20002009 the least and highest number of annually reported new ss + tb cases over the period were 583 cases in 2001 and 965 cases in 2008, respectively [table 2 ]. unlike the pattern of all tb cases which generally showed a rising trend (except the declines in 2001 and 2004), that of the new ss + tb cases declined significantly (trend x = 7.37 ; p = 0.007). the highest annual increase for new ss + tb was observed from 2007 to 2008. there were increased reports for all tb and smear negative (-ve) tb cases in 2009 while registered new ss + tb cases showed a decline [table 2, figure 1 ]. on the other hand, the proportion of ss + among the new ptb cases showed a consistent downward trend from 76% in 2005 to 63% in 2009 (trend x = 37.83 ; p < 0.001). similar pattern also applied to the proportions of new ss + of all tb cases [figure 1 ]. trends of ptb detection in enugu state, 2000 - 2009 after 2004, the number of reported extra - pulmonary tb cases increased markedly [table 2 ]. the median number of extra - pulmonary tb cases detected over a 5-year period of 2000 - 2004 was 36 while that for 2005 - 2009 was 150, which represents a four - fold rise (trend x = 51.43 ; p < 0.001). the annual number of relapsed cases fluctuated across a narrow range of 28 - 63 over the period with a median of 43 cases [table 2 ]. likewise, the median number of treatment failure cases was 8 (range : 4 - 17). the summary of treatment outcomes (in percent) for the new ss + ptb cohorts registered at the dots centres in enugu from 2000 to 2008 is shown in table 3. the treatment success rate ranged from 78% in 2006 (and 2007) to 85% in 2001 with a median of 82%. also, the median cure rate was 65% (range : 57 - 72). both treatment success and cure rate showed remarkable increase in 2008 compared to the values of the preceding year. the difference between the treatment success rate and cure rate, which represents treatment completed, showed a near consistent downward trend from 2001 to 2008 (trend x = 17.03 ; p < 0.001). treatment outcome (per cent) of new ss+ptb cases in enugu state the trends of the proportion of new ss + ptb cases that died, defaulted, or failed treatment are shown in figure 2. generally, both treatment failure rates and death rates were consistently below 10% of the registered new ss + ptb cases throughout the period. from year 2006 to 2007, the death rates reduced remarkably while the failure rates increased by a similar margin ; but in 2008, both parameters declined with varied magnitudes. on the other hand, the pattern of default rates among cohorts of new ss + ptb cases appeared to be in the upward direction with occasional declines [figure 2 ]. the highest default rate of 12% was recorded in 2005 ; incidentally, reduction in both failure and death rates also occurred in the same year. trends of unfavourable treatment outcome for new ss + ptb cases in enugu state the proportion of all tb cases reported from enugu state varied from 1.60% in 2007 to 1.97% in 2004 with a median of 1.77% [table 4 ]. likewise, the median proportion of new ss + ptb cases reported from the state was 2.10% ; the lowest value (1.70%) was in 2007 while the highest (2.27%) was in 2004. generally, the trends of the proportions of new ss + ptb and all tb cases reported from the state declined significantly despite the slight increase in proportions observed in 2008 [table 4, p < 0.05 ]. over the 5-year period of 2004 - 2008, 3,945 (59.6%) cases of new ss + ptb were reported in enugu state as against 1,98,748 (52.6%) from the whole nation. the observed difference was statistically significant [p < 0.001, or = 1.33 (95% ci : 1.26, 1.40) ]. percentages of national tb cases reported from enugu state the modal age group for new ss + tb cases in enugu state was 25 - 34 years (29.8%), followed by 35 - 44 years (20.0%) while 0 - 14 years contributed the least (1.4%). with exception of the 0 - 14 age group, the total number of male cases registered per age group for the 5-year period was higher than that of females. details of the age and sex distributions of the new ss + ptb in enugu state, nigeria are shown in table 1. sex and age group distribution of new ss+tb cases in enugu state, 2005 - 2009 the reported annual number of all tb cases increased progressively from 914 cases in the year 2000 to 1684 in 2009 except in 2001 and 2004 when for each year, it was less than that of the preceding year [table 2 ]. from 2004 to 2009, all cases of tb increased at a mean rate of 7.4% per annum (median 5.2%) ; the least annual increase of 1.2% occurred from 2004 to 2005 while the highest (16.1%) was reported from 2007 to 2008. trends of tb case findings in enugu state, 20002009 the least and highest number of annually reported new ss + tb cases over the period were 583 cases in 2001 and 965 cases in 2008, respectively [table 2 ]. unlike the pattern of all tb cases which generally showed a rising trend (except the declines in 2001 and 2004), that of the new ss + tb cases declined significantly (trend x = 7.37 ; p = 0.007). the highest annual increase for new ss + tb was observed from 2007 to 2008. there were increased reports for all tb and smear negative (-ve) tb cases in 2009 while registered new ss + tb cases showed a decline [table 2, figure 1 ]. on the other hand, the proportion of ss + among the new ptb cases showed a consistent downward trend from 76% in 2005 to 63% in 2009 (trend x = 37.83 ; p < 0.001). similar pattern also applied to the proportions of new ss + of all tb cases [figure 1 ]. trends of ptb detection in enugu state, 2000 - 2009 after 2004, the number of reported extra - pulmonary tb cases increased markedly [table 2 ]. the median number of extra - pulmonary tb cases detected over a 5-year period of 2000 - 2004 was 36 while that for 2005 - 2009 was 150, which represents a four - fold rise (trend x = 51.43 ; p < 0.001). the annual number of relapsed cases fluctuated across a narrow range of 28 - 63 over the period with a median of 43 cases [table 2 ]. likewise, the median number of treatment failure cases was 8 (range : 4 - 17). the summary of treatment outcomes (in percent) for the new ss + ptb cohorts registered at the dots centres in enugu from 2000 to 2008 is shown in table 3. the treatment success rate ranged from 78% in 2006 (and 2007) to 85% in 2001 with a median of 82%. also, the median cure rate was 65% (range : 57 - 72). both treatment success and cure rate showed remarkable increase in 2008 compared to the values of the preceding year. the difference between the treatment success rate and cure rate, which represents treatment completed, showed a near consistent downward trend from 2001 to 2008 (trend x = 17.03 ; p < 0.001). treatment outcome (per cent) of new ss+ptb cases in enugu state the trends of the proportion of new ss + ptb cases that died, defaulted, or failed treatment are shown in figure 2. generally, both treatment failure rates and death rates were consistently below 10% of the registered new ss + ptb cases throughout the period. from year 2006 to 2007, the death rates reduced remarkably while the failure rates increased by a similar margin ; but in 2008, both parameters declined with varied magnitudes. on the other hand, the pattern of default rates among cohorts of new ss + ptb cases appeared to be in the upward direction with occasional declines [figure 2 ]. the highest default rate of 12% was recorded in 2005 ; incidentally, reduction in both failure and death rates also occurred in the same year. the proportion of all tb cases reported from enugu state varied from 1.60% in 2007 to 1.97% in 2004 with a median of 1.77% [table 4 ]. likewise, the median proportion of new ss + ptb cases reported from the state was 2.10% ; the lowest value (1.70%) was in 2007 while the highest (2.27%) was in 2004. generally, the trends of the proportions of new ss + ptb and all tb cases reported from the state declined significantly despite the slight increase in proportions observed in 2008 [table 4, p < 0.05 ]. over the 5-year period of 2004 - 2008, 3,945 (59.6%) cases of new ss + ptb were reported in enugu state as against 1,98,748 (52.6%) from the whole nation. the observed difference was statistically significant [p < 0.001, or = 1.33 (95% ci : 1.26, 1.40) ]. percentages of national tb cases reported from enugu state the findings of this study with respect to sex and age patterns of new ss + ptb, are consistent with the documented global epidemiology of the disease.8910 also, the observed modal age group of 25 - 34 years is consistent with the report of a related study from a neighbouring state,11 and the 2008 national report of nigeria.5 the study 's findings are in line with the belief that tb is a disease of adults and that the burden of the disease lies more with the male sex.101112 male to female ratio among the new ss + ptb cases may be associated with the hiv prevalence in the general population and it has been shown that more female than male cases of tb are detected in countries with hiv prevalence of above 1%.8 nevertheless, considering the hiv prevalence in the study area, it is obvious that enugu state of nigeria is a deviation from that general assertion ; this deviation is also applicable to the neighbouring ebonyi state,11 and indeed the whole country.5 currently, there are no clear explanations for the higher notification of tb in males than females;8 the confusion must have been compounded by the female tb case preponderance observed in this study within the 0 - 14 year age group. furthermore, the number of all tb cases reported annually in enugu state showed a rising trend but the proportion of new ss + ptb cases was declining [figure 2 ]. though, these trends were also observed in the annual national reports45 and a related study from ebonyi state,13 it should, however, stimulate further research especially as regards the quality of the microscopic centres within the dots services of the state and nigeria. in contrast to this study finding, a report from northwest turkey where both active and passive tb case finding were practised, showed that tb case notification decreased over the period reviewed;14 it is most likely that the active case detection strategy might have been responsible for the observed tb case detection rate decline. thus, there may be need for the tb programme in nigeria to consider incorporating this strategy. the 4-fold increase in the detection of extra - pulmonary tb cases, which started in 2005, is very remarkable but there is no clear explanation, and similar pattern was not observed in related studies.11131415 it could be that the prevalence of this category of tb increased in the state. the proportion of registered tb patients that were failure or relapse cases declined from 2006 [table 2 ]. this trend may suggest an improved tb management at the dots centres which may be consistent with the reported tb programme 's operational changes that followed the multi - drug - resistant (mdr)-tb study in enugu.16 these operational changes include the creation of awareness among national tb programme personnel on the mechanism of tb drug resistance and its prevention, the introduction of dosage - friendly fixed - dose combination (fdc) anti - tb packs, addition of the regimens 2rhez/4rh for category i treatment.16 however, it is equally likely that the number of the patients registered, did not represent the true picture of these categories of patients in the community. noting that health - seeking behaviour is related to treatment experience by patients and community among other determinants ; it is likely that patients who relapsed or failed treatment may lose confidence in the dots services and seek alternative care from other sectors. according to the 2006 census, the population of enugu state residents contributes 2.3% of the national population,17 which is higher than the proportions of all tb and new ss + ptb from the state [table 4 ]. this may suggest a low tb prevalence in the state or inadequate case detection. however, this study 's finding that a tb case reported from enugu state within the period reviewed was 1.3 times more likely to be new ss + ptb when compared to the whole nation may support low tb prevalence in the state. though the state 's median treatment success rate of 82% falls short of the national target of 85%, it is still higher than 80% in ebonyi state,13 national value of 78%,8 and 80% in southern ethiopia.15 the higher treatment success rate shown in this study may therefore suggest good performance by the state 's tb programme. however, treatment success rate for years 2000 - 2003 were consistently higher than those of 2004 - 2008 and a similar picture was also observed with the treatment default rate [table 3 ], which may imply a reduction in patients compliance within that period. the reasons for the disparity should be explored by the state tb programme so as to improve the disease control. on the other hand, the disparity between treatment success rate and the cure rate may suggest inadequate laboratory support, which appeared to have improved in 2008 when the lowest disparity (10%) was recorded. it is recommended that 1 microscopy centre should serve 100,000 population.8 therefore, assuming that the state 's 2006 census figure of 3,367,837 million and the 2010 estimate of 3,757,159 were correct (annual population growth rate of 2.83%);18 then, enugu state should have at least 38 microscopy centres instead of the 30 recorded in the annual report.5 treatment of tb in nigeria is standardised for each patient category. therefore, since under dosing and risk of monotherapy must have been minimised by the introduction of fdc drugs, it is possible that misclassification of category two patients as category one in the dots centres, as noted in southern nigeria,16 may be the major contributing factor to treatment failures in enugu state. it takes repeated sputum microscopy at specified treatment interval to declare a tb case as a treatment failure and during this waiting period, s / he is a risk to the community. so, proper patient categorisation should be viewed as important as quality control of microscopy centres and a mechanism for the routine monitoring of the performance of tb health staff should be developed and enforced. the study has limitations secondary data from the state 's tb programme was used for the study, therefore, minimal errors could have occurred during data entry and computations but would not have affected the study 's results. most importantly, the disease trends identified in the study only represent cases managed in dots centres, which can not be the true situation in a population where a lot of treatment options exist. the assumption is in line with united nation 's opinion that the tb data reported by ministries in developing countries were usually only a fraction of the real population figures.19 also, the periods of years reviewed for all study objectives were not uniform because of incomplete data, which limited trend assessments for some variables. nevertheless, since the study is essentially the first formal effort at articulating and publishing the tb burden in the state, it is hoped that the study will stimulate and direct policy relevant researches in the subject area. it is also hoped that the study will motivate researchers in other regions of the country to articulate and publicise tb burden and trends in their environment. in conclusion, the study has demonstrated an increasing trend of tb cases in enugu state of nigeria for the 10-year period reviewed. all tb cases and proportion of new smear negative ptb showed a near consistent rising trend, while that of the new ss + ptb was declining. the annual number of extra - pulmonary tb increased by 4-folds in 2005 and persisted thereafter. | background : the burden of tuberculosis (tb) in nigeria is high. unfortunately, the data from the tb programme of the states ministries of health are usually unpublished, which possibly contribute to the prevailing ignorance and poor attitude of nigerians to the disease. this study determined the trends of tb burden and treatment outcome in enugu state, nigeria ; and relate the state 's disease burden to that of the nation.materials and methods : a descriptive study of secondary data from the tb control programme, ministry of health, enugu state, the national annual report of 2008, and world health organisation (who) tb database for the 10-year period of 2000-2009.results:the number of female tb cases was higher than males within the 0 - 14 age group only. the annual number of all tb cases showed a rising trend from 914 cases in the year 2000 to 1684 in 2009 ; but the proportion of new sputum smear (ss+) pulmonary tuberculosis (ptb) cases declined (trend x2 = 7.37, p = 0.007). the average number of extra - pulmonary tb cases increased fourfold from 2000 - 2004 to 2005 - 2009 (36 versus 150 cases). the median treatment success rate was 82% (range : 78 - 85). for the period 2004 - 2008, 2.0% of all new ss + pbt cases reported in nigeria, originated from enugu state. the proportion of new ss + ptb reported in enugu state was significantly higher than national value (59.6% versus 52.6%) [p < 0.001, or = 1.33 (95% ci : 1.26, 1.40)].conclusion : the burden of tb in enugu state of nigeria had increased over the period reviewed. however, the state 's contribution to the disease burden in nigeria was low. |
