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the thalamic reticular nucleus (trn) is hypothesized to regulate neocortical rhythms and behavioral state. using optogenetics and multi - electrode recording in behaving mice, we found that brief selective drive of trn switched thalamocortical firing mode from tonic to bursting and generated state - dependent neocortical spindles. these findings provide causal support for the trn in state - regulation in vivo and introduce a new model for addressing the role of this structure in behavior. |
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squamous cell carcinoma (scc) after ileal pouch - anal anastomosis (ipaa) for ulcerative colitis (uc) is very rare [1, 2 ]. scc is extremely sensitive to chemoradiation therapy (crt), but an ileal pouch is generally considered a contraindication to radiotherapy. few cases of scc in ipaa patients have been described in the literature, with variable treatments and unpredictable outcomes. we report the successful management of an scc of the anal transitional zone (atz) after ipaa and systematically review the literature for ipaa - related scc. the patient has had good functional outcomes but suffered from several episodes of pouchitis. in 2014, he was seen at the outpatient clinic due to a nodule in the anal canal which had rapidly increased in size (fig. the patient underwent flexible pouchoscopy and examination under general anaesthesia with biopsies, pelvis mri (fig. 2a findings were consistent with a moderately well - differentiated scc of the atz, with no evidence of distant disease (t1n0m0). we performed a systematic review of the literature to identify scc arising from the pouch or from the atz of uc patients. inclusion criteria were unequivocal diagnosis of scc that can be reasonably considered as de novo carcinoma, diagnosis of uc, and availability of adequate information. studies including patients with conditions other than uc were only included if uc patients were identifiable. available data of all patients operated on with ipaa between 1978 and 2016 were evaluated. keywords and medical subject headings (mesh) used were : restorative proctocolectomy, ulcerative colitis, squamous cell carcinoma, pouch, ipaa, and scc. the patient has had good functional outcomes but suffered from several episodes of pouchitis. in 2014, he was seen at the outpatient clinic due to a nodule in the anal canal which had rapidly increased in size (fig. the patient underwent flexible pouchoscopy and examination under general anaesthesia with biopsies, pelvis mri (fig. 2a findings were consistent with a moderately well - differentiated scc of the atz, with no evidence of distant disease (t1n0m0). we performed a systematic review of the literature to identify scc arising from the pouch or from the atz of uc patients. inclusion criteria were unequivocal diagnosis of scc that can be reasonably considered as de novo carcinoma, diagnosis of uc, and availability of adequate information. studies including patients with conditions other than uc were only included if uc patients were identifiable. available data of all patients operated on with ipaa between 1978 and 2016 were evaluated. keywords and medical subject headings (mesh) used were : restorative proctocolectomy, ulcerative colitis, squamous cell carcinoma, pouch, ipaa, and scc. the multidisciplinary meeting recommendation for the present case was to proceed with the conventional radiation dose to the expected effects on the ileal pelvic pouch and to the nodal spread. he received a single administration of mitomycin (13.5 mg) and daily capecitabine tablets during the course of radiotherapy (1,300 mg / day). 4) and only suffered from pain and ulceration to the skin during crt. at the 24-month follow - up, the patient has acceptable function, no incontinence nor urgency, and good control of flatus. on endoscopy, the mucosa of the pouch was healthy, but a stricture with inflammation was noticed at the pouch inlet (fig. which did not fit inclusion criteria, only 5 papers reporting on 7 scc were available for analysis (table 1). when matching the series of several centres, including a total of 5,136 patients, only 3 cases of scc have been observed over time, corresponding to a cumulative incidence of 0.058% 25 years after ipaa [3, 4, 5, 6, 7 ]. five scc developed from either the atz or rectal cuff [7, 8, 9 ] and 2 from the mucosa of the pouch [10, 11 ]. out of 2 patients with scc of the pouch, 1 with scc close to the ileal pouch inlet was unsuitable for crt and died 6 months after pouch excision and 1 had pouch excision followed by crt (mitomycin c, 5-fluorouracil, and local radiotherapy) with no response. only 1 paper reported crt for ileal pouch - related scc arising from the rectal cuff. the patient was a 47-year - old woman, who received 5-fluorouracil, mitomycin c, and external beam radiation therapy and appeared to have a clinical response. several cases of ipaa - related cancers have been reported [1, 12, 13 ]. a systematic review of the literature suggested that the cumulative risk of de novo ipaa - related cancer is 0.33% after removing dubious cases, meaning those that should have been considered either persistence or recurrence of previously treated cancer. dysplasia or cancer on the colectomy specimen is the strongest predictor of ipaa - related cancer [1, 4, 7 ]. patients with these features are 8 times more likely of developing ipaa - related cancer, irrespective of mucosectomy. seven [7, 8, 9, 10, 11 ] ipaa - related scc have been described, of which 5 developed from either the atz or rectal cuff [7, 8, 9 ] and 2 from the mucosa of the ileal pouch [10, 11 ] (table 1). dysplasia on the specimen is considered a risk factor for scc, but metaplasia of the ileal pouch could underlie scc, especially squamous metaplasia [10, 11 ]. this is poorly addressed in uc, whereas more attention is paid to the detection of dysplasia for subsequent cancer concern and surveillance. crt is the treatment of choice for anal scc, but it can also achieve complete response in up to 66% of patients with rectal scc. it is largely agreed that pelvic irradiation is contraindicated in patients with a pelvic ileal pouch because of the predictable risk of radiation enteritis, which is likely to cause pouch failure due to structural damage to the mucosa and body of the reservoir. however, no studies have addressed the actual role of radiotherapy in these patients, and these considerations might be even more interesting under the light of newer treatment regimens. one paper reported crt for an early tumour discovered on routine anal biopsy, and no description is provided concerning the stage [8, 11 ]. moreover, the patient suffered from poor function, with seepage and 20 bowel movements per day. when acute toxicity settled down, good function was restored, but the patient required diphenoxylate hydrochloride and atropine sulfate 8 times daily. no data are available concerning the long - term effects of crt and treatment outcomes. the largest series on the topic suggested that patients with rectal cancer can have ipaa, but radiotherapy before pouch surgery impairs subsequent function and increases failure rates. european guidelines currently recommend that radiotherapy should precede pouch surgery and should be avoided after ipaa, even acknowledging that the evidence for that assumption is low. indeed, scc located in the pouch body seem to be more aggressive and might be difficult to treat with radiation therapy. out of 2 patients with scc occurring in the ileal pouch, 1 with scc close to the ileal pouch inlet was unsuitable for crt and died 6 months after pouch excision, and 1 was approached with pouch excision followed by crt (mitomycin c, 5-fluorouracil, and local radiotherapy) but had no response. we were able to manage an scc of the ileal pouch by means of a crt protocol that achieved complete response, without the adverse effects of radiotherapy on pouch function. avoiding excessive worsening of function is important, especially in frail patients, who may suffer from serious dehydration and electrolyte loss secondary to increased ileal pouch - anal output. the early outcome was acceptable, and no particular measures were required apart from loperamide administration. in the long term, the patient did not need any surgical treatment, which is associated with a doubled risk of failure and dysfunction over time in ipaa [19, 20 ]. however, the decision on how to treat patients with scc and anal cancer of the pelvic pouch needs to be individualized, and patients and their relatives should participate in the decision - making process. scheduled follow - up of the pouch is usually not recommended in uc patients without risk factors for cancer. indeed, the risk of colorectal cancer arising from the rectal remnant or atz is lower after ipaa than the lifetime risk of cancer in the general population. however, endoscopic follow - up and surveillance after ipaa probably need to be further elucidated [13, 21 ] because scc may occur at some point even in patients with no known risk factor. early detection is the key to preserve the pouch in patients who develop ipaa - related scc. crt according to our regimen could be considered in scc arising from the rectal cuff or atz. this could be the first - line approach in anal scc after ipaa, but it could also be advocated for other types of cancer, hopefully increasing the success rates and survival. metaplasia of the pouch might play a role in scc, but this remains to be further elucidated. patients with pelvic pouches receiving crt should be followed up with scheduled long - term appointments including pouchoscopy to diagnose recurrence and late adverse effects of radiation. written informed consent for patient information and images to be published was provided by the patient. | backgroundfew cases of pouch - related cancers have been reported in ulcerative colitis (uc), and squamous cell carcinoma (scc) is very rare.methoda systematic review of the literature was performed to identify all unequivocal cases of pouch - related scc in uc patients.resultseight cases of scc developing after ileal pouch - anal anastomosis (ipaa) have been observed since 1978. two arose from the pouch mucosa and 6 from below. the pooled cumulative incidence of scc is below 0.06% after ipaa. many patients had neoplasia on the preoperative specimen, but squamous metaplasia of the pouch or anorectal mucosa may have an important role in scc. these patients are rarely offered chemoradiation therapy and the outcome is poor. selected patients with scc located close to the pouch outlet can be treated with chemoradiation prior to consideration of surgery and salvage their pouch. a chemoradiation regimen is suggested to avoid pouch excision in these patients.conclusionsscc is rare after pouch surgery but associated with extremely poor survival. very low scc can be managed with chemoradiation treatment, preserving the pouch and avoiding surgery, even in older patients. the role of pouch metaplasia, surveillance frequency, and treatment modalities after ipaa need further studying. |
carpal tunnel syndrome (cts) represents the most common compressive peripheral neuropathy, predominantly presenting as a tingling sensation, pain and numbness in the distribution of the median nerve and it is estimated to affect about 1% of the population33,37). cts is usually diagnosed on the basis of the patient 's history and clinical findings, and confirmed by electrophysiological studies. recently, ultrasonography and/or magnetic resonance imaging can also be used to evaluate the median nerve and surrounding structures to complement electrophysiological study in diagnosing cts12,35). when conservative treatment including medications, splinting or local injection of steroid into carpal tunnel fails, simple decompression of the median nerve by division of the transverse carpal ligament is indicated as an effective surgical treatment. as surgical technique, open carpal tunnel release (octr) has been the gold standard method. since the introduction of endoscopic technique by okutsu.32) in 1987 for treatment of cts, endoscopic carpal tunnel release (ectr) has been widely used as a minimally - invasive carpal tunnel surgery22). ectr techniques are divided into two categories depending on the number of skin incision used to access carpal tunnel : single - portal vs. dual - portal techniques. ectr has advantage over octr such as less postoperative pain and more rapid postoperative recovery but there is still major controversy between the two procedures regarding the outcome and the complication rates5,22,31,41). successful results after endoscopic carpal tunnel decompression have been reported with range from 92 to 98%10,22,40). in most cts patients with successful postoperative outcome, preoperative tingling sensation and pain decreased significantly within 1 - 7 days after ectr and completely resolved by one - postoperative month31,41). however, a small number of patients showed delayed improvement after 1 month following ectr. although various studies have reported preoperative prognostic indicators of the outcome of carpal tunnel release8,23,30,43,44), to the knowledge of the authors, there have been no studies that have analyzed the delayed improvement patients after endoscopic carpal tunnel surgery. we analyzed patient subpopulation with the delayed improvement after ectr to investigate the characteristics of those patients and to determine the predictable factors of delayed improvement. we retrospectively reviewed databases of cts patients who underwent ectr at our hospital from 2002 to 2011. the preoperative assessment included a detailed history, symptoms and physical examination and all patients undergoing ectr had electrodiagnostic studies confirming compression of the median nerve at the level of the transverse carpal ligament. however, small number of patients with severe symptoms underwent ectr even though electrodiagnostic studies did not reveal any abnormalities, compatable with cts. patients with postoperative period more than 12 months were analyzed and patients with prior carpal tunnel surgery or patients with diabetes or an underlying disease, related to cts were excluded from the study. the clinical findings in the patient group were classified into 3 grades according to the consistency and severity of symptoms as follows17). grade i : intermittent symptoms with paresthesia at night grade ii : persistent symptoms throughout the day with hypesthesia in the finger of the median nerve distribution grade iii : constant symptoms with weakness or thenar muscle atrophy the electrophysiological study was performed using a cadwell sierra wave (cadwell laboratories, kennewick, wa, usa). the study consisted of motor and sensory median nerve conduction tests using standard techniques according to the practice parameters for the electrodiagnosis of cts of the american association of electrodiagnostic medicine, the american academy of neurology, and the american academy of physical medicine and rehabilitation3). the electrophysiological abnormalities were classified into three grades according to stevens ' classification as follows39). mild : prolonged median sensory distal latency moderate : prolonged median sensory and motor distal latency severe : abnormal needle electromyography other than the above two abnormalities, or no response in sensory and motor distal latency the modified menon 's single - portal method25) was applied to all of the 793 patients as a means of ectr by the same neurosurgeon. ectr was carried out under monitored anesthetic care by intravenous injection of propofol without endotracheal intubation. after exsanguination of the arm and inflation of a tourniquet up to 250 mm hg, a 1.0-cm transverse incision is made along the proximal wrist crease in the ulnar side of the palmaris longus tendon. subligamentous dissection toward the fourth finger is carried out to reflect the synovial tissue from the undersurface of the transverse carpal ligament (tcl) and to create a space for the endoscopic device with a synovial elevator. a washboard sensation could be felt in the undersurface of the tcl with the elevator and its tip could be felt on the palm while it passes the distal part of the tcl. a slotted cannula is introduced into the carpal tunnel and a rigid 30-angled 2.7-mm endoscope is inserted through the cannula. manifestation of reduced transillumination could be observed in the palm while the endoscope is under the tcl. white transversely running fibers of tcl and a fat pad distal to the distal edge of the tcl can be seen on the endoscopic view. after ensuring that there are no intervening structures in the operative field after the tcl is completely cut out, both an escape of palmar fat into the carpal tunnel and retraction of cut edges of the tcl out of the endoscopic view could be observed as well as increased transillumination through the palm. a right - angled probe is used to palpate the divided tcl. the tourniquet is released. wound closure is accomplished in layers following hemostasis and a bulky compressive dressing is applied. clinical severity and electrodiagnostic abnormality were compared between groups using chi - square test and demographic data including age and duration of symptoms was compared using pearson 's correlation analysis. all analyses were performed using spss, version 19.0, commercially available software package (spss inc., chicago, il, usa). we retrospectively reviewed databases of cts patients who underwent ectr at our hospital from 2002 to 2011. the preoperative assessment included a detailed history, symptoms and physical examination and all patients undergoing ectr had electrodiagnostic studies confirming compression of the median nerve at the level of the transverse carpal ligament. however, small number of patients with severe symptoms underwent ectr even though electrodiagnostic studies did not reveal any abnormalities, compatable with cts. patients with postoperative period more than 12 months were analyzed and patients with prior carpal tunnel surgery or patients with diabetes or an underlying disease, related to cts were excluded from the study. the clinical findings in the patient group were classified into 3 grades according to the consistency and severity of symptoms as follows17). grade i : intermittent symptoms with paresthesia at night grade ii : persistent symptoms throughout the day with hypesthesia in the finger of the median nerve distribution grade iii : constant symptoms with weakness or thenar muscle atrophy the electrophysiological study was performed using a cadwell sierra wave (cadwell laboratories, kennewick, wa, usa). the study consisted of motor and sensory median nerve conduction tests using standard techniques according to the practice parameters for the electrodiagnosis of cts of the american association of electrodiagnostic medicine, the american academy of neurology, and the american academy of physical medicine and rehabilitation3). the electrophysiological abnormalities were classified into three grades according to stevens ' classification as follows39). mild : prolonged median sensory distal latency moderate : prolonged median sensory and motor distal latency severe : abnormal needle electromyography other than the above two abnormalities, or no response in sensory and motor distal latency the modified menon 's single - portal method25) was applied to all of the 793 patients as a means of ectr by the same neurosurgeon. ectr was carried out under monitored anesthetic care by intravenous injection of propofol without endotracheal intubation. after exsanguination of the arm and inflation of a tourniquet up to 250 mm hg, a 1.0-cm transverse incision is made along the proximal wrist crease in the ulnar side of the palmaris longus tendon. subligamentous dissection toward the fourth finger is carried out to reflect the synovial tissue from the undersurface of the transverse carpal ligament (tcl) and to create a space for the endoscopic device with a synovial elevator. a washboard sensation could be felt in the undersurface of the tcl with the elevator and its tip could be felt on the palm while it passes the distal part of the tcl. a slotted cannula is introduced into the carpal tunnel and a rigid 30-angled 2.7-mm endoscope is inserted through the cannula. manifestation of reduced transillumination could be observed in the palm while the endoscope is under the tcl. white transversely running fibers of tcl and a fat pad distal to the distal edge of the tcl can be seen on the endoscopic view. after ensuring that there are no intervening structures in the operative field, the tcl is incised proximally to distally with a forward - facing knife. after the tcl is completely cut out, both an escape of palmar fat into the carpal tunnel and retraction of cut edges of the tcl out of the endoscopic view could be observed as well as increased transillumination through the palm. wound closure is accomplished in layers following hemostasis and a bulky compressive dressing is applied. clinical severity and electrodiagnostic abnormality were compared between groups using chi - square test and demographic data including age and duration of symptoms was compared using pearson 's correlation analysis. all analyses were performed using spss, version 19.0, commercially available software package (spss inc., chicago, il, usa). single - portal ectrs were performed in 1194 hands of 793 patients with electrodiagnostically - proven idiopathic cts from 2002 to 2011. among 1194 hands, 570 hands of 388 patients with minimal 1-year postoperative follow - up mean age of the patients was 53 years (range, 24 - 82 years) and mean follow - up period was 23.6 months (range, 12 - 160 months). patients ' clinical outcomes were assessed with postoperative improvement of tingling sensation, pain and numbness at 1, 3, 6, and 12 months postoperatively. among 570 operated hands, 545 hands (95.6%) we divided the 545 satisfied hands into early (group a) and delayed (group b) improvement group according to improvement period of 1 month. the mean age of group a was 53 years (range, 24 - 82 years). the mean age of group b was 52 years (range, 32 - 71 years). mean symptom duration was 42.4 months in group a and 33.4 months in group b. table 1 shows the baseline characteristics of the study groups. in group b, 11 hands improved in postoperative 2-months, 15 hands in postoperative 3-months and 9 hands in postoperative 6-months, respectively. in group a / b, according to clinical severity, 60/1 hands were graded to i, 345/24 hands to ii, 105/10 hands to iii (table 2). in group a / b, based on electrodiagnostic abnormality, 57/3 hands were divided to mild group, 221/11 hands to moderate group and 222/21 hands to severe group with normal findings in 10 hands of group a (table 3). statistical analysis of these results between groups did not reach significance (clinical severity : p=0.191, electrodiagnostic abnormality : p=0.294) but severity of electrodiagnostic abnormalities or clinical severity had a tendency to affect on delayed response. in addition, age and duration of symptoms did not affect the postectr improvement period (age : p=0.478, duration of symptoms : p=0.1). classic symptoms of cts include tingling sensation, pain and numbness in the median nerve distribution of the hand comprising the thumb, index finger, middle finger and radial half of the ring finger. however, sensory symptoms do not always involve the entire median nerve distribution of the hand. in some patients the primary pathophysiologic mechanism of cts development is known as an increase in interstitial pressure in the carpal tunnel, leading to median nerve compression. the normal interstitial pressure in the carpal tunnel is known to be 2.5 mm hg with the wrist in neutral position and it increases to approximately 30 mm hg with the wrist