atrial septal defect (asd) is one of the most common congenital heart diseases encountered in outpatient clinics. surgical closure has traditionally been accepted as the gold standard treatment for an asd with volume overloading. transcatheter device closure was developed as an alternative to surgery by king.1) in 1976 and accepted as a safe and effective treatment replacing surgical closure since the 2001 approval of the amplatzer septal occluder (aso) (st. however, due to the unfavorable anatomy of secundum asd, a detailed evaluation of asd has become crucial for device closure2,3). thus, an evaluation of asd in terms of successful transcatheter device closure is needed. transthoracic echocardiography (tte) is the gold standard for diagnosing asd and has excellent sensitivity in young patients. in general, the subcostal view will definitely show asd, and the apical four - chamber view and the parasternal short axis view are also helpful for making the diagnosis. however, when tte imaging is poor due to obesity, previous thoracic surgery, or large breasts, transesophageal echocardiography (tee) is an alternative that provides excellent images. high - resolution contrast computed tomography (ct) and advanced cardiac magnetic resonance imaging (mri) can also be used to delineate asd. the general indications for closure include evidence of right cardiac volume overload even without symptoms. before deciding on closure, bidirectional shunt and pulmonary hypertension associated with asd should be evaluated with pulmonary vasodilators or the balloon occlusion test4). indications for the closure of asd in adults and children have been published by the american heart association5,6). most asds are asymptomatic, but symptomatic asds should be corrected surgically at any age. elective surgical closure of asymptomatic asds is performed at 3 - 5 years of age7). transcatheter closure of an asd with the aso has been recommended for patients weighing > 15 kg. however, the weight restriction has eased over time, and many authors reported safe and effective device closure in patients weighing < 15 kg8,9). the sinus venosus, coronary sinus, and primum types of asd require surgical closure while most secundum asds are amenable to transcatheter closure. in addition to the size of the asd, the shape, number of defects, and surrounding rims are important for successful transcatheter closure. usually, four or five rims should be evaluated before deciding on device closure. the anterior or retro - aortic rim can be evaluated on a parasternal short axis image. the posterior rim and anterior - inferior rim can be seen in the apical four - chamber view. the posterior - superior and posterior - inferior rims should be evaluated from the subcostal view or right parasternal view. in general although retro - aortic rim deficiency is very common, most asds with deficient retro - aortic rims are closed successfully by the aso11,12). however, an asd with two or more deficient rims should not be closed with a device, as device embolization is highly expected in cases with significantly deficient posterior - inferior rims3). a sufficient rim is an indicator not only of length, but also of the floppiness of the rim tissue. durongpisitkul.13) reported that a posterior - inferior rim length of at least 10 mm is safe for successful device closure of an asd. 1), and tee has been accepted as the best method for a complete evaluation of the defect for the purpose of device closure14) (fig. 2). nevertheless, the evaluation of the posterior - inferior rim may be incomplete in some patients13). by contrast, cardiac ct offers superb spatial and temporal resolution with a short scanning time. it allows for detailed imaging not only of intracardiac structures, but also of coexisting anomalies. in my experience, additive abnormal findings that include extracardiac lesions as well as cardiac lesions can be detected on ct images. my experience includes the detection of silent coronary stenosis and lung problems outside of the asd on ct images. some reports addressed the usefulness of cardiac ct for asd device closure and demonstrated superior resolution and measurement of the surrounding rims using cardiac ct15,16) (fig. however, the radiation hazard posed by ct should not be ignored despite the use of a minimal exposure technique. we recommend cardiac ct for selected cases that have a complicated asd or a large asd with a rim deficiency. very few reports are available on the use of cardiac mri for evaluating an asd prior to device closure17,18). it is not surprising that many asds are not circular in shape, and indeed complex asd shapes have been described by some authors16,19,20,21,22,23). three - dimensional (3d) images should be reconstructed from 2d images for a complete understanding of defect shape. this can be achieved by real - time 3d tee or 3d cardiac ct (fig. the preprocedural evaluation of an asd undergoing device closure should be different from that used before surgical closure. devices used for asd closure do not reflect the various defect shapes, and all asds are assumed to be circular based on the longest diameter. device closure of an asd that is 10 mm in the long diameter and 5 mm in the short diameter can differ from the closure of a nearly circular defect with a 10-mm diameter. song.16) reported that an oval shaped asd with a short diameter that was 75% smaller than the long diameter could be closed safely with an aso smaller than that used for a circular defect. they also reported their experience of a successful transcatheter closure of an asd that was 45 mm in the long diameter and 24 mm in the short diameter with a 38-mm aso16). it is well known that defects within 7 mm of each other can frequently be closed with a single device. we experienced the successful closure of multiple asds with multiple asos or a cribriform device24). two - dimensional echocardiographic images have difficulty in comprehensively defining the shape of an asd or multiple asds. by contrast, the excellence of 3d images has been reported by many interventionists. reconstructed en - face ct or tee images are superior for visualizing the morphology and the number of defects16,22,23,24,25). the morphology and location of the defect may be more easily understood on 3d images obtained from ct than those obtained from tee. however, in the case of an asd with a very thin or floppy interatrial septum, defect size and morphology can easily be distorted on the basis of ct images. even though understanding anatomy and location based on tee images can be complicated, real - time 3d tee is used widely due to the much shorter time required for 3d reconstruction. balloon sizing has been accepted as an integral part of asd device closure with an aso, but several reports described techniques that did not use balloon sizing16,23,26,27). oversizing has been suspected to play an important role in cardiac erosion after implantation and should be avoided during size selection. it is not surprising that a stretched balloon sizing technique results in frequent oversizing, which is why the stop - flow technique has replaced stretched balloon sizing28). several reports have indicated that sizing with 3d tee has similar results to those of the balloon sizing technique29,30). the longest diameter at the end - systole phase has been used to size the device, but defect morphology is often ignored when selecting device size. seo.31) found that devices implanted using the balloon sizing technique in large oval shaped defects were smaller than those used in large circular defects, and suggested that device size could be selected using 3d tee linear parameters. by contrast, song.23) measured defect area by planimetry on an en - face 3d tee image and found that the defect area correlated nicely with the device size selected by the stop - flow technique (fig. these data indicate that oversizing can be avoided by considering defect morphology in 3d images of the asd. 2d tee or intracardiac echocardiography (ice) has been used during the procedure even though tte can be used in some cases. although it necessitates other venous access and a biplane image is not obtainable, ice can be conveniently used by the same operator simultaneously. in particular, ice provides a more accurate image of the posterior - inferior rim that is superior to a tee image. an accurate size evaluation for adequate device size selection during the procedure rigatelli.35) reported their experience with ice during asd device closure, achieving a good outcome even without the use of balloon sizing methods. real - time 3d tee provides several benefits during asd device closure, such as visualization of the catheter and wire course, position of the device, and the interaction between the device and surrounding structures after implantation36). in the case of multiple asds or a multifenestrated asd, wire selection should be monitored for successful complete closure, and real - time 3d tee is very helpful37). although transcatheter closure of an asd is technically simple and straightforward, an imaging study is potentially very important for the successful completion of the procedure. three - dimensional tee or ct reconstructed images are helpful for an accurate understanding of the morphology and preprocedure sizing. in cases of multiple asds | transcatheter closure of atrial septal defects has become a popular procedure. the availability of a preprocedural imaging study is crucial for a safe and successful closure. both the anatomy and morphology of the defect should be precisely evaluated before the procedure. three - dimensional (3d) echocardiography and cardiac computed tomography are helpful for understanding the morphology of a defect, which is important because different defect morphologies could variously impact the results. during the procedure, real - time 3d echocardiography can be used to guide an accurate closure. the safety and efficiency of transcatheter closures of atrial septal defects could be improved through the use of detailed imaging studies. |
the frequency of umbilical cord blood transplantation (cbt) from unrelated donors has increased among adult patients who require allogeneic hematopoietic stem cell transplantation but have no suitable bone marrow or peripheral blood stem cell donors. although very rare, there are reports of patients who have undergone cbt and subsequently developed cutaneous disseminated vzv infection. there is a lack of detailed information on the clinical course and serological findings in such cases. here, we report in detail the clinical course and serological findings of a case of vzv reinfection in an adult post cbt, which appeared clinically to be varicella and followed a severe and prolonged course. a 50-year - old japanese man with a history of cbt for acute lymphoblastic leukemia in march 2009 developed remittent fever and fatigue on 25 august 2010. subsequently, he noticed an itchy skin eruption and visited our dermatology department on 1 september, 2010. physical examination revealed sparsely distributed vesicles, some with umbilication, and small erythematous erosions on the face, anterior chest, upper back, and upper extremities [figure 1a, b ]. histological analysis of a vesicle showed an intraepidermal blister containing degenerated balloon cells [figure 1c, d ]. within a few days, there was widespread eruption of vesicles and bullae over the body [figure 1e, f ] and the fever became continuous. clinical appearance of the skin lesions at day 3 : (a) erythematous erosion in the region of the left eyebrow and (b) an umbilicated vesicle. histological findings in a vesicle on the right forearm : (c) acanthosis with intraepidermal blisters and a mild perivascular infiltration of lymphocytes in the upper dermis (hematoxylin and eosin ; original magnification 40) and (d) an intraepidermal blister containing degenerated balloon cells (hematoxylin and eosin ; original magnification 200). clinical appearance of the skin lesion at day 10 : scattered vesicles and bulla, some of which became erosions on the chest, abdomen (e), and back (f) he was admitted for intravenous acyclovir (10 mg / kg / day) for 5 days, with a reduced dose as the patient had renal dysfunction of an unknown cause. the skin lesions responded promptly to therapy : no new skin lesions developed and the bullous lesions began to scab, though some lesions remained active. the patient was discharged after 5 days of intravenous acyclovir, with oral valacyclovir (20 mg / kg / day) advised for a further 9 days. by september 21, we measured the serum levels of anti - vzv igg and anti - vzv igm by enzyme - linked immunosorbent assay kits (enzygnost anti - vzv / igg and enzygnost anti - vzv / igm, dade behring, deerfield, il, usa) on days 3 and 33 after the appearance of the skin eruption. the values of anti - vzv igg were expressed in international units (i.e., miu / ml, with values 2.0 : positive). serum anti - vzv igg was negative on day 3 and 260 miu / ml on day 33. the former is considered to play a more important role in the host defense against vzv, since vzv is cell - associated during active infection. as cord blood contains naive t - cells but no antigen - specific memory t - cells, primary vzv infections by exogenous vzv are expected to occur frequently in cbt recipients. however, localized herpes zoster is the most frequent clinical presentation in adult patients after cbt. in addition to localized herpes zoster, a rare occurrence of vzv reinfection manifesting as a generalized vesicular eruption without herpes zoster lesions, as was seen in our patient, has been previously observed in patients after cbt. this type of vzv reinfection can be caused by either reactivation of latent virus or exogenous reinfection with vzv. since we did not compare vzv dna between the primary and present infection, it remains unknown whether the infection in our patient was of exogenous or endogenous origin. anti - vzv igg has anti - vzv activity and remains in the body for a long time. considering that our patient had a childhood history of varicella and that the serum anti - vzv igg at day 3 was negative, it appears that humoral immunity against vzv had been lost after cbt, resulting in susceptibility to vzv reinfection. in general, vzv reinfection that appears clinically to be varicella follows a mild clinical course in normal elderly people and immunocompromised hosts. this may be partly explained by the lack of anti - vzv igg at the onset of the skin eruption. however, aisa. have reported a case of post - cbt varicella with a relatively severe clinical course despite the presence of a high anti - vzv igg titer. further investigations are necessary to determine why certain post - cbt patients develop varicella, and to clarify why vzv reinfection in post - cbt patients may be more severe than that in normal aged people and immunocompromised patients. | most varicella - zoster virus (vzv) infections after cord blood transplantation (cbt) present as localized herpes zoster. here, we report a case of vzv reinfection in an adult patient after cbt that appeared clinically to be varicella. a 50-year - old japanese man underwent cbt for the management of acute lymphoblastic leukemia. seventeen months later, he developed a small number of vesicles with umbilicated centers. a skin biopsy showed an intraepidermal blister containing degenerated balloon cells. subsequently, the skin eruption developed over his entire body. the patient was treated with intravenous acyclovir for 5 days, followed by oral valacyclovir for 9 days. it took more than 3 weeks for most of the skin lesions to scab. serum levels of anti - vzv igg on days 3 and 33 after the onset of the skin eruption were negative and 260 miu / ml, respectively. serum anti - vzv igm on days 3 and 33 was not detected. our patient was diagnosed with vzv reinfection. |
vitiligo is the most common depigmenting disorder, with a worldwide occurrence of 0.1 - 2% in general population. multiple conditions have been described colocalized in vitiligo patches, like psoriasis or lichen planus. however, actinic granuloma has not been described in association with vitiligo lesions so far. actinic granuloma was first described by o'brien in 1975 as an uncommon disease, most frequently presented in middle - aged women [6, 7 ]. some authors consider actinic granuloma a subtype of annular granuloma and others a different entity. we report the case of a 50-year - old woman, who was referred to our dermatology department in 2006 with a diagnosis of lip - tip vitiligo. the milky patches were located in the face, on the dorsum of the hands, and on the forearms and legs. no diagnosis of lupus, sarcoidosis or other dermatologic diseases could be elucidated in her medical or family history. she was treated with topical steroids (clovate cream) and topical tacrolimus (protopic 0.1% ointment), with poor response in achral lesions. oral puva therapy with 8-mop was started in 2007, obtaining a moderate repigmentation in distal areas. in june 2009, during a routine follow - up, the patient complained that non - pruriginous lesions had appeared in her hands 2 weeks ago. on physical examination, she presented with multiple asymptomatic, erythematous, annular plaques and solitary papules 5 - 10 mm in diameter, located on the dorsum of both hands, strictly confined to vitiliginous lesions. full blood count, liver and renal function and urine analysis were all in normal ranges. based on the clinical picture, presumptive diagnosis of coincident vitiligo and annular granuloma was made. fibers phagocyted by the histiocytes were seen, too. besides, absence of melanocytes in fontana - masson staining was demonstrated. several articles have suggested that annular and actinic granuloma are different conditions based on histopathological characteristics. different terms have been used to designate this disorder : annular elastolytic giant - cell granuloma (aegcg), o'brien granuloma, multiple [10, 11 ], actinic [7, 8 ] or elastolytic granuloma. multiple granuloma was coined by leiker in 1964, and it is clinically similar to actinic granuloma, being considered the same entity in some studies. a photo - mediated etiology has been proposed in the literature for this subtype [10, 11 ]. o'brien suggested the role of infrared and ultraviolet actinic radiation [6, 7, 13 ]. the importance of a granulomatous chronic inflammation in association with another systemic illness such as sarcoidosis or diabetes mellitus was proposed by hanke. cd4 lymphocytes would act against the solar - damaged elastic fibers, which could be recognized as antigenic products [6, 7, 13 ]. histological characteristics like sarcoidal granuloma or multinucleated giant cells suggest an inflammatory response to small products, like altered elastic fibers. the confinement of the inflammatory reaction to the superficial dermis, corresponding to the area of solar elastosis, supports the actinic theory. this photo - mediated damage could be considered as a trigger factor in our patient and could explain the strict colocalization of elastolytic granuloma on vitiligo patches. vitiliginous skin is more photo - sensible and the actinic damage is more intense than in the rest of the body. in this case, the affected skin on the dorsum of the hands was not only prone to photo - damage as a result of sun exposure, but it was also selectively exposed to puva therapy over the last 2 years in our patient. in this case, we present an uncommon clinical feature with strict colocalization of elastolytic granuloma in vitiliginous areas. the location of the lesions strongly supports the photo - damage hypothesis in our patient. moreover, the patient received puva and sun therapy as an important part of her treatment. to our knowledge, this is the first patient with coexistence and strict anatomical coincidence of actinic granuloma and vitiligo reported in the literature thus far. we conclude that the actinic radiation played an important role in this form of granuloma in our patient. however, further studies to clarify pathogenetic links between vitiligo and actinic granuloma would be necessary. | vitiligo is the most common depigmenting disorder, with a worldwide occurrence of 0.1 - 2% in the general population. multiple conditions have been described colocalized in vitiligo patches, like psoriasis or lichen planus. however, actinic granuloma has not been described in association with vitiligo lesions so far. |
it is estimated that 85 millions pregnancies occurred in plasmodium falciparum malaria - endemic areas. this pathology is among the main causes of the low birth weight (lbw) [2, 3 ], resulting from 3% to 8% neonatal and infant deaths. this led the world health organization (who) to recommend a package of affordable interventions including the use of the insecticide - treated nets (itns), the intermittent presumptive treatment (ipt) with sulfadoxine - pyrimethamine (sp), and the effective case management of clinical malaria with recommended antimalarials. the ipt with sp (ipt - sp) is given free of charge under direct observed therapy (dot) during the antenatal clinics (ancs) at regular predefined intervals of time, with the aim to achieve the clearance of the existing plasmodium falciparum asymptomatic infection (therapeutic effect) and the suppression of new maternal infections (prophylactic effect) over a period of time. at least two doses of ipt - sp are recommended during the second and third trimesters of pregnancy. the occurring of clinical malaria should be treated with quinine or artemisinin - based combinations. quinine is considered to be a safe drug along all the period of the pregnancy, but safety data of artemisinin derivatives during the first trimester of pregnancy are limited, thus it should not be prescribed in this period. ongoing assessments on the compliance of health care providers with national policy on malaria treatment are needed. actually, no published data on prescribing practices in accordance to these new strategies are available in central african republic (car) and elsewhere. since 2006, the car malaria control programme is implementing the who new guidelines on malaria management [4, 6 ]. the training seminars of the health care providers (nurses, midwives and midwives assistants, medical officers) from public and private health centers were achieved. the supervision and the field training visit the aim of this study was to design a preliminary assessment on the compliance of a trained staff in the malaria control new strategies at the main district maternity in bangui. thus, we identified the antimalarials prescribed for possible clinical malaria episodes during the pregnancy and the timing profile of those prescriptions according the gestational age. our study was conducted from june to september 2009 at one of the two main maternities of bangui city, the maternity of the castors government health centre. this health centre is located in the most populated area of the city and is purposely selected during this study as a representative pilot center for the practice of the trained health care providers in bangui, regarding antimalarials prescription to pregnant women. the study involved all women who gave birth at the castors health centre maternity, and who have been clinically followed up in the antenatal care services at this same health centre. in april 2006, the anc staff (2 midwives and 6 midwives assistants) has baseline training in the malaria control new strategies and is regularly supervised once per year accordingly. this was the key indication for us to assess how the implementation of the new prescribing recommendations is progressing. the average proportion of pregnant women in bangui who deliver in this maternity each year is estimated at 35% (2500 deliveries), and more than 95% of those women have their anc visits in this health centre (data checked from the centre registers). after delivery, all the women from whom we got a written informed consent were immediately approached to record the prescribed antimalarials from their antenatal clinics (anc) cards. women who have not done all their anc visits at the castors health centre were excluded from this study. all the sp records were the ipt - sp, which is the only antimalarial treatment given free of charge to the pregnant women (direct observed therapy or dot). this study was undertaken during a preliminary assessment of our project aiming at estimating the burden of hiv infection on the malaria prevention effectiveness during pregnancy in car. this project was reviewed and approved by the scientific committee board of the university of bangui in charge of the validation of scientific protocol studies in car. data were entered in epiinfo software version 3.5.1 (cdc, atlanta, usa). an investigator checked the epiinfo data base and corrected errors identified after referring to questionnaires. data consistency was also verified using excel software (microsoft corporation), and statistical analysis was achieved in stata 8.0 (statacorp lp, usa). proportions were compared with the -test (or fisher test), and a p value at less than 0.05 was considered statistically significant. their mean (sd) age was 24.7 (6.0) years (range : 15 to 45 years). a proportion of 21.8% (n = 123) has been given at least one dose of ipt - sp. only a proportion of 8.5% of them was given more than one dose of ipt - sp, as is recommended by the malaria control programme. overall, 163 pregnant women (28.8%) were prescribed at least once an antimalarial for a possible clinical malaria episode. eight anc cards contained 3 prescriptions (4.9%), and 29 anc cards contained 2 prescriptions (17.8%). overall, four different groups of antimalarials were prescribed to the 163 women during the pregnancy, and the total number of those prescriptions was 208 (the mean number of antimalarials prescription per women was 1.28) : quinine (56.7%), artemisinin - based combinations (26.8%), artemisinin monotherapies (14.4%), and other antimalarials monotherapies (chloroquine, halofantrine, and amodiaquine). the malaria laboratory microscopy diagnosis was available in the anc cards for only 18.9% antimalarials prescriptions (39/208). all those results were positive for a plasmodium falciparum, and the quinine was the only antimalarial prescribed accordingly. antimalarials prescription began in the second month of gestation, reaching a peak at the sixth month of gestation. the global proportion of those prescriptions significantly increased from the first trimester (16.8%) to the second trimester (56.3%) (p value < 10). there were a total of 85 artemisinin components prescriptions during the current pregnancies (30 artemisinin monotherapies and 55 artemisinin - based combinations). among those prescriptions, four (13.3%) prescriptions of artemisinin monotherapies and six (10.9%) artemisinin - based combinations prescriptions occurred in the first trimester of pregnancy the number of quinine prescription in the first trimester is 24/35 (68.6%) and in the second trimester 65/117 (55.5%), while artemisinin - based combinations are 6/35 (17.1%) for the first trimester and 40/117 for the second, and artemisinin monotherapy is 4/35 (11.4%) for the first trimester and 22/117 (18.8%) for the second. within each category of antimalarials, the distribution trend increased from the first trimester to the second trimester : 24/118 (20.3%) to 65/118 (55.1%) for quinine, 6/55 (10.9%) to 30/55 (54.5%) for artemisinin - based combinations, and 4/30 (13.3%) to 22/30 (73.3%) for artemisinin monotherapies. details on the trend of those antimalarials prescription according to pregnancy age are showed on figure 1 (see supplementary material available online at doi : 10.4061/2011/414510). this study showed that nearly three years following the change in national malaria control strategies during pregnancy, more than 80% of prescriptions represented the recommended antimalarials (the quinine and artemisinin - based combinations). the availability and affordability of this antimalarial in the health centre pharmacies (a cure costs less than 2 us$) could have motivated this preference. the quinine is also preferred by the prescribers during malaria management practices in bangui, and the predominant argument is the prompt evidence of its activity on malaria symptoms. antenatal exposures to antimalarial drugs during the pregnancy are a perpetual concern because there is limited information on their safety. indeed, pregnant women are systematically excluded from clinical trials, hence the lack of evidence bases on the toxicity of antimalarial drugs in this population. nevertheless, the obligation to treat (to save lives) led to the use of antimalarials drugs during pregnancy, as the experts agreed on the safety of the quinine along all the period of the pregnancy and the artemisinin - based combinations during the two last trimesters of the pregnancy. thus, the prescription of the artemisinin derivatives which occurred in the first trimester of pregnancy is relevant because it is not advisable [4, 9 ] and may cause embryotoxicity or enhance embryolethality risks. thus, this regimen is no longer recommended by who due to its short plasmatic half - life, which could enhance the spreading of parasite resistance and compromise the efficacy of artemisinin - based combinations. it is also surprising that there were 3 prescriptions of the chloroquine which has been withdrawn from the car market because of its lack of efficacy on malaria. the first limitation was the method used to record the prescriptions of antimalarial drugs from solely anc cards. indeed, other antimalarials taken by the women during their current pregnancies could not have been reported in the anc cards. however, recording antimalarials use through interview could lead to error because some pregnant women may not have accurately recalled the name of the antimalarial prescribed and/or taken, thus leading to biased findings on the health staffs compliance with the national recommended guidelines. the second limitation is that laboratory malaria analysis results were poorly reported at the anc visits cards. indeed, the clinical symptoms have not always the positive predictive value of malaria [14, 15 ]. another limitation is that the findings from this study could be different in other health maternities centres due to changes of staff (new health providers), or specificities related to prescribing habits. the findings of this study from a district maternity in bangui provide the indicator of the antimalarials groups prescribed to pregnant women, during the period of the new malaria treatment guidelines implementation in car. the observation from this study is that there is a satisfying adherence by trained health care providers to the indicated categories of antimalarials during pregnancy from the anc staff of the castors health center of bangui. nevertheless, there are some errors of antimalarials prescription related to the period of gestation and to not recommended antimalarials prescriptions. the activities focusing on the supervision of health care providers should be reinforced to assure adequate malaria treatment during pregnancy according to national guidelines. in particular, our findings helped the national malaria programme to enhance supervision activities to the staff of castors maternity centre in bangui in order to limit the prescription errors in the future. further studies assessing the indicators of the national antimalarial policy implementation to the pregnant population are needed in other areas of the country. | introduction. the aim of this study was to identify the antimalarials prescribed during the pregnancy and to document their timing. method. from june to september 2009, a survey was conducted on 565 women who gave birth in the castors maternity in bangui. the antenatal clinics cards were checked in order to record the types of antimalarials prescribed during pregnancy according to gestational age. results. a proportion of 28.8% anc cards contained at least one antimalarial prescription. the commonest categories of antimalarials prescribed were : quinine (56.7%), artemisinin - based combinations (26.8%) and artemisinin monotherapy (14.4%). among the prescriptions that occurred in the first trimester of pregnancy, artemisinin - based combinations and artemisinin monotherapies represented the proportions of (10.9%) and (13.3%). respectively. conclusion. this study showed a relatively high rate (> 80%) of the recommended antimalarials prescription regarding categories of indicated antimalarials from national guidelines. but, there is a concern about the prescription of the artemisinin derivatives in the first trimester of pregnancy, and the prescription of artemisinin monotherapy. thus, the reinforcement of awareness activities of health care providers on the national malaria treatment during pregnancy is suggested. |