in either maximum flexion or maximal extension26). symptoms of the median nerve compression including tingling sensation develop when carpal tunnel interstitial pressure increases above 30 mm hg, leading to a reduction of epineural blood flow, development of epineural edema and block in axonal transport26). with prolonged compression of the median nerve at higher carpal tunnel interstitial pressure, manifestations of the median nerve compression generally progress over a period of months to years to develop more severe sensory symptoms and even thenar muscle weakness. there are numerous causes of this increase in carpal tunnel pressure but most cases of cts do not have identifiable cause42). however, idiopathic cts is believed to be caused by combination of an anatomically small carpal tunnel and changes in the tenosynovium of the flexor tendons in the carpal tunnel such as edema, vascular sclerosis and fibrous hypertrophy26). secondary causes of cts include neuropathic factor with diabetes, metabolic and physiologic factor such as hypothyroidism, rheumatoid arthritis and pregnancy, and space - occupying lesion with tumor or cyst42). in addition, risk factors in the general population that contribute to cts include repetitive movement of wrist extension and flexion, obesity, rapid dieting, short height, hysterectomy without oophorectomy and recent menopause11). we excluded the cases with secondary cts from the study because the predisposing conditions of an identifiable cause that contribute to cts may affect the surgical outcome, other than known preoperative prognostic indicators8,23,30,43,44). decompression of the carpal tunnel is recommended when symptomatic discomfort interferes with work or activity of daily living despite conservative treatment or advanced cts with thenar muscle weakness even when sensory symptoms are not disabling. in these cases, decompression of the carpal tunnel can be used to improve clinical symptoms and physical findings of cts, to prevent progression and loss of hand function and to eventually minimize loss of time from work. two different types of surgical procedures are in use for the treatment of cts : open and endoscopic release of the transverse carpal ligament. octr is the traditional option and is easy to perform with good symptomatic improvement and a low complication rate. since the introduction of a less invasive carpal tunnel surgery using surgical endoscope by okutsu.32) in 1987, endoscopic procedures have been widely used as an alternative treatment to octr, and single - portal agee technique1) and two - portal chow9) technique are the two most commonly used procedures. we used modification of single - portal menon technique25) using a forward - facing knife to divide the transverse carpal ligament. it is beyond the scope of this study to describe details of each technique so we do not discuss it in this article. since the introduction of endoscopic carpal tunnel surgery in the late 1980s, thereis still controversy regarding the efficacy, safety and complication rate of open and endoscopic procedures. jimenez.22) reported that ectr surgery offer similar success and complication rate compared to open surgery in an extensive review of published articles on ectr surgery covering six different types of techniques. in addition, he described patients undergoing ectr experienced less postoperative pain and earlier return to work or activity of daily living but endoscopic technique need a learning curve and surgical experience for a successful outcome. ashwoth5) also described in a critical review of 57 review articles regarding ectr that ectr surgery, compared to open surgery, seemed equally effective at improving symptoms and hand function in short and long outcome, and it may be more effective at reducing time to return to work. endoscopic surgery was reported to have more transient nerve injuries such as neuropraxia, numbness and paresthesia but open surgery to have more wound problems including scar tenderness, hypertrophic scar and infection. he also reported endoscopic surgery is more resource - intensive and demands a higher surgical skill than open technique. several studies have reported preoperative prognostic factors, affecting the outcome after surgical release of transverse carpal ligament and preoperative predictors included duration and severity of symptoms, grade of electrodiagnostic abnormalities, worker 's compensation and work activity8,23,30,43,44). good outcome has been associated with preoperative tingling other than anesthesia or weakness as well as good response to corticosteroid injection into carpal tunnel and poor result has been correlated with preoperative weakness or atrophy, associated diseases such as diabetis and thyroid disease, worker 's compensation and heavy or repetitive work activity13,18,23,24,29,36,43,44). the finding of delayed improvement in our series demonstrates late postoperative improvement group had more proportion of patient with severe clinical grade, but clinical severity did not differ significantly between early and late improvement groups. even though we can expect poor outcome in patients with severe clinical grade of cts, it is recommended that postoperative follow - up up to 6 months is necessary in idiopathic cts patient without improvement. the duration of cts has been shown to have no significant correlation with clinical outcome in patients undergoing carpal tunnel surgery19,43), whereas, other studies found worse outcome with longer duration2,13,24,27,29,30,44). pauda.34) reported that the main positive prognostic factors of idiopathic cts without treatment are short duration of symptoms and young age. our study has indicated that the chronicity of cts did not affect the time of improvement following ectr. the role of ncs in predicting the outcome of carpal tunnel surgery is not clearly determined2,4,8,21,27,44). schrijver.38) noted that ncs improved significantly at postoperative 12 months but only a modest correlation existed between relief of preoperative symptoms after surgery and the improvement of ncs. however, other studies showed that ncss improve after surgery28). in severe cts cases, nerve conduction studies results may not correlate with the clinical findings due to the varying nature of the impairment in different nerve fibers. in addition, nerve conduction studies will not accurately predict the recovery following release of the carpal tunnel, though neither do any of the other investigations predict this with any certainty6). our study has shown that grade of electrophysiologic abnormalities does not correlate with the time course of improvement after carpal tunnel surgery, which means ncss are not helpful to predict the improvement period. several studies demonstrate that improvements after carpal tunnel release decreased in older patients who were at risk of severe cts, compared to a younger population14,15,16,20). therefore, we can expect older patients to show a more delayed improvement than younger patients but we found no significant differences in the time of improvement following ectr between early and delayed improvement group. in addition, worker 's compensation can affect postoperative outcome and worker 's compensation group showed diminished outcome, compared to patients without worker 's compensation19). in our study, worker 's compensation did not affect postoperative improvement period but we think it may delay postoperative improvement period. therefore, worker 's compensation status can be considered as a possible factor of delayed postoperative improvement in patients with worker 's compensation. endoscopic decompression of carpal tunnel is effective as a primary or alternative surgical technique for the treatment of cts, demonstrating symptomatic improvement within postoperative one - month in most patients. therefore, it is advised that postoperative observation for at least 6 months is necessary in the patients without symptomatic improvement to expect the delayed response. | objectivein most patients with carpal tunnel syndrome (cts), pain and/or paresthesia disappeared or decreased in a month after endoscopic carpal tunnel release (ectr). however, subpopulation of patients showed delayed improvement following ectr. we analyzed the delayed improvement hands to investigate the characteristics of those patients and to determine the predictable factors of delayed improvement.methodssingle-portal ectrs were performed in 1194 hands of 793 cts patients from 2002 to 2011. five - hundred seventy hands with minimal 1-year postoperative follow - up were included. we divided the 545 satisfied hands into early (group a) and delayed (group b) groups according to improvement period of 1 month. demographic data, clinical severity and electrodiagnostic abnormality were compared between groups.resultsgroup a included 510 hands and group b included 35 hands. in group b, 11 hands improved in 2 months, 15 hands in 3 months and 9 hands in 6 months, respectively. in group a / b, according to clinical severity, 60/1 hands were graded to i, 345/24 hands to ii, 105/10 hands to iii. in group a / b, based on electrodiagnostic abnormality, 57/3 hands were classified to mild, 221/11 hands to moderate and 222/21 hands to severe group. statistical analysis between groups did not reach significance but electrodiagnostic or clinical severity had a tendency to affect the delayed response.conclusionit is difficult to predict the factors contributing to postoperatively - delayed response in subpopulation of cts patients. however, we recommend that postoperative observation for at least 6 months is necessary in patients without symptomatic improvement. |
our synthesis was initiated with chromone 12 which was prepared in greater than 50 gram batches and purified by recrystallization (figure 2). utilizing diisopropylsilyl ditriflate to activate chromone 12 to a siloxybenzopyrylium species, vinylogous addition of 2-trimethylsiloxyfuran was achieved which was followed by hydrogenation to afford chromone lactone 13 on adecagram scale (89 % yield, dr > 10:1) after purification by trituration (supplementary information s4s19). further treatment of 13 with nah in thf led to the production of epi - blennolide c 14 through dieckmann cyclization. due to the propensity of thevinylogous acid moiety of tetrahydroxanthone 14 to both epimerization and tetrahydroxanthone - to - chromone lactone rearrangement, we utilized the neutral methylating reagent trimethylsilyldiazomethane to obtain the desired o - methylated product 16 in 61 % yield along with the separable isomer 15 (20 % yield). after phenol - directed ortho - iodination of 16, the derived aryl iodide 17 was subjected to kinetic acylative resolution using birman 's catalyst on a gram scale (s factor > 200). chiral monomer ()-17 was isolated in 46 % yield (99 % ee) and the o - acylated product (+) -18 was obtained in 46 % yield (99 % ee) after recrystallization. after obtaining the two enantiopure tetrahydroxanthone monomers, our initial plan was to employ our stannane dimerization strategy which successfully led to syntheses of secalonic acids a and d. however, after evaluation of various conditions, we observed only trace stannylation of iodides 17 or 18 (table 1) which may be due to steric hindrance of the aryl iodide substrate. gratifyingly, iodide ()-17 was found to be stable under basic conditions and to produce an inseparable mixture of dimeric products (dr = 88 : 12) under one - pot suzuki coupling conditions using pd(oac)2/sphos and bis (pinacolato) diboron. we subsequently attempted to establish the stereochemistry of the major atropisomer by nmr analysis and by acid - mediated deprotection of both atropisomers 19 and 20 to 1 and 23 (figure 4). however, both efforts failed as the axial chirality center is remote from existing stereocenters leading to almost identical h nmr spectra for both a tropisomers of rugulotrosin a. fortunately, a single crystal of the major atropisomer ()-19 was obtained by recrystallization (ch2cl2/meoh) from its mixture with 20. x - ray crystal structure analysis of ()-19 (ccdc#1022610) was achieved which verified that the relative stereochemistry was identical to that found in natural rugulotrosin a. during our evaluation of dimerization conditions, we found that water was essential and that the ratio of atropisomers was dependent on ligands for palladium rather than solvent, temperature, or reaction time. accordingly, we focused our efforts on the influence of ligands on atropisomeric selectivity (table 1). after extensive ligand screening, we found that monophosphines were crucial for the success of there action, and that bidentate ligands such as xantphos or dpephos (not shown) failed to provide the desired dimeric products. besides sphos(l1), the related ligand ruphos (l2) provided dimeric products in even higher atropselectivity (93:7). interestingly, we found when the 2 ' position of the monophophine ligand did not contain an oxygen atom (e.g. carbon atom in xphos l2 or a nitrogen atom (davephos, not shown)), dimeric products were obtained in trace amounts. after experiments with the achiral ligand l4, which provided similar results to sphos, we anticipated that related chiral ligands may achieve efficient control of atropselectivity. we began this investigation with (r) and (s)-bi - dime ligands l5, which recently have shown excellent atropselectivities in biaryl couplings. however, neither ligand was able to perform the one - pot dimerization of ()-12. gratifyingly, chiral versions of the sphos ligands, s - cy - mop (l6) and r - cy - mop (l7), both successfully produced dimeric products. the ligand r - cy - mop provided a satisfactory dr (95.5:4.5) and the mismatched ligand s - cy - mop afforded a lower dr (81:19), both favoring the same major atropisomer. even with the increased bulk of ligand l8, the dr did not noticeably improve. in order to enhance product yield, we prepared the buchwald 3rd generation precatalysts with sphos, r - cy - mop, and s - cy - mop. indeed, use of the precatalyst increased yields for these cases and maintained at ropselectivity. use of bis (neopentyl glycolato) diboron ortetrahydroxydiboron as boron transfer reagents led only to trace amounts of dimeric products. the acylated, enantiopure monomer (+) -18 was also investigated in the suzuki dimerization which generated a separable 3:1 mixture of atropisomersin 36 % yield (figure 3). fortunately, an x - ray crystal structure of the minor atropisomer ()-22 (ccdc#1022611) was obtained to verify relative and absolute stereochemistry. introduction of a bulk yester onto the secondary alcohol of the monomer (e.g. ocoph) did not affect the ratio of atropisomers. after obtaining and establishing stereochemistry for several protected dimers, we anticipated that we could access all atropisomers and enantiomers of rugulotrosin a. dimer ()-19 was treated with 3 m hcl / meoh for 10 min which afforded rugulotrosin a (()-1) in 89 % yield (figure 4, a). in a similar manner, (+) -rugulotrosin a was obtained from acidic hydrolysis of (+) -21 (figure 4, b). with both enantiomers of rugulotrosin a in hand, natural rugulotrosin a was unambiguously assigned as (+) -1 though comparison of rotation data. similarly, atrop - rugulotrosin a ()-23 and entatrop rugulotrosin a (+) -23 were synthesized using similar protocols from the protected precursors ()-22 and (+) -22 (figure 4, c and d). to verify the stability of the axially chiral biaryl moiety, we heated rugulotrosin a (+) -1 in toluene (1 mg / ml) at 100 c for 12 h and 150 c for 3 h. under both conditions, we did not observe formation of atrop rugulotrosin. this is supported by a high barrier of rotation of rugulotrosin a, calculated as 47.7 kcal / mol using the b3lyp/6 - 31g(d) level of theory (see supplementary information s52s58). in order to rationalize the axial chirality selectivity observed in the dimerization, we modeled the geometry of the expected intermediate diaryl palladium (ii) complexes using sphos (l1) as ligand (figure 5). conformational analysis was carried out by systematic six - fold rotation about the c - pd bonds, followed by optimization of candidate structures at the b3lyp / lanl2dz level of theory. the three lowest energy structures 24a 24c all have dihedral angles (c1, c2, c2, c1) for the monomer fragments which would afford atropisomer ()-19 (precursor to rugulotrosin) after stereospecific reductive elimination (figure 5,a). the next two higher energy structures 25a and 25b display a reversed dihedral angle which should lead to atropisomer 20 (table 1). space filling models of structures 24a - c and 25a - b (supplementary information s34s52) show a more crowded arrangement of the latter, in part because of steric interactions between the methyl ester and the cyclohexyl group. the distances between the hydrogen of the methyl ester to the hydrogen on the cyclohexyl of the sphos ligand are 2.4 and 2.5 for conformers 25a and 25b, respectively. based on literature precedent, we assume that these catalyst - reactant complexes are under thermodynamic equilibration ; additional computational studies will be required to predict barriers to interconversion. it thus appears that product stereochemistry is determined by reactant and catalyst assembly in the pd (ii) complex ; this provides a basis for prediction of stereochemistry in future atropselective couplings. after assigning the absolute configuration of rugulotrosin a, we employed our library of synthetic rugulotrosins to determine whether penicillium nov. (mst - f8741) was capable of producing atrop - rugulotrosin a. to this end, separate 21 day fermentations of mst - f8741 were extracted with either mecn (figure 4,e(a)) or meoh (figure 4, e(b)) and were subsequently analyzed using an hplc - dad method optimized for the resolution of rugulotrosin a (figure 4, e(c, d)) and atrop - rugulotrosin a (figure 4, e(e)). to further enhance sensitivity, these analyses were also carried out using hplc - dad - esim sprotocols augmented by single ion extraction (sie) (supplementary information, s22). as these analytical studies failed to detect any trace of atrop - rugulotrosin in mst - f8741 extracts, we conclude that the rugulotrosin biosynthetic pathway present in penicillium nov. a had previously been reported to exhibit antibacterial activity, we also tested the synthetic rugulotrosins ((+) -1, ()-1, ()-23, and (+) -23) against several strains of gram - positive and gram - negative bacteria. consistent with earlier reports,(+)-1 exhibited antibacterial activity against bacillus subtilis (atcc 6633) (ic50 = 2.1 m) and staphylococcus aureus (atcc 25923) (ic50 = 6 m). of note, the enantiomer ()-1 was also active against b. subtilis (atcc 6633) (ic50 = 0.7 m) and s. aureus (atcc 25923) (ic50 = 18 m), whereas the antibacterial activity of the atropisomer ()-23 was weak and limited to b. subtilis (atcc 6633) (ic50 = 10 m) and (+) -23 lacked any appreciable antibacterial activity. none of the rugulotrosins (+) -1, ()-1, ()-23, or (+) -23 exhibited activity against the gram - negative bacteria escherichia coli (atcc 25922) and pseudomonas aeruginosa (atcc 27853), nor did they exhibit cytotoxicity (ic50>30 m) against human colon (sw620) and lung (nci - h460) cancer cells. in conclusion, we have developed a concise, atropselective approach to rugulotrosin a and stereoisomers through point - to - axial chirality transfer which has facilitated determination of the absolute configuration of rugulotrosin a. computational studies modelling the geometry of intermediate diaryl palladium (ii) complexes have provided a rationale for the atropselectivity observed in the key suzuki dimerization. these studies highlight the utility of sphos and related bulky monophosphine ligands in palladium couplings both to bring monomers in proximity and enhance remote steric effects leading to atropselectivity. through hplc analysis of fungal extracts and synthetic samples,. moreover, the atropisomers and enantiomers of rugulotrosin a were found to have different activities against gram - positive bacteria illustrating the importance of stereochemistry on target selectivity. further studies regarding atropselective syntheses of dimeric natural products are currently under investigation and will be reported in due course. | chiral, dimeric natural products containing complex structures and interesting biological properties have inspired chemists and biologists for decades. a seven step total synthesis of the axially chiral, dimeric tetrahydroxanthone natural product rugulotrosin a is described. the synthesis employs a one - pot suzuki coupling / dimerization to generate the requisite 2,2'-linked biaryl linkage. highly selective point - to - axial chirality transfer was achieved using palladium catalysis with achiral phosphine ligands. single x - ray crystal diffraction data was obtained to confirm both the atropisomeric configuration and absolute stereochemistry of rugulotrosin a. computational studies are described to rationalize the atropselectivity observed in the key dimerization step. comparison of the crude fungal extract with synthetic rugulotros in a and its atropisomer verified that nature generates a single atropisomer of the natural product. |