chronic myelogenous leukemia (cml) is a chronic myeloproliferative disorder which remains in chronic phase for a variable period followed by accelerated phase and later blast crisis. various drugs have been used in the management of cml and these are busulfan, alpha - interferon, hydroxyurea, and imatinib, the last mentioned, a tyrosine kinase inhibitor. in last three decades, the incidence of human immunodeficiency virus (hiv) infection has been on the rise. the immunodeficiency state makes the patient vulnerable to various infections. with the introduction of anti - retroviral therapy and modern antibiotics, the long - term survival of hiv patients has certainly improved and now hiv is considered as chronic infection. however, it is also observed that there has been an increase in the incidence of cancers in hiv afflicted patients. there are few case reports about development of cml in hiv-1 patients in adult and pediatric age groups. we report a case here, where cml was diagnosed earlier and while the treatment was ongoing, hiv infection was detected. it was observed that the patient not only responded favorably to hydroxyurea, as far as cml was concerned, but is maintaining good health in the presence of hiv infection. the first case where both these conditions were coexisting was reported in 1995. in this patient, record a case of congenital hiv infection who had cml for more than 10 years. this patient was treated with antiretroviral agent, zidovudine for hiv and alpha - interferon for coexisting cml webb studied 10 patients in whom cml and hiv was coexistent. the response to imatinib and combination anti - retroviral therapy (cart) was excellent. combination of imatinib with highly active retroviral therapy was shown to have a good control of cml and hiv over a period of 69 months. in 1997, complete cytogenetic response was documented with a combination of alpha - interferon and zidovudine in an asymptomatic patient of cml and hiv. before tyrosine - kinase inhibitors appeared on the scene, hydroxyurea was used extensively for two reasons. one, the cost of the drug was affordable and secondly it offered good control of cml. in early 1980s, patients with hiv infection were treated with hydroxyurea alone and later this was combined with didanosine or stavudine as first line therapy. however, results of monotherapy with hydroxyurea in hiv infection were inferior to that of haart. in addition peripheral neuropathy was observed in 5.8% when hydroxyurea was used as a single agent. these patients due to altered immunological status often contracted pulmonary tuberculosis and isoniazid used for treating mycobacterial infection further enhanced the chances of peripheral neuropathy. a 50-year - old male patient was a known case of diabetes mellitus and was being treated for that since 1989. he was ; however, quite irregular with this treatment and his diabetes was often out of control. in november 2005 the fever was low grade and there was no history of hemoptysis, dysuria and hematuria. routine laboratory investigations showed high white blood cell count which was 59,000/mm, and there were premature leucocytes on peripheral smear. his diabetes was out of control with fbs level 264 mg / dl and ppbs value 372 mg / dl. a provisional diagnosis of cml with uncontrolled diabetes mellitus was made and he was further investigated. he was given hydroxyurea 500 mg 3 times a day and subsequently the doses were titrated with reference to wbc count. during the follow - up that spanned over next several months for this, he was treated with antibiotics and insulin which controlled the infection promptly. in october 2010, he complained of occasional fever, cough with slight expectoration and loss of appetite, weakness and weight loss. he was diagnosed to have pulmonary tuberculosis on the basis of his clinical picture and positive tb elisa test (igm) for which he received anti tubercular treatment for next 10 months. in february 2011 the treadmill test was strongly positive for exercise induced coronary insufficiency. as his symptoms and stress test suggested coronary heart disease, he was referred to a cardiac center for further evaluation. 2d - echo was carried out which showed echo - concentric lvh, normal resting systolic function, presence of diastolic dysfunction, no regional wall motion abnormalities, and normal pa pressure. coronary angiography was done on 1 march 2011 which showed lvef 50% and triple vessel disease. revascularization was contemplated ; however, his routine investigation showed a positive hiv test ; hence no further interventional treatment was carried out. cd3, cd4 and cd8 count results were 2940 cells / mm, 393 cells / mm and 2477 cells / mm respectively. viral load was tested on 5 november 2011and it showed 43356 hiv-1 rna copies / ml. he was advised to start anti - retroviral therapy along with imatinib, however, patient declined to take this treatment due to lack of resources. patient continues to take hydroxyurea regularly and in spite of advanced hiv he has not developed any major complications of hiv or cml during his follow - up period. its incidence in india is recorded from 6 population based cancer registries and varies from 0.8 to 2.2/100,000 for males and from 0.6 to 1.6/100,000 populations for females. studies based on hospital patients show a higher incidence which ranges from 40% to 82 % of leukemia cases in adults. majority of patients (90 - 95 %) present in an initial chronic phase of variable duration (average-5 - 5 years). when initial treatment is started in chronic phase of cml, approximately 95% of patients achieve complete hematological response. they are : acquired immunodeficiency syndrome (aids) defining cancers which include non - hodgkin lymphoma, invasive cervical cancer and kaposi sarcoma. this patient of cml with hiv-1 infection shows a different clinical course than either presenting singly. his cml was diagnosed 7 years ago and so far has not shown any evidence of accelerated phase or blast crisis. the presence of hiv infection was detected 5 years after the diagnosis of cml and though he has not received triple drug therapy, he remains clinically stable. if imatinib was used for cml, he would also need triple drug anti - retroviral therapy because the tyrosine kinase inhibitor does not have any effect on hiv-1. therefore for cml coexistent with hiv-1 infection, hydroxyurea as a single agent seems to be useful at least in this case. in fact, before the advent of effective anti - retroviral agents, many hiv patients were treated with a combination of hydroxyurea and didanosine as hydroxyurea has a synergistic effect with reverse transcriptase inhibitors such as didanosine. hydroxyurea significantly enhances the antiretroviral effects of the adenosine analog reverse transcriptase inhibitor dideoxyinosine by reducing intracellular de - oxyadenosine triphosphate concentrations due to inhibition of ribonucleotide reductase enzyme. as newer antiviral agents such as protease inhibitors, integrase inhibitors, entry blockers and other agents from reverse transcriptase class, were introduced the use of hydroxyurea in the management of hiv-1 became less and less. though, it was easily available at a lower cost, severe hematological toxicity and associated neuropathy, hydroxyurea was later omitted from the guidelines for the antiretroviral therapy. he is taking only one agent, hydroxyurea since the diagnosis of cml and has good performance status in spite of having a very low cd4/cd8 ratio. he was treated for tubercular infection but did not acquire other opportunistic infections in last several months. he has significant longer survival without an ideal treatment for cml or hiv infection. it is observed that when a single antiretroviral agent is used in the treatment of hiv-1 infection, drug resistance develops in a short time. this could probably be the first report of a patient who had cml first and later developed hiv. this case does not fit in conventional classification of hiv associated cancers. as per this definition, patients have retroviral infection first and later due to infections such as hhv8, eb virus or other unknown viruses and/or mechanisms develop different types of cancer at a later date. at the time of preparing this report (december 2012) the patient was taking hydroxyurea and the cml is fairly well controlled. his performance status remains at zero (ecog score) and he regularly visits for follow - up. | association of cancer and hiv infection is seen in practice. commonly observed cancer in hiv infected patients are non - hodgkin 's lymphoma, cervical cancer and kaposi sarcoma, coexistent chronic myelogenous leukemia (cml) and hiv infection are rare. we report a case where these two diseases were found in a patient and were treated with a single agent hydroxyurea. |
dental profession is not only restricted to examination, investigation, diagnosis and treatment of oral- and oro - facial lesions of local origin but also to serve in other community services and legal matters. dentist has a pivotal role in the identification of person as mouth provides with infinite evidence because of the distinctive features of teeth, lips and palate. the wrinkles and grooves on labial mucosa, called as sulci labiorum forms a characteristic pattern called as lip prints. (from the greek word cheilos : lips, e skopein : see) is the name given to lip print studies. the importance of cheiloscopy is linked to the fact that lip prints are unique to one person except in monozygotic twins. the second prints of interest, which are highly individualistic and forms the basis for personal identification in forensic examinations are fingerprints. each individual possesses a unique set of minute raised ridges on volar pads called friction ridge skin. these clear and apparent unique outlines of the ridges are called fingerprints. due to the immense potential of fingerprints and lip prints as an effective method of identification an attempt has been made in the present work to investigate whether the lip prints are unique to any fingerprint in the population under investigation and to see if this association will help in the identification of the person at the scene of crime. the study sample comprised 100 students randomly selected from bapuji dental college hospital, davangere, karnataka, 50 males and 50 females aged between 18 and 20 years. consent for the study was taken from all the individuals and ethical clearance from the institution was obtained for the same. materials used for recording lip prints and thumbprints were : red colored lipstick and lip brush, no. 1 whatman filter paper, magnifying lens. the lips of the individuals were cleaned and outline of the lip was marked with sharp lip liner pencil no 15. lipstick was applied uniformly on the lips by a lipstick applicator brush starting at the midline and moving laterally. the lips were allowed to dry after which lip impression was made on a no. 1 whatman filter paper. the subject was asked to make a lip impression in the normal rest position of the lips by dabbing it in the center first and then pressing it uniformly toward the corners of the lips. while studying the various types of lip prints, each individual 's lips were divided into six compartments, i.e. three compartments on each lip, and were allotted the digits 16 in a clockwise sequence starting from the subject 's upper right quadrant. lip prints were studied in all the quadrants and the type of pattern which was repeated maximum number of times was considered as described by acharya and sivapathasundharam. first digits of left hands were cleaned, and the red - colored lipstick was rolled over the left thumbs. 1 whatman filter paper laid down on a pressure pad, and primary patterns (loops, whorl and arches) were observed. in this study, the classification of patterns of lines on the lip proposed by tsuchihashi y has been followed as this is the most widely used classification in the literature. tsuchihashi y classification of lip prints is as follows : type i - a clear - cut groove running vertically across the liptype i - partial - length groove of type itype ii - a branched groovetype iii - an intersected groovetype iv - a reticular patterntype v - grooves do not fall into any of type i iv and can not be differentiated morphologically (undetermined). type i - a clear - cut groove running vertically across the lip type i - partial - length groove of type i type ii - a branched groove type iii - an intersected groove type iv - a reticular pattern type v - grooves do not fall into any of type i iv and can not be differentiated morphologically (undetermined). chi - square test was used to see the association and proportions was used to see the percentage. the study sample comprised 100 students randomly selected from bapuji dental college hospital, davangere, karnataka, 50 males and 50 females aged between 18 and 20 years. consent for the study was taken from all the individuals and ethical clearance from the institution was obtained for the same. materials used for recording lip prints and thumbprints were : red colored lipstick and lip brush, no. 1 whatman filter paper, magnifying lens. the lips of the individuals were cleaned and outline of the lip was marked with sharp lip liner pencil no 15. lipstick was applied uniformly on the lips by a lipstick applicator brush starting at the midline and moving laterally. the lips were allowed to dry after which lip impression was made on a no. 1 whatman filter paper. the subject was asked to make a lip impression in the normal rest position of the lips by dabbing it in the center first and then pressing it uniformly toward the corners of the lips. while studying the various types of lip prints, each individual 's lips were divided into six compartments, i.e. three compartments on each lip, and were allotted the digits 16 in a clockwise sequence starting from the subject 's upper right quadrant. lip prints were studied in all the quadrants and the type of pattern which was repeated maximum number of times was considered as described by acharya and sivapathasundharam. first digits of left hands were cleaned, and the red - colored lipstick was rolled over the left thumbs. the smeared finger was printed on a no. 1 whatman filter paper laid down on a pressure pad, and primary patterns (loops, whorl and arches) were observed. the lips of the individuals were cleaned and outline of the lip was marked with sharp lip liner pencil no 15. lipstick was applied uniformly on the lips by a lipstick applicator brush starting at the midline and moving laterally. the lips were allowed to dry after which lip impression was made on a no. 1 whatman filter paper. the subject was asked to make a lip impression in the normal rest position of the lips by dabbing it in the center first and then pressing it uniformly toward the corners of the lips. while studying the various types of lip prints, each individual 's lips were divided into six compartments, i.e. three compartments on each lip, and were allotted the digits 16 in a clockwise sequence starting from the subject 's upper right quadrant. lip prints were studied in all the quadrants and the type of pattern which was repeated maximum number of times was considered as described by acharya and sivapathasundharam. first digits of left hands were cleaned, and the red - colored lipstick was rolled over the left thumbs. 1 whatman filter paper laid down on a pressure pad, and primary patterns (loops, whorl and arches) were observed. in this study, the classification of patterns of lines on the lip proposed by tsuchihashi y has been followed as this is the most widely used classification in the literature. tsuchihashi y classification of lip prints is as follows : type i - a clear - cut groove running vertically across the liptype i - partial - length groove of type itype ii - a branched groovetype iii - an intersected groovetype iv - a reticular patterntype v - grooves do not fall into any of type i iv and can not be differentiated morphologically (undetermined). type i - a clear - cut groove running vertically across the lip type i - partial - length groove of type i type ii - a branched groove type iii - an intersected groove type iv - a reticular pattern type v - grooves do not fall into any of type i iv and can not be differentiated morphologically (undetermined). chi - square test was used to see the association and proportions was used to see the percentage. in this study, the classification of patterns of lines on the lip proposed by tsuchihashi y has been followed as this is the most widely used classification in the literature. tsuchihashi y classification of lip prints is as follows : type i - a clear - cut groove running vertically across the liptype i - partial - length groove of type itype ii - a branched groovetype iii - an intersected groovetype iv - a reticular patterntype v - grooves do not fall into any of type i iv and can not be differentiated morphologically (undetermined). type i - a clear - cut groove running vertically across the lip type i - partial - length groove of type i type ii - a branched groove type iii - an intersected groove type iv - a reticular pattern type v - grooves do not fall into any of type i iv and can not be differentiated morphologically (undetermined). chi - square test was used to see the association and proportions was used to see the percentage. the overall patterns of lip and left thumbprints in both the sexes are given in figures 1 and 2, respectively. type ii pattern of lip print was predominant in both males (60%) and females (59), followed by type iii pattern, 31% in males and 26% in females, type iv, 6% in males and 8% in females and type v 1% in both males and females [figure 3 ]. loop fingerprint pattern was most commonly seen a pattern in both males (68%), and females (61%) followed by whorl (39% females and 27% males) and arch 5% in males [figure 4 ]. (a) type i, (b) type ii, (c) type iii (d) type iv patterns of the left thumb prints. (a) loop (b) whorl (c) arch graph showing gender wise distribution of lip prints graph showing gender - wise distribution of thumbprints the overall correlation of lip prints with thumbprints in males is given in figure 5. in males, type ii and although it was associated with all three finger patterns, type ii lip print associated with loop fingerprint is highly significant (68%) (p < 0.001) followed by arch fingerprint pattern and whorl fingerprint pattern. type iii lip print showed a significant relationship with whorl type of fingerprint (69%) (p < 0.001). relationship between lip print and thumbprint in males in females, highly significant relationship was seen between type ii lip print and loop type of thumbprint (68%) (p < 0.001) followed by whorl type of thumbprint [figure 6 ]. the overall correlation of lip prints with thumbprints in males is given in figure 5. in males, type ii and type iii of lip pattern showed statistical significance. although it was associated with all three finger patterns, type ii lip print associated with loop fingerprint is highly significant (68%) (p < 0.001) followed by arch fingerprint pattern and whorl fingerprint pattern. type iii lip print showed a significant relationship with whorl type of fingerprint (69%) (p < 0.001). relationship between lip print and thumbprint in males in females, highly significant relationship was seen between type ii lip print and loop type of thumbprint (68%) (p < 0.001) followed by whorl type of thumbprint [figure 6 ]. various established techniques have been utilized in the personal identification. to accurately identify an individual, the association between two variables in the forensic science is of utmost importance as it can possibly be a supplementary tool along with the routinely used techniques. lip print is unique of an individual and hence beholds the potential for identification purpose. synder was one of the france 's greatest criminologists who first recommended the use of lip print in personal identification and criminalization. they are considered to be most important forms of transfer evidence and are analogous to fingerprints. it is possible to identify lip patterns as early as the 6 week of in uterine life. from that moment lip print pattern the lipstick marks produce persistent lip prints that can be recovered for investigations even after a lapse of few days. alvarez and associates have shown that these prints can be developed and visualized using agents such as aluminum powder and magnetic powder. ball states that vermilion border has minor salivary glands and the edges of the lips have sebaceous and sweat glands. the secretions of oil and moisture from these enable development of latent lip prints in most crime scenes, analogous to latent fingerprints, where there was a close contact between the victim and culprit. it has been noted that lip prints recover after undergoing alterations such as minor trauma and inflammation. however, major trauma to the lips may lead to scarring and the surgical treatment rendered to correct the pathosis may affect the size and shape, thereby altering the pattern and morphology of the grooves, but the use of lip prints in criminal cases is limited as compared to fingerprints because the credibility of lip prints has not been firmly established in our courts. the analysis of fingerprints as a form of identification dates back to prehistoric times. fingerprints of an individual have been used as one of the vital parts of identification in both civil and criminal cases, because of their unique properties of absolute identity. no two fingers are found to have identical prints, and it is an overwhelming mathematical probability that no two ever will be found to match. the ridge patterns are formed in the human fetus before birth and remain the same throughout a person 's life and even after death until they are lost through decomposition. fingerprints proved important due to the fact that unlike most human traits ; dermal ridges and the configurations formed by them are not affected by age. the detailed structure of individual ridges is extremely variable, and throughout postnatal life they are not affected by environment. the present study aimed to correlate the lip pattern with that of finger pattern for personal identification. many studies have been done till now on lip prints for gender identification showing varied results. sharma. had concluded that undetermined lip pattern (27.5%) in males, vertical and partial vertical lip patterns in females (25%), are common. reported that intersecting pattern was most common both in males (39.5%) and females (36.5%) and their finding was similar to that of sivapathasundharam. in the study done by gondivkar. criss - cross lip pattern was reported in 51.05% males and 37.06% branched lip pattern in females. showed type i to be predominant in females and types i and iv to be predominant in males. reported predominant pattern to be branched type, 49% in males and 40% in females. our study also showed branched type to be most common one, 59% in females and 60% males. among the fingerprints, our observation showed loop pattern to be most common, followed by whorl pattern, arch pattern, which was similar to many other studies. identification by lip print only, was of meager use, so a correlative study was designed between lip prints and finger prints to have a broader view for personal identification. there was significant relationship seen between the type ii lip print and loop type of thumbprint in both males and females. an association was seen between type iii lip print and whorl type of thumbprint in males which was not seen in females. this finding will help in identification of gender at crime scenario, thus helping in ruling out or positively identifying the suspect or victim. a study done by nagasupriya. showed a significant relationship between branching type of lip print and arch type of thumbprint followed by loop type thumbprint in males and females they found an association between type i and arch type of thumbprint. this difference in results may have occurred due to heterogeneous group of population, our study population included subjects of different ethnic background and also because of interobserver variation in classification of lip print types. a lot of scientific researches are based on lip prints and finger prints, but a study comparing and correlating these two variables is minimal. detecting and identifying lip prints at the crime scene may provide important evidence, and its correlation with thumbprint found in this study will act as an additional tool in forensic science for personal identification. the association found between type iii lip print and whorl type of thumbprints in this study may be used as an effective tool in gender identification thus helping to prove facts in crimes where there are few evidence available. | context : identification of person living or dead using diverse characteristics is the basis in forensic science. the uniqueness of lip and fingerprints and further, association between them can be useful in establishing facts in legal issues.aims:the present study was carried out to determine the distribution of different lip print patterns among subjects having different thumbprint patterns and to determine the correlation between lip print patterns and thumbprint patterns.materials and methods : the study sample comprised 100 students randomly selected from bapuji dental college hospital, davangere, karnataka, 50 males and 50 females aged between 18 and 20 years. red colored lipstick was applied on the lips by a lipstick applicator brush. lip and thumb impressions were made on no. 1 whatman filter paper and visualized using magnifying lens. three main types of fingerprints (loop, whorl and arch) were identified ; tsuchihashi y classification of lip print patterns was followed in the study. chi - square test was used to see the association between lip and thumbprints.results:the correlation between lip and left thumb print patterns for gender identification was statistically significant. in both males and females, type ii lip pattern associated with loop finger pattern were most significant and in males, type iii lip pattern with whorl type of finger pattern showed statistical significance.conclusion:we conclude that the correlation found between lip print and thumbprint can be utilized in the field of forensic science for gender identification. |
ewing 's sarcoma (es) was first described as an osteolytic bone tumor composed of malignant, small round cells by james ewing in 1921. extraosseous ewing 's sarcoma was first described by tefft in 1969 ; it is a rare, malignant mesenchymal tumor similar to intraosseous es. since its characterization in 1980s, this tumor has increasingly been reported from diverse sites including the oral cavity, salivary glands, subcutis, lung, heart, pericardium, biliary tract, kidney, urinary bladder, uterine corpus and cervix, gonads, pancreas, vagina, rectovaginal septum, prostate, esophagus, and stomach. to the best of our knowledge, no reports of its occurrence in the lesser sac have been documented in the literature. a 47-year - old woman presented with a short history of abdominal pain of 15 days duration. physical examination revealed an epigastric mass measuring 7 8 cm, which was firm in consistency and moving with respiration. examination suggested a large, well - defined, heterogenously enhancing mass measuring 12 15 cm with an epicenter in the lesser sac and loss of fat planes with the body and part of the tail of the pancreas and posterior wall of stomach. the possibility of an exophytic pancreatic mass or exophytic gastrointestinal stromal tumor (gist) from the posterolateral wall of the stomach was proposed (fig. 1). the patient underwent an exploratory laparotomy, which showed a tumor in the lesser sac abutting the left dome of the diaphragm dorsally, the splenic hilum to the left, the transverse mesocolon inferiorly, and the posterior wall of stomach anteriorly. the tumor extended posterior to the stomach and was firmly adherent to the pancreatic tissue. excision of the tumor with a distal pancreatectomy and splenectomy was performed and the specimen was received in our laboratory for histopathological examination and diagnosis. grossly, the tumor was well - circumscribed, partly encapsulated, measured 10 15 cm, and weighed 830 g. the tail of the pancreas was compressed by the tumor and was identified near the splenic hilum. cut section of the mass showed a grey tan hemorrhagic tumor with large areas of necrosis (corresponded to the cystic changes seen on ct) (fig. 2). microscopy revealed a fairly well - circumscribed tumor with a fibrous pseudocapsule composed of sheets of small round cells with enlarged round to oval nuclei, fine stippled chromatin, and moderately clear to amphophilic cytoplasm, which was periodic acid - schiff stain positive. geographic areas of necrosis with focal peritheliomatous proliferation of tumor cells around the blood vessels, increased mitosis, prominent apoptosis, and nuclear moulding were noted. in some areas, peripherally compressed pancreatic tissue was seen and no tumor infiltration was discerned (figs. 3 and 4). the tumor cells were cd99 positive, while cytokeratin (ck), desmin, synaptophysin (syp), and chromogranin (chr) were negative (fig. a final diagnosis of extraosseous ewing 's sarcoma / primitive neuroectodermal tumor (es / pnet) of the lesser sac was made. she was scheduled for alternating ie (ifosfamide and etoposide) and vac (vincristine, adriamycin, and cyclophosphamide) chemotherapy. currently, the patient has completed two cycles of chemotherapy with no further complaints and is receiving regular follow - up care. es and pnet are characterized by the same cytogenetic alterations (t(11;22) (q24;q12) which forms ewsr1-fli1 fusion product) and comparable morphologic and immunophenotypic features. they are hence classified under the same group of lesions - the es / pnet family of tumors. in their extensive review on ectopic tumors, wick and nappi attributed the origin of these tumors to ectopic neural and neuroectodermal proliferations. they further suggested that although neural tissues are ubiquitously distributed throughout the body, the occurrence of tumors related to this lineage is extremely uncommon in some topographic sites. es / pnet is a poorly differentiated tumor that is integrated in the morphologic category of ' small round cell ' tumors. the various entities that have ' small round cell ' morphology occurring at this site are lymphoma, pancreatic endocrine tumor (pet), pancreatoblastoma, extra - renal wilm 's tumor, extra - adrenal neuroblastoma, hepatoblastoma, rhabdomyosarcoma, desmoplastic small round cell tumor (dsrct), and visceral small cell neuroendocrine carcinoma (scnc). specifically, there is a broad spectrum of tumors having a similar morphology that includes sheets of small, round blue cells. this problem is markedly enhanced when the tumor site of origin is uncertain, as observed in the present case. differential diagnoses of dsrct, scnc, pet, and pancreatoblastoma were entertained based on histomorphology in this case. dsrct 's are usually multicentric tumors involving the peritoneal cavity, although extraperitoneal neoplasms have been described. the cellular phase does not have much desmoplasia and can resemble soft tissue es. since the tumor cells were negative for ck and desmin, dsrct was ruled out. cell to cell molding was noted in the present case, which is usually seen in scnc. since the cells were negative for syp and chr, small cell neuroendocrine carcinoma and malignant pet were ruled out. pancreatoblastomas are rare in adults and microscopically show islands of squamoid morules amongst the neoplastic cells, which was not observed in this case. strong membrane positivity for cd99 was observed in all of the cells confirming the diagnosis of extraosseous es / pnet. unlike intraosseous es, the radiologic findings of extraosseous lesions are non - specific. in the present case, this confusion may be partly due to the unanticipated occurrence of the tumor in a previously undescribed site. in conclusion, extraosseus es / pnets are malignant, highly aggressive tumors, with a poor patient outcome. even though it is rare, this entity should be considered in the differential diagnosis of intraabdominal, extraintestinal masses. | extraosseous ewing 's sarcoma / primitive neuroectodermal tumor (es / pnet) is an uncommon, aggressive, and malignant tumor with a poor patient outcome. its occurrence in the lesser sac is a rare event and to the best of our knowledge, has not been previously described. the present case was clinically and radiologically misdiagnosed as a pancreatic tumor / gastrointestinal stromal tumor. histopathology revealed a tumor with " small round cells " that were positive for cd99, confirming the diagnosis of es / pnet. this report highlights the importance of considering ewing 's sarcoma in the differential diagnosis of intraabdominal, extraintestinal masses. |
stress / strain redistributions occurring within mandibular bone as a result of prosthodontic treatments are highly complex and an understanding of the biomechanical factors (strains) initiating bone remodelling due to prosthodontic procedures has not been conclusive so far. for this reason an in - depth understanding of the biological activity in the supporting abutments and bone structures is required as a means for possible improving the outcomes of such restorations. therefore a detailed biomechanical model becomes essential, especially in typical clinical cases, in order to develop a computational biomechanical simulation capable of identifying the quantitative mechanical response to fixed prosthodontic treatment. fixed partial dentures are commonly used as a conservative prosthodontic treatment option in restorative dentistry with proven clinical reliability [2, 3 ] and have played an integral role in the rehabilitation of oral function for years. however, the rigid construction of fpd systems changes the local biomechanical status, whereby bone may model and remodel to accommodate a new loading environment. thus a critical factor that determines the long - term success of fpds is how occlusal forces are transferred to the surrounding root abutments, periodontal ligament (pdl), and bone. bone remodelling is dependent on the maximum load experienced throughout its load history ; however, remodelling is also characterised by the number of daily cycles that are consistent with mastication [5, 6 ]. in typical three - unit fixed partial dentures, the tooth root abutment - bone interface must be able to tolerate changes in occlusal force behaviour without instigating adverse bone tissue responses. in this sense, understanding the effect of biomechanics on biological response is a key step to optimal design of an fpd. in general, the three main biomechanical issues related to an fpd are (1) mechanical loading, (2) transmission of the load to the interfacial tissues, and (3) biological reactions of surrounding tissues to the transmitted load. frost 's mechanostat theory is one of such which defines a threshold minimum effective strain (mes) as a mechanical stimulus to trigger bone remodelling. an mes remodelling threshold has been suggested in the range of 0.00080.002 unit bone surface strain, below which it is suggestive of bone resorption and above which it is indicative of bone apposition. clinically, an fpd can be established immediately upon tooth extraction or more often upon extraction socket healing. resorption is an inevitable consequence of extraction of a natural tooth due to local bone disuse. although the resorption may not be eliminated completely, its severity can be reduced by ensuring that the prosthesis transmits mechanical loads to the underlying bone structure properly [13, 14 ]. previous investigations [15, 16 ] conducted on fixed prosthodontics have mainly focused on technical complications and stress peaks within the prosthetic devices. while in other clinical scenarios, tooth - implant systems incorporating an fpd has been evaluated [1719 ] for assessing bone - implant osseointegration. despite their relevance, limited studies have been reported on the biomechanical responses of mandibular bone as a consequence of fixed prosthodontics. indeed, the complexity of biological reactions surrounding an fpd have made