the term synapse has been defined as a specialized structure allowing the functional interaction between neurons. this structure has morphologic and functional characteristics capable of directing and modulating neuronal signals. following the classical studies of furshpan and potter, it was recognized that beside the chemical synapses, there was another type of neurotransmission in the nervous system denominated as electrical synapses. in the well - known chemical synapses, the communication is carried out through the release of neurotransmitters from the presynaptic neuron that bind to specific receptors, causing changes in the ionic permeability of the postsynaptic membrane. by contrast, in the electrical synapses, the communication is done by gap junctions (gj) that are clusters of intercellular channels allowing the bidirectional transit of ions directly into the cytoplasm of adjacent cells [2, 3 ]. gj are formed by two assembled hemichannels, each one located in the neighboring membranes and constituted by six subunits of integral membrane proteins called connexins (cx). the cx gene family is represented by 21 members expressed in human and 20 in the mouse genome, and the proteins encoded by these genes have been classified and named by its molecular weight [4, 5 ]. the localization of electrical synapses in the brain has been extrapolated from the expression patterns of cx. therefore, it has been determined that approximately half of the cx isoforms are present in the mammalian brain. astrocytes are characterized by the high levels of the subunits cx30 and cx43, which change their expression in an age dependent manner. it has been established that cx30 and cx43 are necessary for different cellular processes including neurogenesis and long - term synaptic plasticity. recent evidence indicates that the presence of cx32 and cx47 in oligodendrocytes is related to the gap junctional coupling with astrocytes and also directly with other oligodendrocytes [10, 11 ]. because oligodendrocytes are crucial for the myelination process, several human demyelinating disorders have been described which are caused by mutations in the genes that encode for cx32 and cx47. the first studies related to gj located in neuronal cells revealed that cx36 is highly expressed in neurons during the early postnatal stages, however, the abundant immuno- reactivity decreases in the adult brain [13, 14 ]. subsequent studies about the expression patterns of cx in neuronal cells showed that cx45 is relatively abundant in both neonatal and adult mouse neurons. moreover, it has been described recently that neurons in the spinal dorsal horn express cx45. due to its extensive expression in the nervous system, both gj and cx seem to be essential elements in the development and physiology of the brain. several studies have proposed that gj and cx participate in several brain processes. in this regard, one hypothesis indicates that electrical coupling mediated by gj plays a role in the generation of highly synchronized electrical activity [17, 18 ]. because the hypersynchronous neuronal activity is a distinctive characteristic of convulsive events, it has been postulated that enhanced gap junctional communication is an underlying mechanism involved in the generation and maintenance of seizures [19, 20 ]. the electrical synapses allow the bidirectional transit of ionic currents and, therefore, can produce changes in the membrane potential of neighboring neurons. these features provide velocity and reciprocity to the communication allowing the synchronization of neuronal networks. although excitatory and inhibitory chemical transmission could be enough to synchronize neuronal activity, it has been observed that such activity is modified in the absence of gj. specifically, the elimination of electrical coupling in interneurons and pyramidal cells in the ca3 area and dentate gyrus of cx36 knockout mice has been explored, and these cx36-deficient mice showed a decrease of gamma frequency oscillations induced by kainate and carbachol in the ca3 region of the hippocampus. despite the fact that synchronization in seizures is a very complex phenomenon that depends on several factors, there is some consensus about the importance of the participation of gj. therefore, several studies have shown alterations of cx expression in both animal seizure models and epileptic patients. recently, akbarpour and colleagues evaluated the expression levels of cx, in astrocytes and oligodendrocytes, during kindling epileptogenesis. they found that cx30 was upregulated in the hippocampus after the first amygdaline electrical stimulation. with the same seizure experimental model, other authors investigated mrna and protein levels of cx in neurons, and described that cx36 was upregulated in the hippocampus of rats with partial seizures. also, it has been described that after repeated seizures the levels of mrna of cx32, cx36 and cx43 significantly increased at the epileptic foci located in the somatosensory cortex. contrariwise, another study demonstrated that injection of 4-aminopyridine induced systemic seizures related to decreased cx36 expression levels in the hippocampus of mice. the relation between seizures and cx expression has also been explored in humans, and the most common result has been the increase of cx43 in the hippocampus obtained from epileptic patients [28, 29 ]. these results not only support the hypothesis about the participation of gap junctional communication on seizures, but also show that the expression pattern of cx strongly depends on the animal model, brain region, and seizure duration. in the literature, there is a variety of chemical compounds characterized as gj blockers. although the mechanisms are not well defined, the efficacy of the gj blockers has been principally evaluated using techniques to measure dye transfer and electrical conductance. after such characterization, the designation as gj blocker has been given to some chemical compounds that have shown the ability to disrupt the gap junctional intercellular communication. in comparison to in vivo experiments, gj blockers have been used in a broader variety of in vitro studies. however, although the data are limited, the behavioral, cognitive and electrophysiological effects of many gj blockers have been reviewed in the past. there is the postulate of the enhanced gap junctional intercellular communication as an underlying mechanism involved in the generation and maintenance of seizures [19, 20 ]. for this reason, this review has focused in analyzing the available data concerning the effects of gj blockers specifically in animal seizure models. unfortunately, the medical use of cbx has been limited because of the several side effects associated with the mineralocorticoid - like actions. interestingly, it was demonstrated that cbx produced inhibition of the gap junctional intercellular communication but without a clear selectivity for particular subtypes of cx [34, 35 ]. after this discovery, many studies focused on evaluating cbx in diverse models of processes related to the gap junctional intercellular communication in the brain [36, 37 ]. the first reports that studied the relationship between cbx and epileptiform activity were carried out in hippocampal slices. some studies described that cbx delayed the induction and reduced the well - established epileptiform activity induced by adding 4-amynopiridine or omitting mg from the slices perfusate [38, 39 ]. also, kaglund. confirmed that cbx also reduced both the frequency and amplitude of epileptic field bursts induced by cesium or low calcium in hippocampal slices. more recently, two studies using genetic and pharmacological models of seizures determined that cbx significantly decreased the amplitude and duration of seizure - like activity in thalamocortical slices [41, 42 ]. these in vitro reports established the basis for the subsequent evaluation of cbx in in vivo models. administration of cbx (40 - 300 mg/ kg) delayed the onset of seizures and reduced the duration of clonic seizures induced by pentylenetetrazole (ptz) [43, 44 ]. similar results but with low doses of cbx (5 - 30 mg / kg), were observed in audiogenic seizures in a genetic model of epilepsy - prone rats. conversely, the same research group reported that neither intravenous nor i.p. administration of cbx had any effect on the number nor duration of spike - wave discharges in a genetic animal model of absence epilepsy. by contrast, gigout. using a similar genetic animal model, described that systemic doses of cbx (100 mg / kg) significantly decreased the duration of spike - wave discharges. administration of cbx protects against tonic seizures induced by maximal electroshock reducing both the incidence of tonic hindlimb extension (thle) and the duration of the thle (fig. some studies have tested the application of cbx directly into the brain to identify specific anatomical substrates where cbx could be exerting its anticonvulsant effects. in this regard, some authors evaluated the effects of cbx directly in the epileptic focus induced by the application of 4-aminopyridine or tetanus toxin on the somatosensory or entorhinal cortex. they found that the direct blockage of gj with cbx in the epileptic focus decreased both the duration of seizures and the amplitude of seizures discharges [26,48 - 50 ]. interestingly, several studies have revealed that microinjection of cbx into the inferior colliculus, substantia nigra, inferior olivary complex, basolateral amygdala and hippocampus reduced the duration and severity of seizures. additionally, it has been reported that bilateral microinjection into ventro- posteromedial thalamic nucleus did not produce any significant effect. by contrast, in a model of absence seizures, cbx microinjected into the reticular nucleus of the thalamus decreased the duration of seizures. likewise, results recently obtained in our laboratory have showed that cbx microinjected into the reticular formation reduces the incidence of generalized tonic - clonic seizures and prevented the epileptiform activity induced by ptz. in conclusion, all of these data have showed that cbx has anticonvulsant effects when administered in different seizure experimental models. some historical records suggest that quin has been used for the treatment of malaria since almost 400 years ago [54, 55 ]. however, due to some adverse effects, the world health organization recommended avoiding the use of quin as a first - line treatment reducing its use only to cases of severe malaria. the first studies concerning quin and excitable cells revealed that this antimalarial drug could block ionic channels and, therefore, suppress some ionic conductances [57, 58 ]. also, it was showed that quin modulated the activity of hemichannels in neurons from the vertebrate retina, and also in oocytes expressing some isoforms of cx [59, 60 ]. subsequently, srinivas. demonstrated for the first time that quin blocked gj formed by cx36 and cx50 in a reversible and dose - dependent way. since cx36 is expressed in mammalian neurons [13 - 14 ] few studies have explored the possible effects of quin on in vitro and in vivo experimental models of seizures. the first report in this subject described that quin blocked the excitability of ca3 pyramidal neurons in hippocampal slices, through the modification of the properties of the membrane resting potential, and the duration and amplitude of spikes. also using hippocampal slices, uusisaari. induced synchronous bursting in pyramidal neurons exposing slices to gabab receptor antagonists and discovered that quin reversibly blocked the denominated gabaergic ictal - like events. interestingly, another study tested the in vitro effects induced by quin in the neocortex of patients with temporal lobe epilepsy. this study showed that quin decreased irreversibly the occurrence of synchronous field potential activity produced by the application of 4-aminopirydine. paradoxically, there is a report that describes excitatory effects of this gj blocker in rat slices. specifically, it was found that quin increased the frequency and amplitude of seizure - like activity induced by low magnesium in neocortical slices. however, it was also described that at high doses, quin caused a biphasic effect characterized by an initial excitatory phase followed by an apparent reduction in seizure - like activity. some in vivo reports have established that quin could have effects on seizure experimental models. administration of quin (25 - 100 mg / kg), reduced both the incidence and duration of seizures and modified the latency to the tonic and myoclonic phases in mice administered with ptz. however, they did not find significant effects when analyzed the effects of quin on maximal electroshock seizures. administration of quin (60 - 240 mg / kg) only elicited a slight protection against the incidence and duration of thle induced by maximal electroshock (fig. thus, in rats, quin (200, 400 and 1000 nmol) significantly decreased the frequency and amplitude of the epileptiform activity induced by the intracerebro- ventricular (icv) injection of penicillin. likewise, another study reported that icv administration of quin (0.5 and 1 mm) significantly increased the latency and decreased the duration of the generalized tonic - clonic seizures induced by ptz. on the other hand, some studies have tested the effects of quin applied directly to cortical areas, and the results have been consistent.. demonstrated that pretreatment with quin on the somatosensory cortex slightly reduced the epileptogenesis induced by 4-aminopirydine. however, when quin was applied to contralateral and ipsilateral cortical areas after the constant expression of seizures, the duration and propagation of seizures decreased significantly. similarly, two reports have established that microinjection of quin into the entorhinal cortex modified the characteristics of the hippocampal epileptiform activity induced by both 4-aminopirydine and pilocarpine [71, 72 ]. the walter reed army institute of research in the usa developed it in the early 1970s. initially, mfq was indicated for the treatment and prophylaxis of malaria ; however, currently it is no longer considered by the world health organization as a first - line drug to eliminate the parasite causing malaria. before its proposal as gj blocker, some studies had reported that mfq has the capability to shorten action potential duration, decreasing currents through l - type calcium channels. later, it was demonstrated that mfq reduced, in a concentration - dependent manner, the intercellular gap junctional currents in n2a cells selectively expressing cx36 and cx50. consequently, mfq was evaluated on the brain due to the expression of cx36 in neuronal cells. therefore, it was determined that mfq reduced, in a time and concentration - dependent manner, the electrical coupling of interneurons in neocortex slices. there is a significant decrease in the excitatory activity of the hippocampus after the application of mfq. however, another report described that mfq induced increases in both the amplitude and frequency of seizure - like events in cortical slices of rats. although this is a still unexplored issue, it has been proposed that the opposing effects observed could be related to the doses of mfq used. voss. reported that, in mouse neocortical slices, mfq (25 m) has no effects on the seizure - like activity induced by perfusion with low concentrations of magnesium. on the other hand, more recently it was described that mfq (10 m) significantly decreased the amplitude and frequency of seizure - like activity induced by the coadministration of 4-aminipyridine and bicuculline in thalamocingulate mice slices. due to the lack of in vivo studies, we have worked on clarifying the controversy about mfq and seizures. significantly decreased the incidence of seizures and the total spectral power of the epileptiform activity induced by ptz. we have also observed a significant reduction of the duration of thle induced by maximal electroshock (fig. most likely, the lack of in vivo studies is because some reports have suggested that mfq could cause undesired neurological effects at doses around 187 mg / kg. we have observed that mfq (40 mg / kg) elicited some protection against the seizures induced by two acute models of seizures (fig., it seems reasonable to propose that mfq acts in a dose - dependent manner, showing anticonvulsant effects at small doses and neurotoxic effects at high doses. qnd is another compound derived from the chinchona tree bark and chemically related to quin. the use of qnd dates since 1914 when it was demonstrated that this compound could be beneficial in the treatment of cardiac arrhythmias. in addition to its antiarrhythmic effect, the center for disease control and prevention has established that qnd gluconate is the only antimalarial agent available for parental administration in the united states. some classical studies reported that the application of qnd to neurons provoked a clear block of ionic conductances though na+, k+ and ca+ channels, and consequently caused modifications of action potentials properties [81 - 83 ]. however, after a meticulous analysis, malchow. proposed for the first time that qnd could modify the gap junctional communication between neurons. more recently, this hypothesis was explored using n2a cells transfected with cx50 channels, and it was demonstrated that qnd (300 m) significantly inhibited the currents in gj formed by cx50. one study has reported that qnd induces an increase in the frequency of seizure - like events caused by the addition of low concentrations of magnesium to rat cortical slices. by contrast, it has been showed that qnd abolishes the ictal - like activities induced by 4-aminopyridine in rat thalamocortical slices. evaluated the effects of qnd but in slices obtained from the neocortex of temporal lobe epileptic patients ; they found that qnd decreases irreversibly the frequency of occurrence of the synchronous field potential activity induced by 4-aminopirydine. using in vivo models, steriade and stoica performed well - designed experiments and reported that qnd (4 - 30 mg / kg) caused significant anticonvulsant effects in experimental models such as electroshock and cortical application of penicillin. in contrast, we have observed that higher doses of qnd (120 mg / kg) are necessary to generate some protection against the thle induced by maximal electroshock (fig. these previous information has led us to suggest that the relation between qnd and seizure experimental models is nowadays underrepresented, and that it is necessary more in vivo studies to explore more in depth this topic. ana and ole are members of the cannabinoid family and these endogenous molecules bind and activate the cannabinoid receptor 1. some reports have demonstrated that these endocannabinoids have effects on ca+ channels and significantly increase the intracellular concentration of ca in different cell types [87, 88 ]. interestingly, it has been described that both, ana and ole, are potent inhibitors of intercellular gap junctional communication in glial cells, and ana also blocked calcium wave propagation in striatal astrocytes [89, 90 ]. following the effects of ana and ole on gap junctional communication, some authors have evaluated their effects on animal seizure models, including two reports that have described ana role in the modulation of seizures induced by maximal electroshock [91, 92 ]. specifically, one report described a modest anticonvulsant activity with ana alone (50 mg / kg). however, the combination of ana (300 mg / kg) with an inhibitor of amidohydrolase, which rapidly metabolize ana, generated a complete protection against seizures induced by electroshock. similar studies have demonstrated that inhibition of amidohydrolase increased the levels of ana in the brain, and reduced the severity of seizures induced by application of kainic acid [93, 94 ]. ana has also been evaluated in other animal seizure models such as ptz and a genetic model of absence epilepsy. interestingly, mannea and umathe have reported that icv administration of ana showed dose - dependent effects ; namely, at small doses (10 - 40 g) ana produced anticonvulsant effects, while at high doses (80 - 160 g) ana increased the percentage of animals with seizures induced by application of ptz. more recently, it has been reported that icv administration of ana significantly decreases in a dose - dependent manner, the presence and duration of spike - wave discharges related to absence seizures. there are few studies relating ole and seizures ; however, they are consistent with those using ana. hence, it has been showed that ole (43 - 700 mg / kg, i.p.) inhibited the seizures induced by ptz but not those induced by picrotoxin or strychnine [97, 98 ]. in addition to the substances previously mentioned, more compounds have been characterized as gj blockers. some of these compounds include meclofenamic acid, niflumic acid, flufenamic acid, heptanol, octanol, glycyrrhetinic acid and retinoic acid. these chemicals have shown ability to block hemichannels and gj composed of cx26, cx32, cx36, cx40, cx43, cx46 and cx50 [99 - 105 ] (table 1). the meclofenamic, niflumic and flufenamic acids are grouped in the fenamates family, which is a group of drugs used as anti - inflammatories and analgesics. of these three compounds, both meclofenamic and flufenamic acids have been evaluated in relation to seizures, and it has been shown that i.p. administration of meclofenamic acid increases the latency to the onset of seizures induced by ptz. additionally, in rats with epileptic focus caused by tetanus toxin, a significant reduction in seizure duration was observed when meclofenamic acid was applied directly to the cortex. similarly, schiller stimulated neocortical slices with a gabaa receptor blocker to evoke seizure - like events, and observed that addition of flufenamic acid (100 - 200 m) reversibly eliminated the seizure - like events. heptanol and octanol are long carbon chain n - alkanols capable of modifying the gap junctional intercellular communication [102, 103 ]. therefore, it was demonstrated that perfusion of entorhinal / hippocampal slices with octanol blocked the primary afterdischarges produced by the tetanic stimulation of schaffers collaterals. using the same model, dantuono. showed that octanol blocked theta activity of the epileptiform afterdischarges induced by the application of picrotoxin. heptanol has also been evaluated in hippocampal slices with similar results to those exhibited by octanol. namely, heptanol significantly depressed the spontaneous field burst and the repetitive population spikes evoked by fimbrial stimulation. to date, there are few studies reporting data about the use of octanol on in vivo seizure experimental models. relatedly, it was showed that octanol reduced seizure induction and seizure discharges when it was applied directly to the epileptic focus in the somatosensory cortex. additionally, it has been reported that icv administration of octanol significantly reduces the frequency and amplitude of epileptiform spikes, as well as the epileptic behavioral score induced by the icv administration of penicillin. interestingly, it has been shown that this chemical displays some anti - tumoral, anti - allergic and anti - inflammatory effects. the first studies concerning glycyrrhetinic acid in the context of epileptogenesis were carried out by de curtis. ; they described that glycyrrhetinic acid eliminated the spontaneous interictal spikes in an in vivo model of focal epileptogenesis. more recently, using rats implanted with a cannula into the reticular nucleus of the thalamus, it was found that glycyrrhetinic acid significantly decreased the duration of atypical absence seizures. interestingly, despite the fact that glycyrrhetinic acid is a compound that is chemically related to cbx, its effects on animal seizure models are not yet fully explored. retinoic acid, a product of the metabolism of vitamin a, has also been reported as a gj blocker. specifically, it has been observed in retinal cells that retinoic acid was able to reduce, in a dose - dependent manner, the amplitude of the gap junctional conductance. evaluated the infusion of retinoic acid directly into the amygdala of rats, and they observed that retinoic acid significantly reduced both the afterdischarge duration and the seizures generated in rats electrically stimulated in the amygdala. although these results seem consistent with the blockage of gj, other recent evidence has suggested that retinoic acid not only increased the expression of cx32 and cx43 but also increased the gap junctional intercellular communication [115, 116 ]. an alternative approach to precisely block gj and cx hemichannels consists of using specific antibodies or small peptide fragments corresponding to intracellular amino acid sequences of diverse cx. thus, the first studies done by moore and burt described that cx - specific antisense oligodeoxynucleotides could reduce the frequency of unitary conductances in cells expressing cx40 and cx43. later, it was established that cells incubated with a synthetic oligopeptide corresponding to a segment of the second extracellular loop of cx43, showed decreased dye coupling and dual whole - cell voltage clamp, indicating a reduction of the cell - to - cell coupling. the cx - mimetic peptides have been proposed as specific and reversible blockers of gj and cx hemichannels ; however, it has been reported that these short amino acid sequences have the ability to inhibit currents from channels constituted of proteins topologically similar to cx, called pannexins (panx). from all the synthesized peptides, gap26 and 27 are the most widely used because they correspond to specific sequences within the extracellular loops of cx37, 40 and 43. although the detailed mechanism of action of the cx - mimetic peptides is unknown, it has been suggested that they interact with the extracellular loops, disrupting the docking of the hemichannels and, therefore reducing the assembly of functional gj [120, 121 ]. to evaluate the effects of cx - mimetic peptides concerning epileptiform activity, samoilova. studied the administration of gap27 to hippocampal slices and detected that spontaneous recurrent epileptiform activity was diminished but only after 10 hours of gap27 treatment. on the other hand, it has been reported that application of a mimetic peptide in hippocampal slices, targeted to the extracellular loop two of cx43, evoked a dose- and