it very difficult to draw general conclusions about the prognosis of restorative treatments with fpds in general dental practice. this paper aims to establish the stress / strain patterns in mandibular bone to identify the initial status of remodelling stimulus, thereby correlating the finite element (fe) results with clinical observation. in order to achieve a precise quantitative analysis of the initial mechanical responses to loading on an fpd, a biomechanical model is essential as the detailed anatomical configuration of dental structures could largely affect their behaviours [21, 22 ]. for this reason, computerized - tomograph- (ct-) based 3d finite element analysis (fea) techniques will be employed in this study to understand the biomechanics in the pdl - bone interface contiguous to abutments of an fpd. it is expected that an increased knowledge in this region can help establish a quantitative relationship associated with biological reactions such as bone remodelling. this study primarily focuses upon mandibular bone predictive or modelled strain responses as a direct result of mastication. three - dimensional computational models of a section of the mandible with teeth were established in this study, representing the right mandibular premolar, first and second molars, and their supporting dental apparatus with / without a three - unit fixed partial denture. the finite element models employed in this study were constructed primarily using computerized tomography (ct) images, digital edge detection technique, and computer aided design (cad) methods [2325 ]. in the present paper, models a and b are of prefixed prosthodontics initially upon first molar extraction and following a healing period of 12-months without fpd construction, respectively. models c and d are presented following the three - unit fpd established upon initial tooth extraction and after a 12 month healing period. the extracted tooth model (models a and c) simulated the removal of the first right molar and the wounded bone structure, immediately after extraction. bone morphology upon extraction results in the localised extraction sockets which vary between 2.0 and 4.5 mm with a width decrease up to approximately 50% as well as an unchanged the mesial / distal attachment. the healed tooth model (models b and d) incorporates the first right molar bone socket area of extraction after a 12-month healing period. the fresh bone socket is healed with a pocket depth of 1.1 mm, width of 7.6 mm, and mesial / distal attachment levels of 0.3 mm. this study will compare the mechanical strains within mandibular bone between the extracted and healed scenarios with prefixed prosthodontics and fpd situations. models a, b, c, and d (figure 1) were the basis for the comparative fe analyses that were conducted in abaqus 6.6.1 (abaqus, inc, providence, ri). the models consisted of a 10-node quadratic tetrahedral solid mesh. to establish these four models, relevant convergence tests were performed to determine the best balanced accuracy and efficiency of numerical simulation, as in, which led to a global element edge length of 1 mm to ensure the sophistication of the models and an optimal computational cost. finally, models a, b, c, and d consisted of 124 196 (dof : 897 516), 115 675 (dof : 836 202), 124 520 (dof : 900 975) and 116 781 (dof : 847 851) quadratic elements, respectively. the models were subjected to occlusal forces which functionally varied for each tooth and the fpd. loads of 50 n, 100 n, and 150 n were applied to the second premolar, first molar, and second molar, respectively (figure 1). vaillancourt suggested that an adequate functional loading of 50 n is sufficient for a premolar. schwarz stated that the molar region can endure mastication forces of up to 3 times greater than the force experienced in the canine region. thus, mastication forces in this study are classified within an upper range of normal bitting forces [30, 31 ]. three - dimensional surface - to - surface contact with solid foods was modelled with a friction coefficient of 0.2, as defined in abaqus (figure 1). all the materials were presumed linear, elastic, homogeneous, and isotropic for the analyses as widely adopted in existing literature [33, 34 ] (table 1). the periodontal ligament (pdl) stress - strain experienced in this paper is well fitted to the linear elastic range for the strains of higher than 5% but less than 20%. although pdl is viscoelastic in nature, the isotropic elastic properties were assigned as the load response lies within the linear elastic range. bone in this study is also modelled isotropically like previous studies [26, 36 ]. isotropic models of the mandible were able to distinguish meaningful strain differences when replicating functional loading, which have been widely accepted by clinicians when evaluating patients. the fe analyses of models a, b, c, and d primarily focused upon the stresses and strains within the alveolar and cortical bony tissues. the biomechanical differences due to mastication in the pre - fpd and fpd cases are evident within the contiguous bone. numerical values for strains in the extracted and healed cases were taken from distal, mesial, lingual, and buccal sites. comparisons between the models were made through the von mises stresses, principal stresses, and equivalent strains. firstly, von mises stresses were evaluated to provide distortion energies as an indicator to overall tissue deformation. then, the first and third principal stresses were characterised to highlight tension and compression behaviours in these specific sites, where the nature of these stresses may affect bone remodelling. finally, the equivalent strains were acquired to enable a measure of quantifying instigators into bone remodelling. equivalent strain represents an aggregate elastic distortion within the bony tissues, which can be calculated from components of principal strains (e 1, e 2, e 3) as follows : (1)e=(12[(e1e2)2+(e2e3)2+(e3e1)2])1/2. according to frost 's remodelling theory, the equivalent strain is considered one of the most appropriate indicators of effectively predicting bone remodelling. the finite element analyses indicated that the elevated von mises stresses occurred in the cortical ridges of functionally loaded teeth, suggesting high distortion energy distributions there. the maximum von mises stresses are located around the second molar roots at the lingual site in model a and the distal region in models b, c, and d. their corresponding values are 37.5 mpa, 36.2 mpa, 27.3 mpa, and 23.3 mpa, respectively. the maximum first principal stresses in mandibular bone of models a and b (without fpd) were situated at the cortical ridge about the mesial aspect of the second molar, which were 10.2 mpa and 11.5 mpa, respectively. it is noted that the freshly extracted case has a slightly lower tensile stress peak than the healed case. the maximum first principal stresses in models c and d (with fpd) were located about the distal region of the second molar and were 25.1 mpa and 21.9 mpa, respectively, in which the fpd in the healed case presents a 15% lower tensile stress peak. it is noted that all these first principal stress peaks are positive, indicating a tension in the surrounding areas. the maximum third principal stresses in the mandibular bone were situated in the lingual region of the cortical ridge in models a, b, c, and d, where the peak values were 58.1 mpa, 57.7 mpa, 16.9 mpa, and 15.5 mpa, respectively. it is noted that all these third principal stress peaks are consistently negative, indicating a compressive nature at these sites. a comparison of the first and third principal stress peaks about the first molar are summarised in table 2. as shown in figure 2, the peak equivalent strains on the ridged regions of the cortical bone were located in the lingual area in models a and b (without fpd), showing 0.00132 and 0.00131, respectively. in models c and d (with fpd), the maximum equivalent strains were situated around the root apex of the second premolar, yielding 0.00785 and 0.00667, respectively. the minimum equivalent strains models a, b, c, and d are 9.46 10, 1.334 10, 9.48 10, and 2.267 10. it is noted that the equivalent strain in the ridged regions of the cortical bone is of primary interest in determining initiation of bone remodelling. the corresponding average values for the equivalent strain in the mesial sides around the first molar region in models a, b, c, and d were 0.0002, 0.0006, 0.0005, and 0.0012, respectively, while they were 0.0006, 0.0008, 0.0013, and 0.002 on the distal sides, respectively. tooth extraction significantly altered the equivalent strain concentrations around the first molar (figure 3). the average lingual strains in models a, b, c, and d were 0.0002, 0.0005, 0.0002, and 0.0004, while the average buccal strains were 0.0001, 0.0002, 0.0002, and 0.001, respectively. in order to gain understanding of the consequences of fpd treatment, it is essential to establish a sound knowledge of the physiological characteristics of all the supporting tissues within such an oral environment. a fundamental design criterion for an fpd is to institute compatibility with its surrounding living tissues. the vitality of bone about an fpd is of primary importance as the condition of bone can in turn affect the stability of the fpd considerably. from the clinician 's point of view, it is imperative that the selected abutment teeth for the fpd are supported by adequately healthy alveolar bone [41, 42 ]. however, it has remained unclear how the alteration of local oral condition induced by extraction of natural tooth and construction of fpd could affect the alveolar bone. therefore there is a need to quantify the mechanical responses of alveolar bone due to construction of fpd. in this study the stress and strain distributions were examined in the pre - fpd and post - fpd cases within the mandible. tooth extraction leads to immediate changes in local bone morphology and loadings, which consequently alter the biomechanical responses in the surrounding bone bed. frost 's mechanostat theory suggests that the minimum effective strain (mes) in the range of 0.00080.002 enables the dynamics of bone turnover to reach equilibrium. in this study, the equivalent strains in the different scenarios yielded the strains within, above, and below frost 's bone adaptive mes remodelling range. the fe analyses showed that the equivalent strains in the cortical ridge of the first molar in the mesial, distal, and buccal regions in model d (healed with fpd) were within the equilibrium range (figures 4 and 5). this is a realistic indication that an fpd treatment could better maintain an appropriate bone remodelling equilibrium, thereby preserving a healthy status of bone. it is also seen that models c and d incorporating the fpd undergo overall higher equivalent strain than models a and b (figure 2). the higher magnitude of equivalent strains is evident and logical as the two abutment teeth are supporting a mastication load suitable for three native teeth. thus the strains within the abutments will be significantly greater than its prebridgework counterparts as the loading condition has substantially increased. from figure 2, the cortical strains in the freshly extracted tooth models a and c are observed to be much lower than their native counterparts due to removal of part of mastication load, suggesting that resorption may occur around the cortical ridge. the resorption is an expected outcome as upon extraction a reduction in ridge height subsequently follows. figure 5 also displays a considerable increase in strains around the apical third tooth root and root apex of the extracted tooth in models a and c. this higher strain concentration suggests that it would be a site of bone apposition. this is a probable scenario as clinically upon extraction the process of bone healing involves the formation of bone within the extraction socket. it can also be noted that with the presence of bridgework, the strains within the mandibular bone are noticeably higher due to restoration of normal masticatory function (figure 2). figure 4 relates the equivalent strains to the mes remodelling range suggested by frost, where significant difference can be observed between the counterparts with or without the fpd construction. model a suggests that resorption may occur in the mesial / distal / lingual / buccal areas of the cortical ridge, but apposition in root apex and about the root surface within the apical third. model b indicates that resorption may appear in the mesial / lingual / buccal regions, making the buccal - lingual ridge thinner and lower. model c (fpd) implies resorption in the mesial / lingual / buccal sites and remodelling equilibrium around distal region. model d appears to perform best in terms of the effective strain level and shows possible occurrence of resorption on the lingual side only. since pdls are of special importance to bone remodelling, figures 6 and 7 provide the equivalent strain values observed within the pdls of the premolar and molar in all the models. it is seen that the regions of bone resorption and apposition can correlate to those strains developed in the pdl. nevertheless, the equivalent strains within the pdl are higher than those observed within the mandibular bone due to its much lower young 's modulus. to better observe the strain distribution in pdl, figure 8 plots the equivalent strains in the different regions versus the distance from the root apex. it is shown that in all dentitions, the highest equivalent strains are at the root apex. in addition, the equivalent strains in the prebridgework models are considerably lower than those in the fpd - models. this is a realistic outcome as the two abutments are supporting a masticatory load of three dentitions after the fpd treatments. quantifying the response within the pdl can provide the indicators as to whether the appropriate mechanical signals are indicative to bone remodelling. furthermore, the anticipation of bone resorption in the extracted models a and c can be seen as a step in the healing process of extraction as alveolar bone atrophy posttooth extraction is a well - known phenomenon [44, 45 ]. from figure 3, it is noted that the fpd treatment results in the fresh extraction site experiencing a considerably higher equivalent strain, thereby somewhat better preserving the ridge height and reducing bone loss. as summarized in table 3, considerable better apposition volumes (in percentage) can be anticipated in the bridgework models c and d. much greater bone volumes, 45.93% and 40.57%, respectively, in the freshly - extracted fpd and healed fpd models, reach the equilibrium of bone turn - over, compared with much higher resorption volumes of 86.20% and 86.97% in the corresponding non - fpd models. this clearly indicates the primary importance of timely fpd treatment, not only for restoring the normal masticatory function but also for maintaining bone quantity and quality. the present study defines the initial biomechanical responses and possible adaptive changes within surrounding bone with or without construction of fpd. it is revealed that the application of an fpd leads to a noticeable alteration in normal stress / strain patterns undergone within the alveolar bone. as a consequence, this paper suggested that the response of the bone - fpd interface (pdl and adjoining bone) to functional load is crucial to the long - term success of the prosthetic treatment. this initial status of biomechanics can be associated with specific biological cellular reactions as a consequence of biomechanical stimuli. the results provide supportive evidence that an fpd treatment in a healed extraction site would help maintain a proper equilibrium of bone turnover. enumerating the adaptive ability of bone to multiple respective loading situations attained by using remodelling processes and bone remodelling algorithms will be our future work. | an understanding of functional responses in oral bone is a crucial component of dental biomechanics. the purpose of this study was to investigate the potential biological remodelling response during mastication on the mandibular pre- and post - insertion of a fixed partial denture (fpd). a series of three - dimensional (3d) finite element analysis (fea) models were presented pre- and postextraction to determine the biomechanical responses to masticatory loading in the anterior mandible. equivalent strains were analysed at lingual / buccal and mesial / distal areas of the premolar to molar region and quantified to anticipate bone remodelling response. mandibular bone incorporating an fpd experienced substantially greater stress / strain magnitudes than that prior to placement of fixed prosthodontics, which is suggestive of engagements of bone remodelling. the results suggest similar outcomes to those reported clinically. developing a simulation reflecting the outcomes of restorative treatment can provide meaningful insight into restorative treatment planning, clinical outcomes, and fixed prosthodontics designs. |