exposure time - dependent response, preventing the seizure - induced neuronal death caused by the application of an antagonist of gaba - a receptors. based on these reports, it has been suggested that blockage of gj and hemichannels constituted by cx43 could prevent the neuronal damage induced by the epileptiform neuronal activity. unfortunately, the medical use of cbx has been limited because of the several side effects associated with the mineralocorticoid - like actions. interestingly, it was demonstrated that cbx produced inhibition of the gap junctional intercellular communication but without a clear selectivity for particular subtypes of cx [34, 35 ]. after this discovery, many studies focused on evaluating cbx in diverse models of processes related to the gap junctional intercellular communication in the brain [36, 37 ]. the first reports that studied the relationship between cbx and epileptiform activity were carried out in hippocampal slices. some studies described that cbx delayed the induction and reduced the well - established epileptiform activity induced by adding 4-amynopiridine or omitting mg from the slices perfusate [38, 39 ]. also, kaglund. confirmed that cbx also reduced both the frequency and amplitude of epileptic field bursts induced by cesium or low calcium in hippocampal slices. more recently, two studies using genetic and pharmacological models of seizures determined that cbx significantly decreased the amplitude and duration of seizure - like activity in thalamocortical slices [41, 42 ]. these in vitro reports established the basis for the subsequent evaluation of cbx in in vivo models. administration of cbx (40 - 300 mg/ kg) delayed the onset of seizures and reduced the duration of clonic seizures induced by pentylenetetrazole (ptz) [43, 44 ]. similar results but with low doses of cbx (5 - 30 mg / kg), were observed in audiogenic seizures in a genetic model of epilepsy - prone rats. conversely, the same research group reported that neither intravenous nor i.p. administration of cbx had any effect on the number nor duration of spike - wave discharges in a genetic animal model of absence epilepsy. by contrast, gigout. using a similar genetic animal model, described that systemic doses of cbx (100 mg / kg) significantly decreased the duration of spike - wave discharges. administration of cbx protects against tonic seizures induced by maximal electroshock reducing both the incidence of tonic hindlimb extension (thle) and the duration of the thle (fig. some studies have tested the application of cbx directly into the brain to identify specific anatomical substrates where cbx could be exerting its anticonvulsant effects. in this regard, some authors evaluated the effects of cbx directly in the epileptic focus induced by the application of 4-aminopyridine or tetanus toxin on the somatosensory or entorhinal cortex. they found that the direct blockage of gj with cbx in the epileptic focus decreased both the duration of seizures and the amplitude of seizures discharges [26,48 - 50 ]. interestingly, several studies have revealed that microinjection of cbx into the inferior colliculus, substantia nigra, inferior olivary complex, basolateral amygdala and hippocampus reduced the duration and severity of seizures. additionally, it has been reported that bilateral microinjection into ventro- posteromedial thalamic nucleus did not produce any significant effect. by contrast, in a model of absence seizures, cbx microinjected into the reticular nucleus of the thalamus decreased the duration of seizures. likewise, results recently obtained in our laboratory have showed that cbx microinjected into the reticular formation reduces the incidence of generalized tonic - clonic seizures and prevented the epileptiform activity induced by ptz. in conclusion, all of these data have showed that cbx has anticonvulsant effects when administered in different seizure experimental models. some historical records suggest that quin has been used for the treatment of malaria since almost 400 years ago [54, 55 ]. however, due to some adverse effects, the world health organization recommended avoiding the use of quin as a first - line treatment reducing its use only to cases of severe malaria. the first studies concerning quin and excitable cells revealed that this antimalarial drug could block ionic channels and, therefore, suppress some ionic conductances [57, 58 ]. also, it was showed that quin modulated the activity of hemichannels in neurons from the vertebrate retina, and also in oocytes expressing some isoforms of cx [59, 60 ]. subsequently, srinivas. demonstrated for the first time that quin blocked gj formed by cx36 and cx50 in a reversible and dose - dependent way. since cx36 is expressed in mammalian neurons [13 - 14 ] few studies have explored the possible effects of quin on in vitro and in vivo experimental models of seizures. the first report in this subject described that quin blocked the excitability of ca3 pyramidal neurons in hippocampal slices, through the modification of the properties of the membrane resting potential, and the duration and amplitude of spikes. also using hippocampal slices, uusisaari. induced synchronous bursting in pyramidal neurons exposing slices to gabab receptor antagonists and discovered that quin reversibly blocked the denominated gabaergic ictal - like events. interestingly, another study tested the in vitro effects induced by quin in the neocortex of patients with temporal lobe epilepsy. this study showed that quin decreased irreversibly the occurrence of synchronous field potential activity produced by the application of 4-aminopirydine. paradoxically, there is a report that describes excitatory effects of this gj blocker in rat slices. specifically, it was found that quin increased the frequency and amplitude of seizure - like activity induced by low magnesium in neocortical slices. however, it was also described that at high doses, quin caused a biphasic effect characterized by an initial excitatory phase followed by an apparent reduction in seizure - like activity. some in vivo reports have established that quin could have effects on seizure experimental models. administration of quin (25 - 100 mg / kg), reduced both the incidence and duration of seizures and modified the latency to the tonic and myoclonic phases in mice administered with ptz. however, they did not find significant effects when analyzed the effects of quin on maximal electroshock seizures. administration of quin (60 - 240 mg / kg) only elicited a slight protection against the incidence and duration of thle induced by maximal electroshock (fig. thus, in rats, quin (200, 400 and 1000 nmol) significantly decreased the frequency and amplitude of the epileptiform activity induced by the intracerebro- ventricular (icv) injection of penicillin. likewise, another study reported that icv administration of quin (0.5 and 1 mm) significantly increased the latency and decreased the duration of the generalized tonic - clonic seizures induced by ptz. on the other hand, some studies have tested the effects of quin applied directly to cortical areas, and the results have been consistent.. demonstrated that pretreatment with quin on the somatosensory cortex slightly reduced the epileptogenesis induced by 4-aminopirydine. however, when quin was applied to contralateral and ipsilateral cortical areas after the constant expression of seizures, the duration and propagation of seizures decreased significantly. similarly, two reports have established that microinjection of quin into the entorhinal cortex modified the characteristics of the hippocampal epileptiform activity induced by both 4-aminopirydine and pilocarpine [71, 72 ]. the walter reed army institute of research in the usa developed it in the early 1970s. initially, mfq was indicated for the treatment and prophylaxis of malaria ; however, currently it is no longer considered by the world health organization as a first - line drug to eliminate the parasite causing malaria. before its proposal as gj blocker, some studies had reported that mfq has the capability to shorten action potential duration, decreasing currents through l - type calcium channels. later, it was demonstrated that mfq reduced, in a concentration - dependent manner, the intercellular gap junctional currents in n2a cells selectively expressing cx36 and cx50. consequently, mfq was evaluated on the brain due to the expression of cx36 in neuronal cells. therefore, it was determined that mfq reduced, in a time and concentration - dependent manner, the electrical coupling of interneurons in neocortex slices. there is a significant decrease in the excitatory activity of the hippocampus after the application of mfq. however, another report described that mfq induced increases in both the amplitude and frequency of seizure - like events in cortical slices of rats. although this is a still unexplored issue, it has been proposed that the opposing effects observed could be related to the doses of mfq used. voss. reported that, in mouse neocortical slices, mfq (25 m) has no effects on the seizure - like activity induced by perfusion with low concentrations of magnesium. on the other hand, more recently it was described that mfq (10 m) significantly decreased the amplitude and frequency of seizure - like activity induced by the coadministration of 4-aminipyridine and bicuculline in thalamocingulate mice slices. due to the lack of in vivo studies, we have worked on clarifying the controversy about mfq and seizures. significantly decreased the incidence of seizures and the total spectral power of the epileptiform activity induced by ptz. we have also observed a significant reduction of the duration of thle induced by maximal electroshock (fig. most likely, the lack of in vivo studies is because some reports have suggested that mfq could cause undesired neurological effects at doses around 187 mg / kg. we have observed that mfq (40 mg / kg) elicited some protection against the seizures induced by two acute models of seizures (fig., it seems reasonable to propose that mfq acts in a dose - dependent manner, showing anticonvulsant effects at small doses and neurotoxic effects at high doses. qnd is another compound derived from the chinchona tree bark and chemically related to quin. the use of qnd dates since 1914 when it was demonstrated that this compound could be beneficial in the treatment of cardiac arrhythmias. in addition to its antiarrhythmic effect, the center for disease control and prevention has established that qnd gluconate is the only antimalarial agent available for parental administration in the united states. some classical studies reported that the application of qnd to neurons provoked a clear block of ionic conductances though na+, k+ and ca+ channels, and consequently caused modifications of action potentials properties [81 - 83 ]. however, after a meticulous analysis, malchow. proposed for the first time that qnd could modify the gap junctional communication between neurons. more recently, this hypothesis was explored using n2a cells transfected with cx50 channels, and it was demonstrated that qnd (300 m) significantly inhibited the currents in gj formed by cx50. one study has reported that qnd induces an increase in the frequency of seizure - like events caused by the addition of low concentrations of magnesium to rat cortical slices. by contrast, it has been showed that qnd abolishes the ictal - like activities induced by 4-aminopyridine in rat thalamocortical slices. similarly, gigout,. evaluated the effects of qnd but in slices obtained from the neocortex of temporal lobe epileptic patients ; they found that qnd decreases irreversibly the frequency of occurrence of the synchronous field potential activity induced by 4-aminopirydine. using in vivo models, steriade and stoica performed well - designed experiments and reported that qnd (4 - 30 mg / kg) caused significant anticonvulsant effects in experimental models such as electroshock and cortical application of penicillin. in contrast, we have observed that higher doses of qnd (120 mg / kg) are necessary to generate some protection against the thle induced by maximal electroshock (fig. these previous information has led us to suggest that the relation between qnd and seizure experimental models is nowadays underrepresented, and that it is necessary more in vivo studies to explore more in depth this topic. ana and ole are members of the cannabinoid family and these endogenous molecules bind and activate the cannabinoid receptor 1. some reports have demonstrated that these endocannabinoids have effects on ca+ channels and significantly increase the intracellular concentration of ca in different cell types [87, 88 ]. interestingly, it has been described that both, ana and ole, are potent inhibitors of intercellular gap junctional communication in glial cells, and ana also blocked calcium wave propagation in striatal astrocytes [89, 90 ]. following the effects of ana and ole on gap junctional communication, some authors have evaluated their effects on animal seizure models, including two reports that have described ana role in the modulation of seizures induced by maximal electroshock [91, 92 ]. specifically, one report described a modest anticonvulsant activity with ana alone (50 mg / kg). however, the combination of ana (300 mg / kg) with an inhibitor of amidohydrolase, which rapidly metabolize ana, generated a complete protection against seizures induced by electroshock. similar studies have demonstrated that inhibition of amidohydrolase increased the levels of ana in the brain, and reduced the severity of seizures induced by application of kainic acid [93, 94 ]. ana has also been evaluated in other animal seizure models such as ptz and a genetic model of absence epilepsy. interestingly, mannea and umathe have reported that icv administration of ana showed dose - dependent effects ; namely, at small doses (10 - 40 g) ana produced anticonvulsant effects, while at high doses (80 - 160 g) ana increased the percentage of animals with seizures induced by application of ptz. more recently, it has been reported that icv administration of ana significantly decreases in a dose - dependent manner, the presence and duration of spike - wave discharges related to absence seizures. there are few studies relating ole and seizures ; however, they are consistent with those using ana. hence, it has been showed that ole (43 - 700 mg / kg, i.p.) inhibited the seizures induced by ptz but not those induced by picrotoxin or strychnine [97, 98 ]. in addition to the substances previously mentioned, more compounds have been characterized as gj blockers. some of these compounds include meclofenamic acid, niflumic acid, flufenamic acid, heptanol, octanol, glycyrrhetinic acid and retinoic acid. these chemicals have shown ability to block hemichannels and gj composed of cx26, cx32, cx36, cx40, cx43, cx46 and cx50 [99 - 105 ] (table 1). the meclofenamic, niflumic and flufenamic acids are grouped in the fenamates family, which is a group of drugs used as anti - inflammatories and analgesics. of these three compounds, both meclofenamic and flufenamic acids have been evaluated in relation to seizures, and it has been shown that i.p. administration of meclofenamic acid increases the latency to the onset of seizures induced by ptz. additionally, in rats with epileptic focus caused by tetanus toxin, a significant reduction in seizure duration was observed when meclofenamic acid was applied directly to the cortex. similarly, schiller stimulated neocortical slices with a gabaa receptor blocker to evoke seizure - like events, and observed that addition of flufenamic acid (100 - 200 m) reversibly eliminated the seizure - like events. heptanol and octanol are long carbon chain n - alkanols capable of modifying the gap junctional intercellular communication [102, 103 ]. therefore, it was demonstrated that perfusion of entorhinal / hippocampal slices with octanol blocked the primary afterdischarges produced by the tetanic stimulation of schaffers collaterals. using the same model, dantuono. showed that octanol blocked theta activity of the epileptiform afterdischarges induced by the application of picrotoxin. heptanol has also been evaluated in hippocampal slices with similar results to those exhibited by octanol. namely, heptanol significantly depressed the spontaneous field burst and the repetitive population spikes evoked by fimbrial stimulation. to date, there are few studies reporting data about the use of octanol on in vivo seizure experimental models. relatedly, it was showed that octanol reduced seizure induction and seizure discharges when it was applied directly to the epileptic focus in the somatosensory cortex. additionally, it has been reported that icv administration of octanol significantly reduces the frequency and amplitude of epileptiform spikes, as well as the epileptic behavioral score induced by the icv administration of penicillin. interestingly, it has been shown that this chemical displays some anti - tumoral, anti - allergic and anti - inflammatory effects. the first studies concerning glycyrrhetinic acid in the context of epileptogenesis were carried out by de curtis. ; they described that glycyrrhetinic acid eliminated the spontaneous interictal spikes in an in vivo model of focal epileptogenesis. more recently, using rats implanted with a cannula into the reticular nucleus of the thalamus, it was found that glycyrrhetinic acid significantly decreased the duration of atypical absence seizures. interestingly, despite the fact that glycyrrhetinic acid is a compound that is chemically related to cbx, its effects on animal seizure models are not yet fully explored. retinoic acid, a product of the metabolism of vitamin a, has also been reported as a gj blocker. specifically, it has been observed in retinal cells that retinoic acid was able to reduce, in a dose - dependent manner, the amplitude of the gap junctional conductance. lately, sayyah. evaluated the infusion of retinoic acid directly into the amygdala of rats, and they observed that retinoic acid significantly reduced both the afterdischarge duration and the seizures generated in rats electrically stimulated in the amygdala. although these results seem consistent with the blockage of gj, other recent evidence has suggested that retinoic acid not only increased the expression of cx32 and cx43 but also increased the gap junctional intercellular communication [115, 116 ]. an alternative approach to precisely block gj and cx hemichannels consists of using specific antibodies or small peptide fragments corresponding to intracellular amino acid sequences of diverse cx. thus, the first studies done by moore and burt described that cx - specific antisense oligodeoxynucleotides could reduce the frequency of unitary conductances in cells expressing cx40 and cx43. later, it was established that cells incubated with a synthetic oligopeptide corresponding to a segment of the second extracellular loop of cx43, showed decreased dye coupling and dual whole - cell voltage clamp, indicating a reduction of the cell - to - cell coupling. the cx - mimetic peptides have been proposed as specific and reversible blockers of gj and cx hemichannels ; however, it has been reported that these short amino acid sequences have the ability to inhibit currents from channels constituted of proteins topologically similar to cx, called pannexins (panx). from all the synthesized peptides, gap26 and 27 are the most widely used because they correspond to specific sequences within the extracellular loops of cx37, 40 and 43. although the detailed mechanism of action of the cx - mimetic peptides is unknown, it has been suggested that they interact with the extracellular loops, disrupting the docking of the hemichannels and, therefore reducing the assembly of functional gj [120, 121 ]. to evaluate the effects of cx - mimetic peptides concerning epileptiform activity, samoilova. studied the administration of gap27 to hippocampal slices and detected that spontaneous recurrent epileptiform activity was diminished but only after 10 hours of gap27 treatment. on the other hand, it has been reported that application of a mimetic peptide in hippocampal slices, targeted to the extracellular loop two of cx43, evoked a dose- and exposure time - dependent response, preventing the seizure - induced neuronal death caused by the application of an antagonist of gaba - a receptors. based on these reports, it has been suggested that blockage of gj and hemichannels constituted by cx43 could prevent the neuronal damage induced by the epileptiform neuronal activity. the gj blockers show affinity not only for gj but also for hemichannels composed of different subtypes of cx (table 1). the cx family is characterized by 20 isoforms expressed in the mouse genome ; however, it has been determined that approximately half of the cx isoforms are present in the brain. interestingly, following previous reports, it has been observed that gj blockers mainly modify the communication of gj and hemichannels constituted of cx36, cx43 and cx50 (table 1) [36, 37, 61, 75, 89, 90, 99 - 105 ]. of all the cx isoforms expressed in the brain, it has been proposed that cx36 is the major neuronal isoform and consequently plays an essential role in the generation of highly synchronized electrical activity. thus, cx36-ko mice show a decrease of gamma frequency oscillations induced by kainate and carbachol in the ca3 region of the hippocampus. on the other hand, it has been described that cx36 levels are upregulated in the hippocampus and cortex after the expression of epileptic seizures in rodents [25, 26 ]. cbx, quin, and mfq are gj blockers which have certain selectivity for neuronal isoforms such as the cx36 (table 1) [36, 37, 61, 75 ]. therefore, it has been suggested that the effects caused by gj blockers are related to the inhibition of assembled gj constituted of neuronal isoforms. similarly, the anticonvulsant effects produced by these gj blockers are comparable to the marked attenuation of the epileptiform discharges elicited by 4-aminopyridine in slices from cx36-ko mice. in the brain, cell - to - cell coupling mediated by gj occurs between neurons, astrocytes, oligodendrocytes, microglia and ependymal cells. there are several cx isoforms expressed in those cells types ; however, the cx43 isoform is expressed predominantly in astrocytes and located ubiquitously in the brain. specifically, it has been proposed that astrocytes participate regulating the extracellular concentration of ions, such as potassium and calcium, and also releasing and reuptaking gliotransmitters and signaling molecules, for example glutamate and atp. many pathological processes including seizures are characterized by reactive changes in the structure and functionality of astrocytes. thus, some reactive changes in astrocytes comprise modifications in the ultrastructure of cytoskeletal proteins, as well as the down- or up - regulation of many proteins. the role of interastrocytic coupling mediated by cx43 in the pathophysiology associated with epileptogenesis is still unknown. however, it has been well described that cx43 expression levels are altered in human epileptic brain and in seizure experimental models [26, 28, 127 ]. cx and panx have the ability to constitute single functional hemichannels that open to the extracellular space under physiological and pathological conditions. cumulative evidence indicates that astrocytes release atp and glutamate associated with intracellular calcium signaling via cx43 and panx hemichannels. consequently, it has been suggested that dysregulation of hemichannel functionality in astrocytes, could lead to increased calcium influx and calcium waves, and hence to a release of neurotoxic concentrations of glutamate and as a consequence the propagation of epileptiform activity [128 - 130 ]. as previously mentioned, most of the gj blockers modify the communication of gj and hemichannels constituted of cx43 (table 1) [36, 37, 61, 75, 89, 90, 99 - 105 ]. therefore, this could be an essential mechanism that explains the anticonvulsant effects evidenced by the gj blockers. in agreement with all of the previous studies, it can be proposed that gj blockers have two main pharmacological mechanisms, which act together to elicit anticonvulsant effects : a) the blockage of assembled gj constituted of neuronal cx (cx36) and the consequent inhibition of the neuronal synchronization, and b) the blockage of astrocytic hemichannels (cx43), causing a reduction of calcium signaling and as a consequence a decreased release of gliotransmitters. nowadays, many substances have been described as gj blockers. however, in some cases the blockage of gap junctional intercellular communication has been evaluated in cellular types which are different from those located in the brain. because of the complexity of the cellular interconnections in the central nervous system, we propose that the effects of some gj blockers in the brain should be analyzed carefully. properties, the ones that have been the most evaluated on seizure experimental models have been the cbx and quin. these two compounds share anticonvulsant effects typically characterized by the reduction of both amplitude and frequency of the epileptiform activity as well as by modifications of the behavioral parameters related to seizures. in accordance with these observations, another gj blocker (mfq) has showed anticonvulsant effects when administered at low doses. reports have suggested that some gj blockers could induce undesired neurological effects at high doses ; however, we propose that small doses must be prudently selected to avoid neurotoxic effects. although the relationship between gj and seizures is a topic which is progressively growing, it is important to note that some gj blockers have not been evaluated in animal seizure models. consequently, we propose that more studies are necessary to explore this issue, and to contribute to the search for new pharmaceutical alternatives for the treatment of epilepsy. | abstract : backgroundgap junctions are clusters of intercellular channels allowing the bidirectional pass of ions directly into the cytoplasm of adjacent cells. electrical coupling mediated by gap junctions plays a role in the generation of highly synchronized electrical activity. the hypersynchronous neuronal activity is a distinctive characteristic of convulsive events. therefore, it has been postulated that enhanced gap junctional communication is an underlying mechanism involved in the generation and maintenance of seizures. there are some chemical compounds characterized as gap junction blockers because of their ability to disrupt the gap junctional intercellular communication.objectivehence, the aim of this review is to analyze the available data concerning the effects of gap junction blockers specifically in seizure models.resultscarbenoxolone, quinine, mefloquine, quinidine, anandamide, oleamide, heptanol, octanol, meclofenamic acid, niflumic acid, flufenamic acid, glycyrrhetinic acid and retinoic acid have all been evaluated on animal seizure models. in vitro, these compounds share anticonvulsant effects typically characterized by the reduction of both amplitude and frequency of the epileptiform activity induced in brain slices. in vivo, gap junction blockers modify the behavioral parameters related to seizures induced by 4-aminopyridine, pentylenetetrazole, pilocarpine, penicillin and maximal electroshock.conclusionalthough more studies are still required, these molecules could be a promising avenue in the search for new pharmaceutical alternatives for the treatment of epilepsy. |
malaria is caused by protozoan parasites of the genus plasmodium, namely, p. falciparum, p. vivax, p. malariae, p. ovale, and p. knowlesi. patients with p. falciparum infection are prone to develop severe malaria in 30% of cases, which resulted in case fatality rate of 20%. in southeast asia, acute renal failure (arf) is one of the most common complications in adults with falciparum malaria [3, 4 ]. the incidence of arf in patients with severe malaria varies widely ranging from 15% to 48% [511 ] which resulted in a high fatality rate of over 70% in untreated patients. the availability of renal replacement therapy (rrt) and appropriate antimalarial chemotherapy has been shown to reduce case fatality rate as well as enhance the recovery of renal function [13, 14 ]. recently, a few observational studies demonstrated that there was an increased risk for mortality with small increments in serum creatinine ; this finding made the case for the adoption of more sensitive creatinine - based criteria for acute kidney injury (aki) [15, 16 ]. the term arf was replaced by aki which was classified as the risk, injury, failure, loss, and end - stage renal failure criteria (rifle criteria) proposed by the acute dialysis quality initiative (adqi) group. in addition, severity of aki by the rifle criteria can be used for predicting both requirements for rrt and mortality rates particularly in critically ill patients. however, data on diagnosing aki using the rifle criteria among patients with severe malaria are limited due to the commonly used who criteria in this field. this study aimed to determine the incidence of arf by the who 2006 criteria and aki by the rifle criteria as well as their association with requirement for rrt and in - hospital mortality this study was approved by the ethics committee of the faculty of tropical medicine, mahidol university, bangkok, thailand. a retrospective study was conducted at the mae sot general hospital, tak province, thailand. medical records of hospitalized patients with severe falciparum malaria classified according to who 2006 criteria between 2004 and 2008 were reviewed. inclusion criteria were (1) aged 15 years or above, (2) confirmed diagnosis of falciparum malaria by peripheral blood smear microscopy, and (3) had clinical and laboratory features of severe malaria as defined by the who 2006 criteria. exclusion criteria were (1) patients with previous history of chronic kidney disease or (2) patients with mixed infection. patients ' data comprising demographic, clinical features, laboratory data, and outcomes including death and requirement for rrt were reviewed and extracted from medical records and discharge summaries according to the eligible criteria. these data were then transferred into a predefined case record form for further analysis. in our study, the who 2006 criteria for severe malaria were used with slight modification due to the availability of data. these included (1) impaired consciousness defined as glasgow coma scale (gcs) score 10, (2) multiple convulsions defined as patients who developed convulsion 2 times within 24 hours, (3) shock defined as a systolic blood pressure 80 mmhg or required inotropic drugs, (4) pulmonary edema or acute respiratory distress syndrome (ards) assessed by clinical and abnormal chest roentgenography specific for these syndromes, (5) hyperbilirubinemia defined as total bilirubin 3 mg / dl, (6) hypoglycemia defined as blood sugar 3 mg / dl and adequate volume status. patients with aki were classified according to the rifle criteria using decrement in the estimated glomerular filtration rate (gfr), but not serum creatinine increment and urine output in our study. patients with aki were classified as no aki (decrease of estimated gfr 25%), rifle - r (decrease of estimated gfr > 2550%), rifle - i (decrease of estimated gfr > 5075%) and rifle - f (decrease of estimated gfr the categories of rifle including loss (rifle - l) and end - stage kidney disease (rifle - e) were not evaluated. estimated gfr was calculated using the chronic kidney disease epidemiology collaboration (ckd - epi) equation as recommended by the adqi group. the incidence of arf in adults with severe malaria was approximately 30% to 50% by the reports from south and southeast asia [8, 9, 11 ]. data from the hospital registry of the mae sot general hospital showed that 25% of adult patients with severe malaria had arf ; thus, we estimated the incidence of arf in our study to be 25% with the desired statistical power of 80% and the type i error (alpha = 0.05) to detect a 10% difference in incidence of aki between the who 2006 criteria and the rifle criteria. the required sample size of at least 231 medical records of hospitalized patients with severe falciparum malaria was needed in our study. the demographic, clinical, and laboratory data collected in this study were analyzed using the statistical package for the social sciences version 18.0 (spss, chicago, il, usa). quantitative data were tested for normality using the kolmogorov - smirnov test and summarized as median (interquartile, iqr) for nonnormally distributed data. qualitative data were summarized as frequency (percentage) and then analysed by chi square test or fisher 's exact test as appropriate. the chi square for linear trend was used to determine a linear trend of in - hospital mortality in relation to severity of aki. univariate analysis was performed to determine the possible risk factors for in - hospital mortality among severe falciparum malaria patients with aki. any variable with p 0.2 in the univariate logistic regression analysis was included in the stepwise multivariate logistic regression analysis using a backward selection method for determining the independent associated factors for in - hospital mortality among severe falciparum malaria patients with aki. all tests of significance were 2 sided, with a p 12.0 10/l (5.286 (2.60210.736), p 5.5 mmol / l (10.427 (2.73139.808), p = 0.001), bicarbonate level 500 u / l (7.429 (2.64620.858), p 500 u / l (13.875 (1.497128.561), p = 0.021) and albumin 12.0 10/l (3.982 (1.14613.836), p = 0.030) were independently associated with in - hospital mortality. p. falciparum infection is the most common cause of arf in patients with severe malaria [3, 4 ]. it appears that several factors contribute to arf in falciparum malaria which includes parasitized erythrocytes inducing microvascular obstruction and/or causing hemolysis. apart from parasites, glycosyl - phosphatidyl - inositol which is a receptor on monocytes covalently bound to the surface antigens of falciparum malaria parasites. the monocytes are then stimulated to release the tumor necrosis factor, which in turn enhances synthesis of various cytokine cascades and mediators. these mediators also cause changes in blood volume status, vasodilatation, and increase vascular permeability resulting in hypovolemia which contributes to ischemic renal failure. we conducted a retrospective study by reviewing 257 medical records of patients with severe falciparum malaria at the mae sot general hospital, tak province, thailand, in order to determine the incidence of arf using the who 2006 criteria and aki using the rifle criteria as well as their association with rrt requirement and in - hospital mortality. serum creatinine was used to calculate estimated gfr as serum creatinine criteria seemed to show a worse rifle category and provided a better predictor for mortality rate than urine output criteria. however, calculation of estimated gfr has several limitations particularly in aki patients with nonsteady state of serum creatinine. the lack of steady state in serum creatinine in aki patients might lead to an overestimation of gfr in patients with rising serum creatinine and underestimation of gfr in patients with declining gfr. regarding the who 2006 criteria, the incidence of arf in our study (44.7%) was similar to the reports in previous studies (3050%) [8, 9, 11 ]. overall in - hospital mortality of patients with severe falciparum malaria was 22.1% and as high as 31.9% in the group of patients with arf in our study. this figure showed significantly higher in - hospital mortality in the group of patients with arf compared to that with no arf (14%). the cause of death was multiorgan failure, which included shock, cerebral malaria, and metabolic acidosis in most cases. these findings were similar to reports in other referral centers showing that arf occurred in approximately 2550%, and the numbers of organ failures were associated with case fatality rate [7, 8, 11, 14 ]. when rrt requirement was observed, indications for initiation of rrt in patients with severe malaria were similar to the timing for initiation of rrt in critically ill patients and dependent on treating nephrologists. however, the indication for initiation of rrt included severe metabolic acidosis, pulmonary edema, and severe hyperkalemia in our hospital. however, the proportion for the requirement of rrt in our study was lower than those reports in other studies (6080%) [7, 8, 11, 14 ]. these criteria have been shown in hospitalized patients to be quite sensitive in predicting the case fatality rate [1517, 21 ]. there were several reports showing the ability of rifle criteria in predicting hospital mortality of critically ill patients [2225 ]. these criteria have been widely used for diagnosing aki in western countries, but they are rarely used in tropical areas, of which infection is one of the most common causes of aki. infectious diseases in these tropical countries including malaria, leptospirosis, scrub typhus, and salmonellosis are commonly found in association with aki. there has been only one report from india showing that aki diagnosed using the rifle criteria was associated with the requirement for rrt and case fatality rate in patients with tropical acute febrile illnesses such as scrub typhus, falciparum malaria, enteric fever, dengue, and leptospirosis. however, these rifle criteria have never been evaluated among a cohort of patients with severe malaria. in our study, aki was evaluated using the rifle criteria in a large group of patients with severe malaria. we demonstrated that the requirement for rrt and in - hospital mortality increased significantly with severity of aki. severe malaria patients with rifle - i or rifle - f were 4 times at risk for in - hospital mortality compared to those with no aki. risk factors for in - hospital mortality among severe falciparum malaria patients with aki were dopamine (or = 7.172, 95% ci = 1.82728.145) or adrenaline infusion (or = 14.502, 95% ci = 2.87473.166), need for mechanical ventilator (or = 10.806, 95% ci = 2.56945.459), and white blood cell count > 12.0 10/l (or = 3.982, 95% ci = 1.14613.836) in our study. previous studies showed that in - hospital mortality among patients with severe malaria was higher in those with multi - organ failure. furthermore, leucocytosis was associated with mortality among patients with falciparum malaria but not associated with bacteremia. early detection of aki may help in the proper management of cases resulting in better outcomes. other conditions such as cerebral malaria, disseminated intravascular coagulation, and metabolic acidosis may contribute to poor outcomes in patients with severe falciparum malaria [5, 8 ]. in our study, there were several limitations due to the study design which was retrospective in nature and some baseline data of the patients were missing. therefore, further prospective evaluations are needed to properly standardize the rifle criteria for diagnosing aki in malaria patients. in conclusion, rifle criteria could be used in diagnosing aki and predicting both requirements for rrt and in - hospital mortality in patients with severe falciparum malaria similar to who 2006 criteria. early diagnosis and early management of aki may help to improve the outcomes of severe malaria patients in future. | there are limited data on the application of the rifle criteria among patients with severe malaria. this retrospective study was conducted by reviewing 257 medical records of adult hospitalized patients with severe falciparum malaria at the mae sot general hospital, tak province in the northern part of thailand. the aims of this study were to determine the incidence of acute renal failure (arf) in patients with severe falciparum malaria and its association with rrt as well as in - hospital mortality. using the who 2006 criteria, arf was the second most common complication with incidence of 44.7% (115 patients). the requirement for rrt was 45.2% (52 patients) and the in - hospital mortality was 31.9% (36 patients). using the rifle criteria, 73.9% (190 patients) had acute kidney injury (aki). the requirement for rrt was 11.6% (5 patients) in patients with rifle - i and 44.9% (48 patients) in patients with rifle - f. the in - hospital mortality gradually increased with the severity of aki. the requirement for rrt (p < 0.05) and the in - hospital mortality (p < 0.05) were significantly higher in arf patients with severe falciparum malaria using both criteria. in conclusion, the rifle criteria could be used for diagnosing aki and predicting outcomes in patients with severe malaria similar to the who 2006 criteria. |
the hi contribution rates applicable to taxable earnings in each of the calendar years 1981 and later are shown in table 1. after 1983, the automatic increase provisions in section 230 of the social security act determine the maximum taxable amount. the social security act was amended during 1982 by the tax equity and fiscal responsibility act (tefra) and during 1983 by the social security admendments of 1983 (public law 98 - 21). the major provisions among the many affecting the hi program were : tefra changed the method by which medicare reimburses hospitals by replacing the previous per diem limits on routine inpatient costs by limits on total inpatient costs per admission and limits on increases in total inpatient costs per admission expire for cost reporting periods beginning on or after october 1, 1985. medicare coverage is extended to federal employees, who are required to pay the hospital insurance portion of the fica tax as of january 1, 1983. medicare will temporarily cover hospice care for beneficiaries having a life expectancy of 6 months or less. this provision is effective november 1, 1983, and expires october 1, 1986. public law 98 - 21 changes the method by which medicare makes payments to hospitals. hospitals will no longer be reimbursed on a reasonable cost basis for their inpatient operating costs. hospitals will be paid a prospectively determined price per discharge using diagnosis - related groups. this provision is effective for hospital fiscal years beginning on or after october 1, 1983. also, no terminations of coverage by state and local governments or entities will be permitted after april 20, 1983. this provision is effective upon enactment. interfund borrowing among the old age and survivors insurance, disability insurance, and hospital insurance trust funds (authorized in 1981) is extended through 1987 with repayment to be made during 1988 - 1989 in 24 equal monthly payments. beginning june 1983, loans would be repayable when the fund ratio of the borrowing fund exceeds 15 percent. at the end of 1982, 26 million people over 65 years of age and 3 million disabled people under 65 years of age were covered under hi, financed primarily by the contributions of 116 million workers through payroll taxes. payroll taxes during 1982 amounted to $ 34.6 billion, accounting for 90.9 percent of all hi income. about 2.7 percent of all income resulted from reimbursements from the general fund of the treasury for military service credit and benefits for certain uninsured persons. interest payments to the hi fund amounted to 5.4 percent of all hi income for 1982. the remaining 1.0 percent was contributed through premiums paid by voluntary enrollees and taxes collected from railroad workers. of the $ 36.1 billion in hi disbursements, $ 35.6 billion was for payments while the remaining $ 0.5 billion was spent for administrative expenses. table 2 displays the hi fund operations for calendar years 1970 - 1982. in most years, the hi fund has increased. however, the fund ratio (the fund at the beginning of the year divided by disbursements during the year) has declined every year from its peak of 79 percent in 1975 to 45 percent in 1981. the fund ratio increased slightly at the beginning of 1982 primarily because of the increase in the contribution rate in 1981. the board of trustees has adopted the general financing principle that annual income to the hospital insurance program should be approximately equal to annual outlays of the program plus an amount to maintain a balance in the trust fund equal to one - half year 's disbursements. because of the $ 12.4 billion loan to the oasi fund at the end of 1982, the trust fund was far below this desired level. projections were made under four alternative sets of assumptions : optimistic, two intermediate sets (alternatives ii - a and ii - b), and pessimistic. under both sets of intermediate assumptions, the trust fund ratio is projected to remain around 20 to 30 percent in most years until the late 1980 's and then decline rapidly with complete exhaustion of the fund around 1990. under the more optimistic set of assumptions (alternative i), the trust fund is projected to grow until about 1988, then to decline steadily until the fund is completely exhausted in 1996. under the more pessimistic set of assumptions (alternative iii), the trust fund is projected to decrease steadily with complete exhaustion of the fund by 1988. table 3 summarizes the estimated operations of the hi trust fund under the four alternative sets of assumptions. figure 1 shows historic trust fund ratios for recent years and projected ratios under the four sets of assumptions. the adequacy of the financing of the hi program on a long - range basis is measured by comparing on a year - by - year basis the actual tax rates specified by law with the corresponding total costs of the program, expressed as percentages of taxable payroll. the actuarial balance is defined to be the excess of the average tax for the 25-year valuation period (1983 - 2007) over the average cost of the program expressed as a percent of taxable payroll. the average cost of the program under alternatives ii - a and ii - b is 3.97 and 4.11 percent of taxable payroll, respectively. figure 2 shows the year - by - year cost as a percent of taxable payroll for each of the four sets of assumptions, as well as the scheduled tax rates. the cost figures in table 4 and figure 2 include amounts for building and maintaining the trust fund at the level of a half year 's disbursements as recommended by the board of trustees. figure 2 emphasizes the inadequacy of the financing of the hi program by illustrating the divergence of the program costs and scheduled tax rates under each set of assumptions. under all four sets of assumptions used in the 1983 report, the outlook for the hospital insurance trust fund is slightly more optimistic than it was in the 1982 report. this is primarily the result of the major legislation during 1982 and 1983 which will help curtail the rapid increase in hospital costs. table 5 below presents a comparison of the projected experience in the 1982 and 1983 reports. the present financing schedule for the hospital insurance program is barely adequate to ensure the payment of benefits through the end of this decade if the assumptions underlying the estimates are realized. the trust fund is exhausted in 1991 and 1990 under alternatives ii - a and ii - b, respectively. under the more pessimistic assumptions, i, the present financing schedule will result in the fund being exhausted in 1996. in order to bring the hospital insurance program into close actuarial balance, either disbursements of the program will have to be reduced by 30 percent or financing will have to be increased 43 percent. despite the short - term uncertainties, the enactment of tefra in 1982 and public law 98 - 21 has substantially reduced the long - range