although, there is a great evidence of implant failure in compromised jaw quality, the newer designs and approaches suggest that the poor quality is not a contraindication. the density of bone site is a determining factor in treatment planning and initial progressive bone loading during prosthetic reconstruction. amongst the various classifications proposed, the misch 's and lekholm 's and zarb 's categorization of bone quality were more clinically applicable. but, irrespective of the difference, all the bone types were treated with the standard protocol. in 1988, misch described four groups independent of the regions of the jaws, based on macroscopic cortical and trabecular bone characteristics as follows : d1 : dense cortical bone ; d2 : thick dense to porous cortical bone ; d3 : thin porous cortical bone on crest ; d4 : fine trabecular bone and d5 : immature bone. it is difficult to obtain anchorage in the less dense bone which results in low success rate. in a meta - analysis, it was found that bone quality was responsible for 7.7% of all implant losses. compromised bone may cause both early and late failures. with the ever - increasing number of manufacturers with innovative fixture designs and patients who accept the modality as a permanent rehabilitation for missing teeth, the greater research with the improved designs on the soft bone is the need of the hour. the present study was done to determine the survival of maestro in the d3 and d4 jaw bone by evaluating annual peri - implant soft tissue health and the bone height around the implants. because the bone is strongest in compression and 65% weaker in shear, a square thread shape is designed such that it results in 10 times less shearing force than with a conventional v - shaped thread. fourteen patients (10 males and 4 females) were selected from the out patient department of periodontics, bapuji dental college and hospital, davangere, india on a written consent - to - treat agreement. the patients aged in the range of 1850 years with a single missing tooth, well - compliant with the oral hygiene instructions and with good dental and general health ; but who did not present with parafunctional habits, insufficient interarch space, diabetes and heavy smoking were subjected to computerized tomographic (ct) scanning as per the standard protocol. the distance from contiguous tooth and proximity from the vital anatomical structures was assessed first. it is established in the literature that the d3 and d4 type is found mostly in the posterior maxilla, anterior maxilla and posterior mandible. as per the hounsfield units calibration ; six, five and three cases were selected for the anterior maxilla, posterior maxilla and posterior mandible respectively [figures 1 and 2 ]. additionally, the pretreatment records included the periodontal status, diagnostic casts, periapical and panoramic radiographs, clinical photographs and surgical stent. computed tomography case i : scout view and bone density at proposed implant crestal (c), middle (m) and apical (a) level computed tomography case ii : scout view and bonedensity at proposed implant crestal (c), middle (m) and apical (a) level the preliminary impressions were made to fabricate a surgical guide stent. on the surgery day, the patients were covered with a surgical drape and were made to rinse with 10 ml of 0.2% chlorhexidine gluconate solution. this was followed by the surgical extraoral scrubbing with betadine (5% povidine iodine). under the appropriate block the osteotomy was done using a nsk 20:1 gear reduction hand piece attached to atr physiodispenser at 1300 rpm starting speed under copious sterile saline irrigation [figures 3 - 4 ]. case 1 : preoperative with stent, drilled osteotomy site, implant inserted, cover screw placed, crown fabricated on abutment, crown cemented case 2 : pilot drill passed through stent, drilled osteotomy site, implant inserted, cover screw placed, flap repositioned, permucosal extension placed at uncovery stage, abutment milled for crown fabrication, crown delivered in occlusion following the drill sequencing on the manufacturer 's instructions, the maestro implants of selected size was removed from the sterile vial and engaged through a premounted abutment onto a hand piece adaptor. it was taken utmost care not to touch the implant and that no thread on the implant body is visible outside the bone crest. thus, the final primary stability of the implant was checked at the end of the insertion. the particular system was opted for the study as it a root form, fine pitched threaded implant to provide a greater contact surface area. it is hypothesized that functional surface area will affect clinical health in poor - quality bone. a 0.050 hex driver was used to remove the abutment mount and replaced with the cover screw. the flap was then repositioned, sutured and covered by a periodontal pack (coe pak). the cover screws were exposed after 4 and 6 months in mandible and maxilla respectively to be replaced by permucosal extension, followed by impressions with the medium viscosity elite implant (zhermack italy) using a custom tray ; and the cemented metal - ceramic crown was delivered finally. the clinical parameters were analyzed statistically and a 3, 6, 9 and 12 months record of the modified plaque index, gingival index, modified sulcular bleeding index ; and clinical implant mobility scale was done. the standardized long cone periapical radiographs using long cone were recorded similarly [figure 5 ]. iopa radiographs compared at implant placement with 12 months post - operative : case1, case2, case 3, case 4, case 5 ; and case 6 out of the 11 implants placed in the maxillary and three in the mandibular jaw, one failed after 3 months. out of the 13 cases included in the study, one dropped out and was excluded after 4 months. plaque score [table 1 ] and gingival index values [table 1 ] showed a slight reduction at continuous intervals until 12 months postoperatively but was statistically insignificant. the mean sulcus bleeding score [table 1 ] reduced to 0.12 0.17 at the end of 1 year, which was statistically significant. there was a decreased mean implant probing depth [table 2 ] from the baseline. the mean vertical bone level changes (combined mesial and distal) were reported reduced from 1.19 1.35 mm at the baseline to 1.37 0.89 mm at the end of 1 year. [table 3 ]. thus, a significant correlation was analyzed between the plaque index and the peri - implant bone loss [table 4 ] ; and an implant success of 92.3% in low - density bone in the study. posttreatment changes in modified plaque, gi and msbi score posttreatment changes in ipd radiographic peri - implant bone level assessment relationship between clinical and radiographic parameters is said to be successful clinically if it is immobile, with the absence of associated pain, infections, neuropathy, and paresthesia. a predictable survival in all the bone densities was evaluated previously using fixture from the system used in this study. an implant or a tooth diagnosed as a clinical failure is easier to describe than one that is a success. although, implant failure can be attributed to many factors, most variables can be eliminated if accepted surgical procedures are followed. while the very dense bone at the implantation site can affect osseointegration by time of the osteotomy, the compromised bone, on the other hand, may cause both early and late failures. in a landmark study, the authors reported a 35% branemark 's fixture loss in any region of the mouth when bone density was poor. similar results were observed by another author who reported 78% of failures in the soft bone types. however, the present study refuted the hypothesis that implants are the least predictable in compromised host bone that was preassessed with ct scan in all patients. wide implants as were used in this study are advocated for the situation. in a study, the authors claimed that the design alterations in the form of wide diameter fixtures were more suited to treat the areas of inadequate bone height, areas of poor bone quality and for the immediate replacement of nonosseointegrated implants. unlike the original branemark screw, which had a v - shaped thread pattern, the maestro implant has a square crest and a flank angle of 3 that serves to decrease the shear force and increase compressive load. the patient was noncompliant to the attempt at tobacco cessation prior to inclusion in the study. the findings of the study corroborated with the previous retrospective evaluation, in which authors claimed an overall failure rate of 11.3% with branemark systems implants placed in smokers. the reason stated by the authors was that the smokers and ex - smokers had lower trabecular bone mineral content when compared to never - smokers. an overall success of 92.3% and 100% success in posterior sites was evaluated 1 year postoperatively. except for one case, which was deemed a failure, all the implants showed an absence of clinical mobility. a mean mesial peri - implant bone gain mesially, but slight average mean annual bone loss (0.18 mm with a standard deviation of 0.95) was reported. within the limits of the study, a significant correlation between plaque index and the mean peri - implant annual bone loss was shown. a few more studies with the similar implant type and incorporating a larger sample size, as well as greater follow up post loading are needed in the future. | aim : in the less dense bone, it is difficult to obtain implant anchorage. the present study was undertaken to determine the survival rate of maestro implants placed in d3 and d4 bones.materials and methods : fourteen patients (10 males and 4 females) were selected for the study and implants were evaluated for posttreatment changes in at 3, 6, 9 and 12 months from implant placement. the implant probing depth and mobility were recorded 3 and 6 months after prosthesis placement. also, peri - implant bone level was assessed at the baseline and 12 months postoperatively, followed by a statistical analysis.results:the mean plaque and gingival indices showed a reduction at repeated intervals. the mean sulcular bleeding showed a slight reduction which was statistically significant. an overall mean bone loss was observed after 12 months follow - up, which was statistically not significant. the overall survival rate of implants was reported as 92.3%.conclusion : the specific implant used in the study is advantageous in the soft bone condition.clinical significance : although, there is a great evidence of implant failure in compromised jaw quality, the newer designs and approaches suggest that the poor quality is not a contraindication. |
speaking one language rather than another involves selecting and articulating words that meet the intended meaning. the precise way in which this process is controlled may depend on the language context. we contrast a context in which a speaker uses just one of their languages to name objects in an experimental session with one where they are required to switch between languages on the basis of a color cue. for example, blue may signal that the picture is to be named in italian whereas red may signal that it is to be named in german. in principle, only one language need be active in the single language context whereas both languages must be active in the dual language context. however, research indicates that the languages of bilingual speakers can be jointly active (e.g., dijkstra., 1998 ; van hell and dijkstra, 2002 ; von studnitz and green, 2002, see van heuven and dijkstra, 2010 for a review) at least to the level of phonology even in a context that requires just a single language (wu and thierry, 2010b). given joint activation, bilingual speakers may resolve language conflict by suppressing the non - target language at some locus (green, 1986, 1998 ; linck., 2009 ; philipp and koch, 2009 ; macizo., 2010 ; snyder., 2010 ; hoshino and thierry, 2011) or by restricting competition to words within the target language in some other manner (e.g., costa and caramazza, 1999 ; elston - gttler,., 2005 ; finkbeiner. does this mean then that the selection process is identical in the single language and in the dual language context ? we take as our point of departure the idea that anguage control involves a cortical subcortical circuit (abutalebi and green, 2007, 2008) and consider whether or not language context (single or dual) modulates activity in the circuit. a major cue is that regions in the left inferior frontal cortex, anterior cingulate cortex (acc) and caudate respond differently as a function of the experimental context. in a dual language context, where participants are required to use both languages in naming simple objects, all three regions activate during language switching (e.g., abutalebi., 2008). strikingly, this pattern of activity is absent for the same l1 nouns when participants name them in l1 in a context in which they must either name the object or generate a verb associated with it. response in the caudate appears particularly sensitive to language switching because it is not observed when the context does not require switching (e.g., rodriguez - fornells. we find direct corroboration when, as per the injunction in wu and thierry (2010a), bilingual speakers use just one of their languages throughout the entire experimental session. in this single language context, the anterior cingulate and caudate are not significantly activated when participants name pictures and activation in these regions does not differ from that found in monolingual speakers (green., 2011). there is however increased activation in left prefrontal regions compared to that shown in monolingual speakers. we have to distinguish between activation correlated with interference and activation associated with the control of interference. we found that regional activation in left pars opercularis (ba44) and left pars triangularis (ba 45) was inversely related to the size of the stroop interference shown by monolingual participants outside the scanner. such an outcome suggests that these regions are involved in the control of interference. regional activation in bilingual speakers relative to monolingual speakers may also reflect differences in the experienced frequency of words (as in the weaker links hypothesis, gollan., 2008). however, a frequency - based account is an insufficient explanation because it presupposes that only a single language is selected and is silent on how such selection is achieved. how then are we to explain the differential response of the control circuit to the different language contexts ? one possibility is that it reflects the stage at which alternate words are gated from production (early or late). a single language context may afford early selection of the intended word mediated by the inferior frontal cortex. alternate words in the other language are blocked at this stage and other structures such as the caudate are not activated. by contrast, in the dual language context, switch trials may necessarily require late selection. on a switch trial, the object name, consistent with the previous language, may be released for production because the new language task has yet to dominate. response of the circuit is similar to that shown when individuals respond on incongruent trials in the stroop task. here suppressing such interference activates the caudate as well as frontal regions (ali., 2010). the experimental context is not the only relevant factor in the response of the control circuit. two other factors are pertinent : the speaker 's proficiency in their second language and, separately, the behavioral ecology of a proficient bilingual speaker. caudate activity may be expected in picture naming even in a single language context when speakers are not very proficient in their second language because they will tend to generate the first language name by default. however, the claim that context affects the locus of selection predicts that the caudate response will be even greater when such speakers perform in a dual language context. where speakers are proficient in both languages their pattern of language use within their bilingual community may also be important in understanding the response of the control circuit. we expect the contextual prediction to be met when participants are from communities that use one language at a time and switch to their other language when addressing a different addressee or when there is a change in topic. such speakers are practiced at avoiding language conflict. in terms of the abutalebi and green (2007,2008) model they recruit the acc and left frontal cortex to monitor and to control their response (see kerns., 2004). in contrast, in communities where individuals code - switch, language control is different (green, 1998). speakers have no need to avoid switching rather they freely exploit the activation of both languages. their skill lies less in avoiding language conflict than in utilizing the joint activation of both languages and adapting their utterances appropriately. speakers adapt french verbs through the addition of a german particle (-ieren) as in choisieren from the french. for speakers from code - switching communities, speaking in just one language without code - switching is likely to be effortful. we might then expect them to show increased caudate response in the single language context compared to that shown by speakers from a non - code - switching community. fluent code - switching is likely to recruit other structures implicated in language control and so studying it is important. we do not know the neural