deficit of the hi fund. more importantly, the prospective payments of public law 98 - 21 have made the outlays of the hi program potentially less vulnerable to excessive rates of growth in the hospital industry by providing the secretary of health and human services with some discretion over the level of payments to hospitals. the quadrennial advisory council on social security, appointed by the secretary, will be addressing the financial status of the hospital insurance trust fund. the board recommends that congress study carefully the advisory council 's recommendations as it takes further action to curtail the rapid growth in the cost of the hospital insurance program which has occurred in recent years and which is anticipated in the future. at the end of 1982, 26 million people over 65 years of age and 3 million disabled people under 65 years of age were covered under hi, financed primarily by the contributions of 116 million workers through payroll taxes. payroll taxes during 1982 amounted to $ 34.6 billion, accounting for 90.9 percent of all hi income. about 2.7 percent of all income resulted from reimbursements from the general fund of the treasury for military service credit and benefits for certain uninsured persons. interest payments to the hi fund amounted to 5.4 percent of all hi income for 1982. the remaining 1.0 percent was contributed through premiums paid by voluntary enrollees and taxes collected from railroad workers. of the $ 36.1 billion in hi disbursements, $ 35.6 billion was for payments while the remaining $ 0.5 billion was spent for administrative expenses. table 2 displays the hi fund operations for calendar years 1970 - 1982. in most years, the hi fund has increased. however, the fund ratio (the fund at the beginning of the year divided by disbursements during the year) has declined every year from its peak of 79 percent in 1975 to 45 percent in 1981. the fund ratio increased slightly at the beginning of 1982 primarily because of the increase in the contribution rate in 1981. the board of trustees has adopted the general financing principle that annual income to the hospital insurance program should be approximately equal to annual outlays of the program plus an amount to maintain a balance in the trust fund equal to one - half year 's disbursements. because of the $ 12.4 billion loan to the oasi fund at the end of 1982, the trust fund was far below this desired level. projections were made under four alternative sets of assumptions : optimistic, two intermediate sets (alternatives ii - a and ii - b), and pessimistic. under both sets of intermediate assumptions, the trust fund ratio is projected to remain around 20 to 30 percent in most years until the late 1980 's and then decline rapidly with complete exhaustion of the fund around 1990. under the more optimistic set of assumptions (alternative i), the trust fund is projected to grow until about 1988, then to decline steadily until the fund is completely exhausted in 1996. under the more pessimistic set of assumptions (alternative iii), the trust fund is projected to decrease steadily with complete exhaustion of the fund by 1988. table 3 summarizes the estimated operations of the hi trust fund under the four alternative sets of assumptions. figure 1 shows historic trust fund ratios for recent years and projected ratios under the four sets of assumptions. the adequacy of the financing of the hi program on a long - range basis is measured by comparing on a year - by - year basis the actual tax rates specified by law with the corresponding total costs of the program, expressed as percentages of taxable payroll. the actuarial balance is defined to be the excess of the average tax for the 25-year valuation period (1983 - 2007) over the average cost of the program expressed as a percent of taxable payroll. the average cost of the program under alternatives ii - a and ii - b is 3.97 and 4.11 percent of taxable payroll, respectively. figure 2 shows the year - by - year cost as a percent of taxable payroll for each of the four sets of assumptions, as well as the scheduled tax rates. the cost figures in table 4 and figure 2 include amounts for building and maintaining the trust fund at the level of a half year 's disbursements as recommended by the board of trustees. figure 2 emphasizes the inadequacy of the financing of the hi program by illustrating the divergence of the program costs and scheduled tax rates under each set of assumptions. under all four sets of assumptions used in the 1983 report, the outlook for the hospital insurance trust fund is slightly more optimistic than it was in the 1982 report. this is primarily the result of the major legislation during 1982 and 1983 which will help curtail the rapid increase in hospital costs. table 5 below presents a comparison of the projected experience in the 1982 and 1983 reports. the present financing schedule for the hospital insurance program is barely adequate to ensure the payment of benefits through the end of this decade if the assumptions underlying the estimates are realized. the trust fund is exhausted in 1991 and 1990 under alternatives ii - a and ii - b, respectively. under the more pessimistic assumptions, i, the present financing schedule will result in the fund being exhausted in 1996. in order to bring the hospital insurance program into close actuarial balance, either disbursements of the program will have to be reduced by 30 percent or financing will have to be increased 43 percent. despite the short - term uncertainties, the enactment of tefra in 1982 and public law 98 - 21 has substantially reduced the long - range deficit of the hi fund. more importantly, the prospective payments of public law 98 - 21 have made the outlays of the hi program potentially less vulnerable to excessive rates of growth in the hospital industry by providing the secretary of health and human services with some discretion over the level of payments to hospitals. the quadrennial advisory council on social security, appointed by the secretary, will be addressing the financial status of the hospital insurance trust fund. the board recommends that congress study carefully the advisory council 's recommendations as it takes further action to curtail the rapid growth in the cost of the hospital insurance program which has occurred in recent years and which is anticipated in the future. financing for the supplementary medical insurance program is established annually on the basis of standard monthly premium rates (paid by or on behalf of all participants) and monthly actuarial rates determined separately for aged and disabled beneficiaries (on which general revenue contributions are based). prior to the 6-month transition period (july 1, 1983 through december 31, 1983) beginning january 1, 1984, the annual basis will change to the 12-month periods ending december 31. monthly actuarial rates are equal to one - half the monthly amounts necessary to finance the smi program. these rates determine the amount to be contributed from general revenues on behalf of each enrollee. based on the formula in the law, the government contribution effectively makes up the difference between twice the monthly actuarial rates and the standard monthly premium rate. figure 3 presents these values for financing periods since 1974. the extent to which general revenue financing is becoming the major source of income for the program is clearly indicated in this figure. standard monthly premium rates and monthly actuarial rates have been announced for periods through december 31, 1983. for the 6-month period ending december 31, 1983 (transitional semester (ts)), the standard monthly premium rate is $ 12.20, and the monthly actuarial rates are $ 27.00 and $ 46.10 for the aged and disabled, respectively. the major provisions among the many affecting the smi program were : the premium rates applicable july 1, 1983 through december 31, 1983 is frozen at the rate that applied june 1983. some general revenues shall be added from july through december to compensate for keeping the smaller june 1983 premium for that period. from january 1984 through december 1985, the monthly smi premium is set at one - half of the actuarial rate for aged enrollees. after december 1985, the determination of the premium rate will revert to the method used before enactment of this provision and future increases shall apply on a calendar year basis. medicare becomes the secondary payer for employees 65 through 69 years of age (and their spouses of the same age) who are covered by health plan benefits of an employer. the basis upon which provider - based physicians are reimbursed are to be prescribed in regulations which distinguish between (a) professional component, and (b) provider component. in fiscal year 1982 general revenue contributions during 1982 amounted to $ 13.3 billion, accounting for 75.6 percent of all smi income. about 21.7 percent of all income resulted from the premiums paid by the participants, with interest payments to the smi fund accounting for the remaining 2.7 percent. of the $ 15.6 billion in smi disbursement, $ 14.8 billion was for benefit payments while the remaining $ 0.8 billion was spent for administrative expenses. the historical operations of the smi trust fund since fiscal year 1977, as well as the projected operations of the fund for fiscal years through 1985, for both alternative ii - a and alternative ii - b are shown in table 6. as can be seen, income has exceeded disbursements for most of the historical years and the trust fund balance is projected to continue to increase through fiscal year 1985. however, as the report notes, the financial status of the program depends on both the total net assets and liabilities. it is, therefore, necessary to examine the incurred experience of the program since it is this experience which is used to determine the actuarial rates discussed above and which forms the basis of the concept of actuarial soundness as it relates to the smi program. the concept of actuarial soundness, as it applies to the supplementary medical insurance program, is closely related to the concept as it applies to private group insurance. the supplementary medical insurance program is essentially yearly renewable term insurance intended to be self - supporting from premium income paid by the enrollees and from income contributed from general revenue in proportion to premium payments. in testing the actuarial soundness of the supplementary insurance program, it is not appropriate to look beyond the period for which the enrollee premium rate and level of general revenue financing have been established. the primary tests of actuarial soundness, then, are that (1) income for years for which financing has been established be sufficient to meet the projected benefits and associated administrative expenses incurred for that period, and (2) assets be sufficient to cover projected liabilities which will have been incurred by the end of that time but will not have been paid yet. even if these tests of actuarial soundness are not met, the program can continue to operate if the trust fund remains at a level adequate to permit the payment of claims as presented. however, to protect against the possibility that cost increases under the program will be higher than assumed, assets should be sufficient to cover the impact of a moderate degree of projection error. the initial tests for actuarial soundness and trust fund adequacy can be viewed by direct examination of absolute dollar levels. in providing an appropriate contingency or margin for error the relative measure or ratio used for this purpose is the ratio of net surplus or deficit to the following year 's incurred expenditures. figure 4 shows this ratio for historical years and for projected years under one of the intermediate assumptions (alternative ii - b), as well as high- and low - cost sensitivity scenarios. financing for the 12-month period ending june 30, 1983, was established to maintain assets at the same level relative to program expenditures which existed prior to june 30, 1982. the resulting excess of assets over liabilities as of june 30, 1983, represents 9.4 percent of the projected incurred expenditures for the following 12-month period. the actuarial rates for the 6-month period ending december 31, 1983, as implemented, will reduce this excess to a more appropriate level. under more pessimistic assumptions as to cost increases, the financing established through december 1983 is sufficient to cover projected benefit and administrative costs incurred through that time period and to build a level of trust fund assets which is adequate to cover the impact of a moderate degree of projection error. general revenue contributions during 1982 amounted to $ 13.3 billion, accounting for 75.6 percent of all smi income. about 21.7 percent of all income resulted from the premiums paid by the participants, with interest payments to the smi fund accounting for the remaining 2.7 percent. of the $ 15.6 billion in smi disbursement, $ 14.8 billion was for benefit payments while the remaining $ 0.8 billion was spent for administrative expenses. the historical operations of the smi trust fund since fiscal year 1977, as well as the projected operations of the fund for fiscal years through 1985, for both alternative ii - a and alternative ii - b are shown in table 6. as can be seen, income has exceeded disbursements for most of the historical years and the trust fund balance is projected to continue to increase through fiscal year 1985. however, as the report notes, the financial status of the program depends on both the total net assets and liabilities. it is, therefore, necessary to examine the incurred experience of the program since it is this experience which is used to determine the actuarial rates discussed above and which forms the basis of the concept of actuarial soundness as it relates to the smi program. the concept of actuarial soundness, as it applies to the supplementary medical insurance program, is closely related to the concept as it applies to private group insurance. the supplementary medical insurance program is essentially yearly renewable term insurance intended to be self - supporting from premium income paid by the enrollees and from income contributed from general revenue in proportion to premium payments. in testing the actuarial soundness of the supplementary insurance program, it is not appropriate to look beyond the period for which the enrollee premium rate and level of general revenue financing have been established. the primary tests of actuarial soundness, then, are that (1) income for years for which financing has been established be sufficient to meet the projected benefits and associated administrative expenses incurred for that period, and (2) assets be sufficient to cover projected liabilities which will have been incurred by the end of that time but will not have been paid yet. even if these tests of actuarial soundness are not met, the program can continue to operate if the trust fund remains at a level adequate to permit the payment of claims as presented. however, to protect against the possibility that cost increases under the program will be higher than assumed, assets should be sufficient to cover the impact of a moderate degree of projection error. the initial tests for actuarial soundness and trust fund adequacy can be viewed by direct examination of absolute dollar levels. in providing an appropriate contingency or margin for error the relative measure or ratio used for this purpose is the ratio of net surplus or deficit to the following year 's incurred expenditures. figure 4 shows this ratio for historical years and for projected years under one of the intermediate assumptions (alternative ii - b), as well as high- and low - cost sensitivity scenarios. financing for the 12-month period ending june 30, 1983, was established to maintain assets at the same level relative to program expenditures which existed prior to june 30, 1982. the resulting excess of assets over liabilities as of june 30, 1983, represents 9.4 percent of the projected incurred expenditures for the following 12-month period. the actuarial rates for the 6-month period ending december 31, 1983, as implemented, will reduce this excess to a more appropriate level. under more pessimistic assumptions as to cost increases, the financing established through december 1983 is sufficient to cover projected benefit and administrative costs incurred through that time period and to build a level of trust fund assets which is adequate to cover the impact of a moderate degree of projection error. | this summary presents an overview of the information contained in the annual reports of the trustees required under title xviii of the social security act, health insurance for the aged and disabled, commonly known as medicare. there are two basic programs under medicare : hospital insurance (hi), which pays for inpatient hospital care and other related care of those 65 years of age and over and of the long - term disabled.supplementary medical insurance (smi), which pays for physicians ' services, outpatient hospital services, and other medical expenses of those 65 years of age and over and of the long - term disabled.the hi program is financed primarily by payroll taxes, with the taxes paid by current workers used to pay benefits to current beneficiaries. however, the hi program maintains a trust fund that provides a small reserve against fluctuations. this type of financing is generally known as pay - as - you - go financing. by contrast, the smi program is financed on an accrual basis with a contingency margin. this means that the smi trust fund should always be somewhat greater than the claims that have been incurred by enrollees but not yet paid by the program. the trust funds hold all of the income not currently needed to pay benefits and related expenses. the assets of the funds may not be used for any other purpose ; however, they may be invested in certain interest - bearing obligations of the u.s. government.the secretaries of treasury, labor, and health and human services serve as trustees of the hi and smi trust funds. the secretary of treasury is the managing trustee. the administrator of the health care financing administration, the agency charged with administering the medicare program, is the secretary of the board of trustees. |
an alternative approach is active breath - hold, which is a temporary suspension of breathing at a desired respiratory phase. however, it is argued that the suspension mechanism facilitates the protection of the heart better in breast cancer patients than in lung cancer patients who tend to have a smaller breathing capacity. gating was initially proposed in proton therapy for liver tumors using a pressure sensor on the abdominal wall. gated radiotherapy using an external respiratory signal has been clinically implemented using commercially available systems. however, it has been reported that respiratory - gated beam delivery can not result in margin reduction without respiratory - correlated image guidance. for more accurate gated radiotherapy, a gold fiducial marker can be implanted near a lung tumor for real - time diagnostic x - ray fluoroscopy. however, a major problem of any gating technique is longer delivery time leading to lower patient throughput as well as the possibility of reduced tumor control. another approach is dynamic tumor tracking with a gimbaled treatment head that allows continuous beam delivery, where the moving target may be located by an implanted fiducial marker and diagnostic x - ray fluoroscopy in real time. although it is assumed that the spatial relationship between tumor volume and a point marker remains unchanged, this may not always be true due to the mobility of the marker. in addition, accuracy of treatment may ultimately be defined as a volumetric relationship between the tumor and the dose distribution during the entire beam delivery. still another method for managing respiratory motion is the use of an internal target volume (itv) determined by contouring clinical target volumes (ctvs) on 10-phase 4d planning ct images. in this case, the problem of respiratory motion management can be reduced to a tumor registration problem with a larger, but virtually still, target volume on the treatment couch. by constraining the breathing amplitude with the aid of abdominal compression, the itv approach may decrease the target volume and thus the dose to normal lung. using 4d cone - beam ct (cbct), a respiratory - correlated lung tumor volume can be visualized immediately before treatment [79 ]. an advantage of the 4d - cbct - based itv approach is that the above - mentioned volumetric accuracy can be directly confirmed on a cbct workstation prior to each fractionated treatment. to the authors ' knowledge, a clinical workflow using 4d cbct for lung treatment has not previously been established. the purpose of this study was to propose a clinical workflow of stereotactic volumetric modulated arc therapy (vmat) for lung tumors from planning to tumor position verification. a large bore 16-multislice ct scanner, aquillion lb (toshiba, japan), an anzai belt (anzai medical, japan) and a body fixation device, bodyfix (elekta, germany), were employed to obtain 10-phase respiratory - correlated ct data for a lung patient under constrained breathing conditions induced by an abdominal compression plate. the patient was diagnosed with stage iv metastatic lung tumor (squamous cell carcinoma) from an anal cancer. the lung tumor had a major diameter of 15 mm and a minor diameter of 8 mm. the amplitude of the tumor motion was 11.7 mm in the craniocaudal direction, 1.8 mm in the lateral direction and 0.8 mm in the anteroposterior direction. a planning target volume (ptv) was defined by adding a 5-mm margin to an itv created by 10 clinical target volumes, each of which was delineated on each of the 10-phase 4d planning ct images. a single - arc vmat plan was created with a d95 prescription dose of 50 gy in four fractions on the maximum exhalation phase ct images where the photon energy was 6 mv with a maximum dose rate of 580 mu / min. the ptv contours were exported to a kilovoltage cbct x - ray volume imaging (xvi) version 4.5, equipped with a synergy linear accelerator (linac) (elekta, uk). immediately before treatment, 10-phase 4d cbct data were reconstructed with an acquisition time of 4 min, generating animated lung tumor imaging. initial bone matching was performed between frame - averaged 4d planning ct and frame - averaged 4d cbct datasets, where the frame - averaged 4d planning ct data were calculated by in - house software and the frame - averaged 4d cbct data were provided by xvi built - in functionality. subsequently, the imported ptv contours and the animated moving tumor were simultaneously demonstrated on the xvi display monitor, and a manual 4d registration was interactively performed by moving the imported itv and ptv contours using a mouse on the axial, coronal and sagittal views until the moving tumor was symmetrically positioned inside the ptv. after confirmation of the registration results on the display monitor, the patient couch was repositioned and stereotactic vmat beams were delivered. during the delivery, another set of 4d cbct projection data was acquired to verify the tumor position, and phase - sorted volumetric reconstruction was performed using in - house software. this in - house program was needed because the current xvi version does not provide cbct imaging during treatment. the current study is in compliance with ethical guidelines of the hospital and written informed consent was obtained before the treatment was initiated. manual 4d registration using the ptv contours and the animated lung tumor required only a few seconds. figure 1a e show 4d cbct images on the first day overlaid with the ptv (in sky blue) and itv (in yellow) contours after the 4d lung tumor registration. registration required a couch translation of 3.2 mm toward the patient 's right, 1.5 mm toward the caudal direction, and 4.4 mm toward the anterior direction, respectively. the figures show images for five consecutive respiratory phases that cover half a breathing cycle, in which the tumor moves from cranial to caudal direction during the half cycle. 1.4d cbct images on the first day overlaid with ptv (in sky blue) and itv (in yellow) contours after lung tumor registration for five consecutive respiratory phases covering half a breathing cycle, where the tumor moves from cranial to caudal direction during the half cycle. after confirmation of the registration results on the display monitor, stereotactic vmat is ready to run. 4d cbct images on the first day overlaid with ptv (in sky blue) and itv (in yellow) contours after lung tumor registration for five consecutive respiratory phases covering half a breathing cycle, where the tumor moves from cranial to caudal direction during the half cycle. after confirmation of the registration results on the display monitor, stereotactic vmat is ready to run. figure 2a d shows 4d cbct images acquired during vmat delivery on the first day and reconstructed afterwards to verify the in - treatment moving tumor position in reference to the ptv and itv contours. the vmat delivery time was 210 s. for the in - treatment cbct, the number of phase bins was four, which covered an entire breathing cycle. it was confirmed that during the vmat delivery on the first day the moving tumor was also located inside the ptv. 2.4d cbct images acquired during vmat delivery on the first day to verify the in - treatment moving tumor position in reference to the ptv and itv contours. in this case, the number of phase bins was four, which covered an entire breathing cycle. 4d cbct images acquired during vmat delivery on the first day to verify the in - treatment moving tumor position in reference to the ptv and itv contours. in this case, the number of phase bins was four, which covered an entire breathing cycle. it is known that vmat delivery is faster than any other intensity - modulated treatment. in addition, in - treatment cbct can be acquired during vmat delivery for 4d tumor position verification. 2 a d show some blurring, possibly due to the smaller number of phase bins, which may be improved by providing 10-phase in - treatment 4d cbct datasets. a limitation of this technique would be that the amplitude of tumor motion may not be sufficiently reduced by abdominal compression in the case of some tumors having a large breathing motion, possibly leading to a prohibitively large itv volume. the measured respiratory movement in this study was relatively large and could have been reduced by increasing the compression of the plate to the abdominal surface. whereas the dose calculation in this study was based on maximum exhalation phase ct images, the authors are currently updating their procedure using mid - ventilation ct data. we have proposed a clinical workflow of stereotactic lung vmat capable of 4d registration and 4d verification of the tumor position, and initial promising results have been obtained. the authors believe that the proposed 4d workflow for stereotactic lung vmat is valid for respiratory motion management because the vmat delivery is sufficiently fast to maintain respiratory periodicity, but much longer than the patient breathing cycle for obtaining 4d cbct. our lung vmat delivery employs a sequence with a maximum leaf speed of 1 mm / degree, and therefore it is anticipated that doubling the dose rate using flattening filter free techniques, combined with doubled leaf speeds, may reduce the treatment time down to an order of 100 seconds for delivering a prescribed dose of 50 gy in four fractions. | we propose a clinical workflow of stereotactic volumetric modulated arc therapy (vmat) for a lung tumor from planning to tumor position verification using 4d planning computed tomography (ct) and 4d cone - beam ct (cbct). a 4d ct scanner, an anzai belt and a bodyfix were employed to obtain 10-phase respiratory - correlated ct data for a lung patient under constrained breathing conditions. a planning target volume (ptv) was defined by adding a 5-mm margin to an internal target volume created from 10 clinical target volumes, each of which was delineated on each of the 10-phase planning ct data. a single - arc vmat plan was created with a d95 prescription dose of 50 gy in four fractions on the maximum exhalation phase ct images. the ptv contours were exported to a kilovoltage cbct x - ray volume imaging (xvi) equipped with a linear accelerator (linac). immediately before treatment, 10-phase 4d cbct images were reconstructed leading to animated lung tumor imaging. initial bone matching was performed between frame - averaged 4d planning ct and frame - averaged 4d cbct datasets. subsequently, the imported ptv contours and the animated moving tumor were simultaneously displayed on the xvi monitor, and a manual 4d registration was interactively performed on the monitor until the moving tumor was symmetrically positioned inside the ptv. a vmat beam was delivered to the patient and during the delivery further 4d cbct projection data were acquired to verify the tumor position. the entire process was repeated for each fraction. it was confirmed that the moving tumor was positioned inside the ptv during the vmat delivery. |
progress in immunotherapy has provided novel insights into the roles of tumor - infiltrating lymphocytes (tils) in cancer. the prognostic significance of tils has been determined in breast cancers, particularly human epidermal growth factor receptor 2 (her2)positive breast cancers and triple - negative breast cancers. the prognosis of her2-positive breast cancer has dramatically improved because of introduction of trastuzumab, a humanized monoclonal antibody targeting the extracellular domain of her2. immune - mediated mechanisms may play a role in treatment with trastuzumab. in the adjuvant setting, a high level of tils is associated with better response to trastuzumab, and an abundant number of tils is an independent prognostic factor in her2-positive breast cancer. based on this background, a group of investigators around the world convened to discuss the parameters and methodological issues associated with evaluating tils in breast cancers and released recommendations for their evaluation. they recommended that all mononuclear cells, including lymphocytes and plasma cells in intratumoral stroma, be scored as a percentage of the area occupied over the total intratumoral stromal area. the innate immune system lies at the frontline of the cellular response to pathogen infection. pattern recognition receptors sense microbial products, including rna produced during viral infection, and interferon (ifn) expression is then upregulated, resulting in stimulation of the transcription of various ifn - stimulated genes (isgs), including the myxovirus resistance a (mxa) gene, through phosphorylation of signal transducer and activator of transcription 1 (stat1). in addition to its function in innate immunity, isgs are known to be broadly expressed in various types of tumors, and ifns are known to participate in receptor - activated signaling pathways associated with the pathophysiology of malignancies. additionally, some studies have shown that mxa may have a tumor - suppressive function. expression of the mxa gene is regulated by ifns, but not by other cytokines. moreover, mxa gene expression is not induced directly by viral infection, but is dependent on ifn signaling, making mxa an excellent marker for ifn action. activation of the ifn pathway is a common outcome of damage - associated molecular pattern (damp) signaling. a recent report demonstrated that high cytoplasmic expression of high mobility group (hmg) b1 and hmgn1, which are damps associated with innate immunity in the extracellular environment, is associated with a high level of tils in her2-positive breast cancers. we hypothesized that mxa, an effective marker of ifn signaling that is an important component of innate immunity, may play a role in tils in her2-positive breast cancers. in this study, we analyzed the expression of mxa protein in her2-positive breast cancers and the relationship between the expression level of mxa and various histopathologic variables, including til levels. a total of 168 patients with her2-positive breast cancer who underwent surgery for primary breast cancer between 2011 and 2013 at pusan national university hospital were included. expression statuses of estrogen receptor (er), progesterone receptor (pr), and her2 were evaluated for all samples, and immunohistochemistry for these proteins was performed at the time of diagnosis. exemption from informed consent after de - identification of the patients information was approved by the institutional review board of pusan national university hospital. hematoxylin and eosin (h&e)-stained slides were reviewed by two pathologists (a.k. and j.y.k.). histopathologic factors included histologic grade, nuclear grade, necrosis, microcalcification, lymphovascular invasion, lymph node metastasis, tils level, tertiary lymphoid structures (tlss) around the ductal carcinoma in situ (dcis), and tls around the invasive component. tils levels were evaluated based on the recommendation by the international tils working group, and tils level was calculated according to the percentage of stroma in the invasive area (fig. tls is a lymph - node like structure characterized by ectopic aggregation of lymphoid cells with specialized high endothelial venules. lymphoid accumulation containing vessels with high endothelial venule features (plump and cuboidal endothelial cells) was considered to be the tls (fig. we evaluated the ducts involved by dcis with surrounding tlss as a percentage of total ducts involved by dcis and the amount of tlss as a percentage of the total circumference of the invasive edge. to conduct statistical analysis, the tils levels were subdivided into three categories (10%, 20%-60%, and > 60%) or two categories (60% and > 60%), as appropriate. the level of tls abundance was divided into two subgroups (low and high) based on the mean value. to define the representative tumor area, each sample was arrayed with a tissue cylinder 2 mm in diameter using a tissue arraying instrument. to minimize tissue loss and the effects of tumor heterogeneity, tissue sections were stained with an automated immunohistochemical staining device (benchmark xt, ventana medical systems, tucson, az). briefly, 3-m - thick paraffin sections were deparaffinized in ez prep using an autostainer. for antigen retrieval, cell conditioner 1 (ph 8.4 buffer) or cell conditioner 2 (ph 6.0 buffer) was used. optimally formulated antibodies targeting er (1:200, leica biosystems, newcastle upon tyne, uk), pr (1:200, leica biosystems), and her-2/neu (1:8, ventana medical systems) were used, and an anti - mxa antibody (1:1,000, ab95926, abcam, cambridge, uk) was also applied. a her2/cep17 chromosome dual - probe (ventana medical systems) er and pr positivity were defined as at least 1% positive staining in tumor nuclei. tumors that were positive for er or pr were categorized as the hormone receptor (hr)positive group. positivity was defined as an immunohistochemical staining score of 3 or gene amplification as identified by sish. tumors were classified into the following two subtypes according to hr (er and pr) expression status : hr+/her2 + and hr/her2 +. the intensity of the staining and percentage of positive tumor cells were considered together for mxa. the intensity of the immunohistochemistry was graded as 0 (negative), 1 (weak), 2 (moderate), and 3 strong (fig. the immunoscore was generated by multiplying the staining intensity by the percentage of the positive tumor cells. finally, we subdivided the tumors into two groups (low and high expression) according to the mean value to enable statistical analysis. chi - square tests, linear - by - linear association tests, fisher exact tests, and spearman rank correlation tests were used as appropriate. all tests were two - sided, and differences with p - values of less than 0.05 were considered significant. a total of 168 patients with her2-positive breast cancer who underwent surgery for primary breast cancer between 2011 and 2013 at pusan national university hospital were included. expression statuses of estrogen receptor (er), progesterone receptor (pr), and her2 were evaluated for all samples, and immunohistochemistry for these proteins was performed at the time of diagnosis. exemption from informed consent after de - identification of the patients information was approved by the institutional review board of pusan national university hospital. hematoxylin and eosin (h&e)-stained slides were reviewed by two pathologists (a.k. and j.y.k.). histopathologic factors included histologic grade, nuclear grade, necrosis, microcalcification, lymphovascular invasion, lymph node metastasis, tils level, tertiary lymphoid structures (tlss) around the ductal carcinoma in situ (dcis), and tls around the invasive component. tils levels were evaluated based on the recommendation by the international tils working group, and tils level was calculated according to the percentage of stroma in the invasive area (fig. tls is a lymph - node like structure characterized by ectopic aggregation of lymphoid cells with specialized high endothelial venules. lymphoid accumulation containing vessels with high endothelial venule features (plump and cuboidal endothelial cells) was considered to be the tls (fig. we evaluated the ducts involved by dcis with surrounding tlss as a percentage of total ducts involved by dcis and the amount of tlss as a percentage of the total circumference of the invasive edge. to conduct statistical analysis, the tils levels were subdivided into three categories (10%, 20%-60%, and > 60%) or two categories (60% and > 60%), as appropriate. the level of tls abundance was divided into two subgroups (low and high) based on the mean value. each sample was arrayed with a tissue cylinder 2 mm in diameter using a tissue arraying instrument. to minimize tissue loss and the effects of tumor heterogeneity, tissue sections were stained with an automated immunohistochemical staining device (benchmark xt, ventana medical systems, tucson, az). briefly, 3-m - thick paraffin sections were deparaffinized in ez prep using an autostainer. for antigen retrieval, cell conditioner 1 (ph 8.4 buffer) or cell conditioner 2 (ph 6.0 buffer) was used. optimally formulated antibodies targeting er (1:200, leica biosystems, newcastle upon tyne, uk), pr (1:200, leica biosystems), and her-2/neu (1:8, ventana medical systems) were used, and an anti - mxa antibody (1:1,000, ab95926, abcam, cambridge, uk) was also applied. a her2/cep17 chromosome dual - probe (ventana medical systems) er and pr positivity were defined as at least 1% positive staining in tumor nuclei. tumors that were positive for er or pr were categorized as the hormone receptor (hr)positive group. positivity was defined as an immunohistochemical staining score of 3 or gene amplification as identified by sish. tumors were classified into the following two subtypes according to hr (er and pr) expression status : hr+/her2 + and hr/her2 +. the intensity of the staining and percentage of positive tumor cells were considered together for mxa. the intensity of the immunohistochemistry was graded as 0 (negative), 1 (weak), 2 (moderate), and 3 strong (fig. the immunoscore was generated by multiplying the staining intensity by the percentage of the positive tumor cells. finally, we subdivided the tumors into two groups (low and high expression) according to the mean value to enable statistical analysis. all statistical analyses were conducted using the spss ver. 22 (ibm corp., armonk, ny). chi - square tests, linear - by - linear association tests, fisher exact tests, and spearman rank correlation tests were used as appropriate. all tests were two - sided, and differences with p - values of less than 0.05 were considered significant. the patients ranged in age from 23 to 85 years (mean age, 52.62 years). all included patients were female, and the cohort was composed of 93 cases of left breast cancer and 75 cases of right breast cancer. ninety cases of hr+/her2 + tumors and 78 cases of hr/her2 + tumors were included. the clinicopathological characteristics of her2 + breast cancers according to hr expression status are summarized in table 1. high histological grade, high nuclear grade, and high level of tils were significantly associated with hr - negative cases (p 60%) for comparisons with various histopathologic parameters (table 3). the high level of tils was significantly correlated with high histological grade (p=0.001), absence of lymphovascular invasion (p=0.007), absence of lymph node metastasis (p=0.007), absence of hr expression (p 60%) for survival analysis. a high level of tils was associated with better disease - free survival in total her2-positive breast cancers (p=0.028) and particularly in hr/her2 + breast cancers (p=0.019) (fig. however, the expression level of mxa was not a prognostic factor (data not shown). the patients ranged in age from 23 to 85 years (mean age, 52.62 years). all included patients were female, and the cohort was composed of 93 cases of left breast cancer and 75 cases of right breast cancer. ninety cases of hr+/her2 + tumors and 78 cases of hr/her2 + tumors were included. the clinicopathological characteristics of her2 + breast cancers according to hr expression status are summarized in table 1. high histological grade, high nuclear grade, and high level of tils were significantly associated with hr - negative cases (p 60%) for comparisons with various histopathologic parameters (table 3). the high level of tils was significantly correlated with high histological grade (p=0.001), absence of lymphovascular invasion (p=0.007), absence of lymph node metastasis (p=0.007), absence of hr expression (p 60%) for survival analysis. a high level of tils was associated with better disease - free survival in total her2-positive breast cancers (p=0.028) and particularly in hr/her2 + breast cancers (p=0.019) (fig. however, the expression level of mxa was not a prognostic factor (data not shown). ifn genes are transcriptionally silent under normal conditions. production of ifns is transient and triggered with stimulation by pathogen - associated molecular patterns, which are conserved among microbial species, and by damps, which are endogenous molecules released from damaged or injured cells. ifns directly affect t - cell activation as a signal 3 cytokine, which is a specific cytokine signal involved in differentiation of nave t cells into effector and memory t cells. additionally, type i ifn can affect t - cell activation indirectly through mediation of the differentiation of monocytes into mature dendritic cells, which are important antigen - presenting cells, and enhancement of antigen presentation by major histocompatibility (mhc) class ii synthesis and antigen processing. type i ifns can elevate the expression of tumor programmed cell death ligand 1 (pd - l1), and the ifn - driven elevation of pd - l1 is thought to interact with programmed death 1 on t cells, resulting in t - cell exhaustion. moreover, one report demonstrated that high expression of isgs is associated with poor response to therapy and immune suppression. expression of mxa in cancer has been reported in various types of organs. in head and neck squamous cell carcinoma, hypermethylation of mxa was observed relative to lymphocytes from healthy individuals. in the human prostate carcinoma cell line, brown. revealed that silencing of mxa expression enhances cell migration, invasion, and anchorage independent growth, which are the hallmarks of an aggressive tumor. quantitative proteome profiling showed that mxa has a potential role as a marker for lymph node metastasis in colorectal cancer. mxa expression has been reported to be upregulated in cell line and xenograft models of hr - positive breast cancer with anti - estrogen resistance. additionally, johansson. showed that mxa expression was higher in a relapse group than in controls, but that it was not correlated with relapse - free survival and breast cancer - specific survival in er - positive breast cancer treated with adjuvant tamoxifen. conversely, sistigu. showed that mxa expression is positively associated with tumor grade, and that the type i ifn metagene centered around mxa can function as a biomarker for pathologic complete response to neoadjuvant anthracycline - based chemotherapy in breast cancer. moreover, mxa expression predicts metastasis - free survival after adjuvant chemotherapy in the subset of patients of breast cancer with poor prognosis. this study revealed the prognostic value of tils in her2-positive breast cancer and the correlation between mxa expression and til level. it should be noted that this study had several limitations, including a relatively small cohort and a short follow - up period, which may not have been sufficient to draw conclusive results. furthermore, multiple cell types in the tumor microenvironment can show high expression of isgs and affect the therapeutic response in breast cancer. in this study, we only evaluated mxa expression in tumor cells due to constraints with tissue microarray sections. overall, further studies of the effects of ifn signaling on tumor immunology in breast cancer are required to better elucidate the mechanism of tils and develop immunotherapies. the prognostic value of til evaluation with h&e sections was assumed in this study, supporting the recommendations by an international tils working group 2014. several reports have indirectly supported the hypothesis that peripheral blood t and b lymphocytes are marshaled to tlss and that differentiated cells migrate to the tumor, where they develop into tils. in this study, we showed that a high level of tils was significantly associated with the abundance of tlss around both the invasive component and dcis, which also indirectly supported the above hypothesis. there are at least two subtypes of her2-positive breast cancers according to the cancer genome atlas network, the her2-enriched mrna subtype and luminal mrna subtype. moreover, different clinical behaviors and histopathologic characteristics of hr+/her2 + breast cancers and hr/her2 + breast cancers have been reported. in this study, we found that hr/her2 + breast cancers were significantly associated with a higher histologic grade, higher nuclear grade, and abundant amount of tils when compared to hr+/her2 + breast cancers, consistent with previous studies. in summary, evaluation of tils with h&e section can provide prognostic information in routine practice. moreover, high expression of mxa in tumor cells was associated with a high level of tils in her2-positive breast cancers and a high level of tils was a prognostic factor for breast cancer. | purposethe prognostic significance of tumor - infiltrating lymphocytes (tils) has been determined in breast cancers. interferons can affect t - cell activity through direct and indirect mechanisms. myxovirus resistance a (mxa) is an excellent marker of interferon activity. here, we evaluated tils and mxa expression in human epidermal growth factor receptor 2 (her2)positive breast cancers.materials and methodsninety cases of hormone receptor (hr)+/her2 + tumors and 78 cases of hr/her2 + tumors were included. the tils level was assessed using hematoxylin and eosin stained full face sections, and mxa expressionwas evaluated by immunohistochemistrywith a tissue microarray.resultsmxa protein expression was significantly higher in tumors with high histologic grade (p=0.023) and high levels of tils (p=0.002). high levels of tils were correlated with high histological grade (p=0.001), negative lymphovascular invasion (p=0.007), negative lymph node metastasis (p=0.007), absence of hr expression (p < 0.001), abundant tertiary lymphoid structures (tlss) around ductal carcinoma in situ (p=0.018), and abundant tlss around the invasive component (p < 0.001). high levels of tils were also associated with improved disease - free survival, particularly in hr/her2 + breast cancers. however, mxa was not a prognostic factor.conclusionhigh expression of mxa in tumor cells was associated with high levels of tils in her2-positive breast cancers. additionally, a high level of tils was a prognostic factor for breast cancer, whereas the level of mxa expression had no prognostic value. |