bases of code - switching but we know that those who code - switch are adept at adapting words to the current syntactic context though of course there will be individual differences in the inventiveness of such code - switches. neuropsychological data implicate cerebellar structures in the control of morphosyntax (fabbro., 2000). (1994) found that damage to the right cerebellum led to morphological deficits including inappropriate substitutions of bound grammatical morphemes. the fundamental idea is that right cerebellum acts together with left frontal structures as part of a language control circuit (fabbro., 2000). critical support for this notion comes from data showing that damage to the right cerebellum suppresses activation in left frontal cortex and elicits aphasic symptoms. reperfusion of the cerebellum reduces such symptoms (marien., 2001). code - switching involves adapting morphosyntax. given the neuropsychological data, we can predict that such adaptation will involve the right cerebellum. from a functional point of view, since code - switching involves the on - line adaptation of the morphosyntactic resources of each language, fluency in code - switching requires timing and synchronization a role for which the cerebellum is also well suited (see kotz and schwartze, 2010 for an integrative framework). a plausible prediction from existing data is that code - switching is mediated by the co - activation of right cerebellar regions and left frontal cortex. research on code - switching is therefore important for extending our understanding of language control in bilingual speakers and building on the model of abutalebi and green (2007, 2008). if functional demand shapes neural circuits then there may be structural correlates in the regions linked in control. in communities where speakers must switch on demand between their two languages, anterior cingulate and/or left inferior frontal regions may show greater gray matter density compared to monolingual speakers of just one of those languages. in contrast, in communities where bilinguals code - switch, right cerebellar structures may show enhanced gray matter density. recent research points to a bilingual advantage in controlling non - verbal interference (see bialystok., 2009, for a review) attributable to the use of cognitive control processes in resolving language conflict. if code - switching utilizes the joint activation of two languages rather than suppresses one in favor of the other then speakers from code - switching communities may not show such an advantage (see green, 2011). they may be adept more generally at seeing how one verbal form can be transformed into another to yield a novel meaning (e.g., turning a noun such as piano into a verb as in she pianoed him). alternatively, and perhaps more plausibly, cerebellar circuits may be specialized and so skills in adapting words will dissociate from the ability to adapt non - verbal objects (e.g., seeing how a pen might be used as a clothes peg) such specialization indicates a difference in the correlates of frontal versus cerebellar control processes. we suggest, in conclusion, that differences in the control demands on bilinguals from different behavioral ecologies are a further source of individual variance in executive control. the contexts of language use may then temper the cognitive advantages of bilingualism and be relevant to understanding the effects of stroke on speech recovery in bilinguals. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | this paper proposes that different experimental contexts (single or dual language contexts) permit different neural loci at which words in the target language can be selected. however, in order to develop a fuller understanding of the neural circuit mediating language control we need to consider the community context in which bilingual speakers typically use their two languages (the behavioral ecology of bilingual speakers). the contrast between speakers from code - switching and non - code - switching communities offers a way to increase our understanding of the cortical, subcortical and, in particular, cerebellar structures involved in language control. it will also help us identify the non - verbal behavioral correlates associated with these control processes |
a thoracoabdominal aortic aneurysm (taaa) is characterized by enlargement of the aortic segment at the diaphragmatic crura and extends for variable distance proximally and/or distally from this point. historically, open surgical repair of taaas has involved greater operative risk than repairs of aneurysms in other aortic segments. the main sources of morbidity during operative repair of taaas are multiorgan failure, paraplegia and respiratory, cardiac or renal complications. experienced surgical centers now report lower mortality and morbidity rates for taaa repair than they once did, largely because of the use of adjuncts to prevent end - organ ischemia. the recent introduction of endovascular techniques may extend the indications of taaa repair to high - risk patients, whose only alternative is now represented by the best medical therapy. conventional treatment of taaas consists of graft replacement with reattachment of the main aortic branches. a multimodal approach is currently used to reduce the trauma of surgery by maximizing organ protection. the surgical technique used, as the extension of the aneurysm, has a significant impact on the outcome of the procedure. thoracoabdominal incision and aortic exposure the patient is positioned with a beanbag in right lateral decubitus (shoulders 60, pelvis 30). the upper portion of the thoracoabdominal incision is made through the 6th intercostal space ; anterolaterally, the incision curves gently as it crosses the costal margin, reducing the risk of tissue necrosis. the pleural space is entered after single right - lung ventilation is initiated (figure 1). paralysis of the left hemidiaphragm by its radial division to the aortic hiatus would contribute significantly to postoperative respiratory failure, hence after thoracoabdominal incision, a circumferential section of the diaphragm is routinely carried out, sparing the phrenic center. under favorable anatomic conditions, a limited phrenotomy is carried out to preserve the tendinous center of the diaphragm ; this has been shown to reduce respiratory weaning time. the upper abdominal aortic segment is exposed via a transperitoneal approach ; the retroperitoneum is entered lateral to the left colon, and medial visceral rotation is performed so that the left colon, the spleen and the left kidney can be retracted anteriorly and to the right. transperitoneal approach allows direct view of the abdominal organs to evaluate the efficacy of revascularization at the end of aortic repair. left heart bypass (lhb) cross - clamping of the descending thoracic aorta leads to several hemodynamic disturbances, including severe afterload increase and organ ischemia. the rationale of lhb is providing flow to the spinal cord, viscera and kidneys during the aortic cross - clamp period together with the reduction of proximal hypertension and afterload to the heart. in preparation for lhb and aortic clamping, intravenous heparin (1 mg / kg) is administered with a target act (activated clotting time) of 220 - 270 seconds. proximal descending thoracic aorta, left atrium or pulmonary vein are usually cannulated for arterial blood drain that is reinfused through a centrifugal pump (biomedicus) into the subdiafragmatic aorta or the common left femoral artery. flow is initially low (500 ml / min) to avoid retrograde embolization and then increased after aortic clamping to a mean distal aortic pressure of about 70 mmhg, a value that is usually achieved using a flow between 1500 and 2500 ml / min. y bifurcation is connected to the circuit and is provided with two occlusion / perfusion catheters for selective perfusion of visceral vessels (figure 2). left heart bypass and renal perfusion catheters. once the proximal aspect of the taaa is isolated between clamps the descending thoracic aorta is transected and separated from the esophagus (figure 3). the proximal end of the graft is sutured to the descending thoracic aorta using a 2/0 monofilament polypropylene suture in a running fashion. the clamp is then removed and reapplied onto the abdominal aorta above the celiac axis (sequential cross - clamping). reimplantation of intercostal arteries to the aortic graft plays a critical role in spinal cord protection. critical patent segmental arteries from t7 to l2 are selectively reattached to the graft by means of aortic patch or graft interposition. the distal clamp is moved onto the distal abdominal aorta below the renal arteries and the upper abdominal aortic aneurysm is opened. visceral hematic perfusion is then maintained by the pump with occlusion / perfusion catheters (9 fr) inserted selectively into the celiac trunk and the superior mesenteric artery (400 ml / min). selective perfusion of renal arteries is performed with a cold crystalloid solution (ringer 4c + mannitol 18% 70 ml, 6-methylprednisolone 500 mg in 500 ml). for visceral arteries reimplantation, a side cut is tailored in the graft and the celiac trunk, superior mesenteric artery and renal arteries are reattached by means of a carrel patch. this technique has been performed in 82.3% of the patients in our series. in 33.1% of the cases treated by carrel patch the left renal artery has been separately reattached to the graft in a direct fashion or by graft interposition. when the relative distance of the visceral arteries would have required a large carrel patch, a branched graft can be successfully used (vascutek gelweave - coselli thoracoabdominal graft) (figure 4). type ii taaa repair : aortic graft replacement and visceral vessels reattachment by means of carrel patch (left) and coselli thoracoabdominal graft (above). this prosthesis allows single vessel reattachment, reducing the risk of recurrent aortic patch aneurysm. in our series the coselli branched graft has been used in 10.5% of cases. the vascutek triplex graft is a new vascular prosthesis and consists of three layers : an inner polyester graft, an outer eptfe layer and a central layer of elastomeric membrane (figure 5). the vascutek triplex graft consists of three layers : an inner polyester graft, an outer eptfe layer and a central layer of elastomeric membrane. in our preliminary experience with this graft we found good handling and tailoring performances and actually a reduced bleeding from the suture lines. finally, an end - to - end anastomosis with the distal aorta is performed. in some cases (taaa type i) endovascular procedures may be an appealing less invasive approach to the thoraco - abdominal aorta, however, the involvement of the visceral segment of the aorta represents a major challenge for taaa stent - graft repair. although total endovascular treatment with branched stent - graft has made it technically feasible to preserve visceral perfusion, the cost - efficacy and durability of these pioneering techniques are yet to be fully assessed. hybrid taaa repair was first introduced by quiones - baldrich in 1999 and mainly consists of open aortic debranching and revascularization followed by endovascular exclusion of the aneurysm. the inflow site for visceral grafts is a healthy artery, usually the infrarenal aorta, the iliac arteries or an infrarenal graft. visceral and renal arteries are then ligated at the origin to avoid back - flow in the aneurysm and consequent type ii endoleak. the open surgical stage requires a laparotomy and a transperitoneal or extraperitoneal access to the visceral vessels ; however, proximal aortic cross - clamping, thoracotomy, aneurysm exposure and monopulmonary ventilation are avoided (figure 6). the hybrid procedure consisted of infrarenal aortic grafting with single visceral vessels revascularization (center). hybrid taaa repair may be indicated in case of previous descending thoracic aortic repair in which a redo left - sided thoracotomy may be associated with major bleeding, increased rate of postoperative respiratory and organ failure and longer total aortic clamping time. a further advantage of the hybrid treatment is the possibility to reduce organ ischemic time and perform visceral protection techniques by selective cooling. hybrid repair is appealing in case of visceral aortic patch (vap) aneurysm after taaa conventional repair. moreover, vap aneurysms have ideal straight and long in - graft proximal and distal necks where the stent - graft can be safely delivered. with this technique, the aortic branches are anastomosed separately and virtually no native aortic remnants are left in situ, thus avoiding the risk of recurrences. from literature, mortality and morbidity rates after taaa conventional repair remain significant even in high - volume centers (table 1 and 2). these data could be not totally representative of the actual outcomes of taaa surgical repair. analized data from the nationwide inpatient sample (nis), dividing centers where taaa surgical repair has been performed (1988-'98) in low volume (1 - 3 cases / year), medium volume (2 - 9 cases / year) and high volume (5 - 31 cases / year). annual surgeon volume has been defined as low (1 - 2 cases) or high (3 - 18 cases). conclusions were that the results of low - volume centers and surgeons were significantly different from those of high - volume centers and surgeons. results after taaa conventional repair - universit vita - salute, scientific institute san raffaele, milan, italy. graphs show in - hospital mortality rates in function of annual hospital volume (left) and annual surgeon volume (right). in particular, in a specific subset of high - risk patients, the outcomes are associated to higher morbidity and mortality rates. as a result, these outcomes have encouraged some centers to consider the hybrid repair as the treatment of choice. the data reported in literature regarding classification and extension of pathology, patient s overall clinical conditions, surgical technique and results of taaa hybrid repair are still very heterogeneous (table 3). further studies are needed to assess the safety, efficacy and long - term survival associated to the hybrid treatment of thoraco - abdominal aortic aneurysms. the etiology of spinal cord ischemia during thoracic aortic procedures is multifactorial, and the risk of paraplegia is a debated concern. conrad. demonstrated the impact of paraplegia / paraparesis on survival of patients who underwent surgical or endovascular taaa repair : 5-year survival rate was 25% in paraplegic / paraparetic patients versus 51% in patients with no spinal complications. five - year survival rate was 41% in paraparetic patients, while no paraplegic patients survived for more than 5 years (figure 8). graphs show the impact of paraplegia/ paraparesis on survival of patients who underwent surgical or endovascular taaa repair (sci : spinal cord ischemia ; scid : spinal cord ischemia deficit ; scid i : flaccid paralysis ; scid ii : muscle function 50%) extensive coverage of the thoraco - abdominal aorta could be identified as the cause of a higher rate of spinal complications. greenberg. compared total stent - graft length in patients that did and did not develope neurological deficit, demonstrating a significant association with the lenght of aortic coverage. bckler. demonstrated that in the animal model endovascular repair is associated to lower spinal cord ischemia and paraplegia rates than aortic cross - clamping. during hybrid taaa repair, the avoidance of supraceliac clamping and the shortened duration of visceral ischemia should lead to greater perioperative hemodynamic stability compared with that during conventional open repair of taaa, and the risk of spinal cord ischemia open surgical repair of thoracoabdominal aortic aneurysms has evolved significantly over the last decades thanks to technical improvements, especially in the area of organ protection. however, despite adjunctive strategies, morbidity and mortality rates are still not negligible. surgical taaa repair is to be performed in high - volume centers by experienced surgeons. the hybrid treatment is currently indicated in a subset of patients, however morbidity and mortality are significant. further studies are needed to assess the safety, efficacy and long - term benefits of this technique. | conventional treatment of thoracoabdominal aortic aneurysms (taaas) consists of graft replacement with reattachment of the main aortic branches. over the past 20 years a multimodal approach has gradually evolved to reduce the trauma of surgery by maximizing organ protection, allowing experienced surgical centers to have better outcomes than previously reported. however, mortality and morbidity associated to taaa open repair remain significant. hybrid repair, consisting of open aortic debranching and revascularization followed by endovascular exclusion of the aneurysm, may extend the indications of taaa repair to high - risk patients that can not benefit from surgery, however results are still under evaluation. aim of this paper is to illustrate the management and results of thoracoabdominal aortic aneurysms surgery with open techniques of organ protection and hybrid approach in our center. |
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