insects : adults of l. f. fossarum (p generation) were collected from ishigaki island (2422 - 27n, 1249 - 12e), okinawa prefecture, ryukyu archipelago, japan. twenty - six females and 16 females were obtained on 3 april and 13 august 2012, respectively. collected adult females were maintained with d. melanogaster as food and allowed to lay eggs at 25 c under a 16:8 (l : d) h photoperiod in the laboratory of kyoto prefectural university, and obtained eggs were used for the subsequent experiments. we also established a laboratory population from two females sampled in spring and 16 females sampled in summer 2012, and used these for the experiments. as food insects, two species of drosophila (d. melanogaster and d. hydei sturtevant) were maintained on a medium jazz - mix (fisher scientific inc., tokyo, japan) at 25 c under a 16:8 (ld) h photoperiod in the laboratory. we maintained two wild types (oregon - r and canton - s) and two mutants, curly (cy) and vestigial (vg) of d. melanogaster, and randomly used these as food for l. f. fossarum. in addition to the two drosophila species, p. interpunctella were collected near the campus of kyoto prefectural university (shimogamo, sakyo, kyoto, japan). adult pairs of p. interpunctella were introduced to single clear plastic containers (135 85 30 mm) containing brown rice as larval food and maintained at 28 c under a 16:8 (ld) h photoperiod in the laboratory. emerged adults of all these food insects were killed by freezer and used as prey in experiments of food insect combinations. general rearing method : eggs from each collected wild female or the laboratory population were incubated on a water - soaked filter paper in plastic petri dishes (52 mm diameter, 13 mm depth). newly hatched nymphs were introduced to translucent plastic containers (60 mm 60 mm 45 mm) in groups of up to five individuals from the same parent. these nymphs were then transferred individually to other clear plastic containers (60 mm 60 mm 95 mm ; plant box, # cul - jar300, agc techno glass co., ltd., tokyo, japan) at the third or fourth instar depending on the subsequent experimental design. each container was filled with 5 mm water that had been dechlorinated by being incubated over 24 h in the laboratory. a cut piece of extruded polystyrene plate was added on the water surface as a resting site. all food insects, polystyrene plates, and water in the rearing containers were replaced daily regardless of what food insects were provided to the water striders. eggs and nymphs were maintained at 25 c under a 16:8 (ld) h photoperiod in all experiments. food insect combinations : to assess effective food insect combinations for rearing l. f. fossarum successively, we estimated viability from f1 eggs to adults, fertility of f1 adults (i.e. germination rates of f2 eggs), hatchability of f2 nymphs, and viability of f2 nymphs until the second instar or adults with the following four food insect experiments. detailed names of food insect species and the amount of each food insect used per day in each experiment are shown in table 1. table 1. species and amount of adult food insects fed to each stage of l. f. fossarumrearing methods1st2nd3rd4th5thadultexperiment 1dm+dhdm 1 dm 2 dm 3dh 1dh 2dh 3experiment 2dmdm 1 dm 2 dm 2 dm 3 dm 3 dm 4dm+pi1, 2 dm 1, pi 1/3 dm 2, pi 1/2 dm 2 dm 3 dm 3 dm 4experiment 3dm+pi1, 2, 3 dm 1, pi 1/3 dm 2, pi 1/2 dm 2, pi 1 dm 3 dm 3 dm 4dm+pi1, 2, 5 dm 1, pi 1/3 dm 2, pi 1/2 dm 2 dm 3 dm 3, pi 1 dm 4experiment 4dm+pi1, 2, 5 dm 1, pi 1/3 dm 2, pi 1/2 dm 2 dm 3 dm 3, pi 1 dm 4dmc+pi1, 2, 5dmc 1, pi 1/3dmc 2, pi 1/2dmc 2dmc 3dmc 3, pi 1dmc 4 dm, dh, and pi indicate drosophila melanogaster, d. hydei, and plodia interpunctella, respectively. numbers after abbreviations indicate daily amounts of each food - insect species per l. f. fossarum larva. under lines indicate periods in which l. f. fossarum were reared in groups. species and amount of adult food insects fed to each stage of l. f. fossarum dm, dh, and pi indicate drosophila melanogaster, d. hydei, and plodia interpunctella, respectively. numbers after abbreviations indicate daily amounts of each food - insect species per l. f. fossarum larva. f1 offspring derived from 26 females from the spring samples (collected on 3 april 2012) were used in this experiment. hatched f1 nymphs were fed on two species of drosophila (dm + dh treatment, table 1). f1 viability was estimated as the percentage survival of individuals until adulthood out of all eggs obtained. f1 offspring which successfully survived to adulthood were crossed with other adults from the same parent (i.e., full - sib mating) and f2 eggs were obtained. thus, we checked for the presence of eyespots on each egg 6 d after oviposition, and the presence of eyespots was regarded as an indicator of successful egg germination. experiment 2 : p. interpunctella for 1st and 2nd instars in addition to d. melanogaster for all instars. f1 offspring from 16 females from the summer samples (collected on 13 august 2012) were used for this experiment. to assess the effect of adding p. interpunctella as a food insect, 20 eggs each from 16 females were split 1:1 into two food treatments. in the first treatment (dm treatment), l. f. fossarum nymphs were fed only on d. melanogaster for all stages ; in the second treatment (dm + pi1, 2 treatment), the nymphs were fed daily, alternating between d. melanogaster and p. interpunctella, until the end of the second instar and then fed only on d. melanogaster until adulthood (table 1). in each treatment, we estimated f1 viability as the mean percentage of individuals surviving until each instar or adulthood of 16 sibling groups and compared the viabilities of the two treatments in each stage. we also compared f1 developmental periods from hatching to adult eclosion using f1 offspring that successfully became adults. we crossed f1 offspring from four randomly selected sibling groups and obtained 50 f2 generation eggs from each group. we counted fertile eggs of the f2 generation using the same method as in experiment 1. we assessed f2 hatchability and viability until second instar only in the dm + pi1, 2 treatment because all from the dm treatment were sterile. experiment 3 : p. interpunctella for third or fifth instars in addition to the dietary conditions from experiment 2. in this experiment, six females that had started oviposition (p generation) were isolated individually into plant boxes and were allowed to lay eggs to produce f1 offspring. to assess to which instars it would be most effective to provide p. interpunctella as food, 20 f1 eggs each from six females were split 1:1 into the dm + pi1, 2, 3 treatment and dm + pi1, 2, 5 treatment (table 1). in the f1 generation, we recorded viability until adulthood from eggs, length of developmental period from first instar to adult, and fertility of f1 adults. in the f2 generation, for each treatment, we recorded nymph hatchability and viability until the second instar in the same manner as in experiment 2. each parameter was compared between the dm + pi1, 2, 3 and dm + pi1, 2, 5 treatments. we only assessed f2 viability until adulthood in the progeny of the dm + pi1, 2, 5 treatment due to the very low survivorship of nymphs from the dm + pi1, 2, 3 treatment (see results). experiment 4 : d. melanogaster reared on cholesterol - added medium for all instars and p. interpunctella for first, second, and fifth instars. this experiment also used the laboratory population of l. f. fossarum and five females that had started oviposition (p generation) were isolated individually into plant boxes and were allowed to lay eggs to produce f1 offspring. to assess the effect of using d. melanogaster reared on a cholesterol - added medium (table 2) as a food insect, 20 f1 eggs each from five females were split 1:1 into the dm + pi1, 2, 5 treatment and dmc + pi1, 2, 5 treatment (table 1). we recorded the same parameters as in experiment 3 (viability until adulthood from eggs, length of developmental period from first instar to adult, and fertility of adults in the f1 generation ; nymph hatchability, viability until the second instar, and viability until adulthood in the f2 generation), and each parameter was compared between the dm + pi1, 2, 5 and dmc + pi1, 2, 5 treatments. ingredients in the cholesterol - added medium for drosophila melanogaster in the experiment 4 (amounts per 100 culture bottles)materialamountmedium (jazz - mix)68 gcholesterol0.36 gdistilled water340 ml fisher scientific inc., illinois, usa. ingredients in the cholesterol - added medium for drosophila melanogaster in the experiment 4 (amounts per 100 culture bottles) fisher scientific inc., osaka, japan. statistical analysis : we used student s t - test in all analyses for split - design experiments in experiments 24. however, we used welch s t - test for the hatchability and viability of the f2 generation in the experiment 4 due to the unequal variances between the dm + pi1, 2, 5 and dmc + pi1, 2, 5 treatments. in every analysis, we used arcsine square - root - transformed the viability, fertility and hatchability per sibling group in the f1 or f2 generation in order to satisfy the requirement of normality. we also analyzed the effect of food - insect combinations on wing morphs (macropterous, micropterous, and apterous) by using the all statistical analyses were carried out using the r version 3.2.3 (r developmental core team 2015). experiment 1 : d. melanogaster and d. hydei : of 658 f1 eggs, only 102 individuals survived until adulthood (viability 15.5%). although 1218 f2 eggs were laid by 14 f1 females, there were no fertile eggs under these food conditions. thus, we could not assess the hatchability and viability of the f2 generation, suggesting that we could not rear l. f. fossarum continuously using only drosophila species as food insects. experiment 2 : p. interpunctella for 1st and 2nd instars in addition to d. melanogaster for all instars : in the dm + pi1, 2 treatment, over 70% of individuals successfully survived to adulthood (fig. on the other hand, viability decreased suddenly after the fourth instar mainly due to failure to molt, and in 10 of 16 sibling groups, no individuals survived until adulthood. nymphal viability was significantly higher in the dm + pi1, 2 treatment than in the dm treatment starting in the fourth instar (fig. 1 ; until second instar t = 0.206, p = 0.839 ; until third instar t = 0.124, p = 0.902 ; until fourth instar t = 2.33, p = 0.0270 ; until fifth instar t = 6.25, p = 6.97e7 ; until adulthood t = 11.8, p = 8.52e13 ; df = 30 for all t - tests). fig. 1. comparison of survival rates between f1 generations fed on plodia in first and second instars (dm + pi1, 2) and fed only on drosophila in all instars and adulthood (dm). p > 0.05, p 0.05, p 0.05, p 0.05, p 0.05, p 0.05, p < 0.05 (welch 's t - test). in f2 generation, hatchability and viability until each instar and adulthood are consistently higher in the dmc + pi1, 2, 5 treatment than in the dm + pi1, 2, 5 treatment, although significant differences between the two treatments are detected in the viability until adulthood alone (fig. 3 ; hatchability t = 1.003, p = 0.346, df = 7.825, viability until second instar t = 1.727, p = 0.146, df = 4.891 ; until third instar t = 1.357, p = 0.243, df = 4.185 ; until forth instar t = 1.386, p = 0.233, df = 4.352 ; fifth instar t = 2.353, p = 0.0515, df = 6.877 ; until adulthood t = 2.484, p = 0.0381, df = 7.935 ; welch 's t - test). there was no significant difference in the mean larval developmental period between the dmc + pi1, 2, 5 treatment (42.4 2.54 d [sd, n = 5 ]) and the dm + pi1, 2, 5 treatment (44.2 2.36 d [sd, n = 3 ]) (t = 1.00, p = 0.355, df = 6, t - test). effect of food - insect combinations on wing morphs : model comparison indicated that the model consisting of only the food - combination effect was selected as the best fit model in experiments 2 and 4 (supp table s1 [online only ]), whereas the best fit model contained only the blood effect of water striders in experiment 3 (supp table s1 [online only ]). the present results show that the combinations of food insects used to rear l. f. fossarum in the laboratory drastically alter viabilities in progeny generations. although the origin of parental populations varied among the experiments, we compared the viability of offspring using a split design in each experiment ; thus, the differences in viabilities between treatments with different food insect combinations are ascribed to the effect of the food insect combination. the developmental period is also influenced by food insects, suggesting the importance of the food insects in shortening the generation time. the present results further indicate that the combinations of food insects in larval periods indeed affect wing morph determination of adults in l. f. fossarum (supp table s1 [online only ]) as reported in other water striders (harada and nishimoto 2007). they strengthen the importance of establishing a continuous rearing method under constant diet conditions for future genetic studies using l. f. fossarum. for genetic studies such as linkage analysis and quantitative traits loci (qtl) mapping, establishing segregating generations (e.g. f2 or backcross generations) we have demonstrated that the viability of f2 progenies depended on the f1 (i.e., parents of f2 progenies) instars that fed on p. interpunctella adults, and the majority of f2 individuals from the dm + pi1, 2, 3 treatment died before the second instar due to failure to successfully hatch or consume food insects in the first instar. in contrast, the fact that nearly 80% of nymphs successfully developed to the second stadium in the dm + pi1, 2, 5 treatment strongly indicates that not only the combination of food insects but also instars feeding on p. interpunctella in the f1 generation are very important in order to obtaining a sufficient number of individuals in the segregating generation in l. f. fossarum. (1991) reported that the viability of the broad shouldered water strider, microvelia douglasi scott (hemiptera : veliidae), which is a polyphagous predator, is also affected by the combination of food insects in the nymphal stage. nymphs of m. douglasi that fed on both d. melanogaster and nilaparvata lugens (stl) (which belong to different insect orders : diptera and hemiptera) develop faster and recover higher viability until adulthood than do nymphs that fed only on either d. melanogaster or n. lugens (sonoda. several other studies have also demonstrated that a mixed - prey diet in nymphal stages improved the adult body size, fecundity, and the hatching success in offspring (toft 1995 ; grundy. 2014), and such advantages of mixed diets have been already explained by the toxin - dilution hypothesis (freeland and janzen 1974 ; toft and wise 1999) and/or the redress - nutritional imbalance hypothesis (mayntz. thus, the present results add to the growing number of cases showing the suitability of mixed - prey diets for predatory arthropods. crickets (e.g., acheta domestica (l.) or gryllus firmus scudder) are often used as food insects for water striders in addition to dipteran species (e.g., fairbairn and king 2009). our present rearing method using p. interpunctella with d. melanogaster recovered the same viability until adulthood when compared to the use of a. domestica with d. melanogaster in the same split design, although we conducted only one replicate in f1 generation (60% in both combinations, data not shown). we usually introduce two adult pairs of p. interpunctella into a plastic container (135 85 30 mm) filled with ca. 10 mm brown rice to generate offspring and generally obtain 50120 adult offspring within 1 month without any subsequent care after introduction (hirooka pers. p. interpunctella is widely used as factitious prey with other pyralid moths (e.g., corcyra cephalonica (stainton) and ephestia kuehniella zeller) to maintain predatory heteropteran insects for biological control purposes (for a review, see de clercq. this study further shows that p. interpunctella could also be a useful food insect for rearing water striders like crickets. the present results clearly indicate that adding nutrients to a drosophila medium improves the viability of water striders. when l. f. fossarum is reared by our present method with d. melanogaster reared on the normal medium, one of the most frequent causes of death in nymphal stages is failed molting. because cholesterol is the precursor to ecdysone (an insect molting hormone) (lang. 2012 ; niwa and niwa 2014), adding cholesterol to the medium for drosophila could aid the synthesis of ecdysone in water striders, resulting in successful molting. although the viability until adulthood in the dmc + pi1, 2, 5 treatment in the experiment 4 was about 20% in the f2 generation, this low viability could be due to the use of laboratory lines showing inbreeding depression. indeed, results of experiment 3 that used less inbred individuals recovered above 50% \viability until adulthood in the f2 generation even in the dm + pi1, 2, 5 treatment. thus, the more than 50% viability in experiment 3 coupled with the consistently higher viabilities in the dmc + pi1, 2, 5 treatment than in the dm + pi1, 2, 5 treatment in experiment 4 suggest that adding cholesterol to drosophila medium is one of the effective ways to obtain sufficient number of individuals in segregating generations. mayntz and toft (2001) have also reported that the nutrient composition of rearing medium for a food insect (d. melanogaster) can affect predator viability in the wolf spider paradosa amentata (clerck) (araneae : lycosidae). therefore, adding nutrients to drosophila medium could be a universal method for rearing predacious insects. this study does not intend to assess the genetic backgrounds of wing polymorphisms in l. f. fossarum, but we preliminarily infer the contribution of genetic components (i.e., broad sense heritability, h) using the present rearing method (dm + pi1, 2, 5). we calculated the broad sense heritability of wing polymorphism as h=2 (among sibling groups)/[(among sibling groups) + (within sibling groups) ] (ueno. we used the four inbred lines established by one - time sib - mating and we split the offspring from each sibling group fifty - fifty into two photoperiods (16:8 (ld) h and 10:14 (ld) h). the mean square of variance obtained between sibling groups (163.4) is higher than that within sibling groups (102.7), although we do not have enough sibling group replications to test statistical significance. the estimated broad sense heritability is 46%, and this indicates that a substantial amount of variation of wing polymorphism is determined by genetic factors in l. f. fossarum. thus, further improvement of the present rearing method, especially for the nutrients to be included in the drosophila medium, will contribute to elucidating the genetic mechanisms determining wing polymorphisms and make l. f. fossarum a model organism for studying the genetic background of wing polymorphism. | the water strider limnogonus fossarum fossarum (f.) (hemiptera : gerridae) shows a macropterous, micropterous, and apterous polymorphism. although a long photoperiod condition induces winged morphs, preliminary studies have revealed that crossing between winged morphs increased the proportion of macropterous individuals, suggesting that the genetic factors also affect wing - morph determination in this species. assessing the genetic backgrounds of wing polymorphism requires constant and repeatable methods for rearing. this study attempts to establish a continuous rearing method for l. f. fossarum under constant diet conditions. initially, we maintain the water striders with two drosophila species as a food, but viability until adulthood is less than 20%. we then add the storage pest plodia interpunctella (hbner), which are readily reared in the laboratory, to the diets. as a result, nymphs fed on p. interpunctella (even only until the second instar) show significantly higher viability and shorter developmental period than nymphs fed on drosophila alone. moreover, feeding on d. melanogaster (meigen) reared on cholesterol - enriched medium instead of a normal medium significantly increases viability in the next generation. this means that only the two food - insect species are enough for establishing a substantial number of individuals in segregating generations (f2 and backcross), limiting dna and rna contaminations from food insects with genome information. thus, the present rearing method opens the way to elucidating the genetic backgrounds of the wing polymorphism in l. f. fossarum. |
a total of 160 fecal samples were collected in june and july 2014 from large - bodied gulls at two sites from each of two general locations within southcentral alaska 1). middleton island is a remote location in southcentral alaska, approximately 115 km offshore from the mainland in the gulf of alaska. middleton island was once the site of an active military installation (ca., 19581963) but is now uninhabited by humans with the exceptions of small field crews of scientists conducting research activities on the island during summer months and federal aviation administration employees (typically two) that work on the island approximately half - time year round. middleton island is occupied by large numbers of gulls each summer including multiple breeding colonies of glaucous - winged gulls (larus glaucescens). fecal samples were obtained during 2226 june 2014 from glaucous - winged gulls at sites on the northern (59.46n, 146.29w) and southern (59.4159.42n, 146.33146.36w) margins of middleton island, near nesting colonies prior to hatch. individual fecal samples were collected by swirling a sterile swab in fresh feces and placing in bacteria freeze media in cryovials. samples from middleton island were kept cool with icepacks for 48 days until frozen at 80c. map depicting sampling locations for large - bodied gulls (circles) in southcentral alaska. the approximate location of anchorage, the most populous city in alaska, is indicated with a star. the kenai peninsula is a landmass extending south of anchorage, alaska, the most populous city in the state, and supports numerous communities and an extensive road system. communities with the highest human population on the kenai peninsula are soldotna and kenai, both located adjacent to the kenai river below skilak lake. the kenai river is the largest waterway on the kenai peninsula and supports commercial, sport, and personal - use salmon fisheries during summer. in addition to a year - round population of approximately 58,000 residents as of 2015 (united states census bureau), the kenai peninsula also receives a considerable influx of tourists each summer as well as recreational users from the nearby city of anchorage. samples were collected from glaucous - winged gulls, herring gulls (larus argentatus), and potentially, hybrids of these two species on the kenai peninsula at the tidal flats at the mouth of the kenai river (60.54n, 151.26w) and at the soldotna landfill (60.44n, 151.11w) on 24 july 2014. fecal samples were collected as previously described and kept cool until frozen at 80c within 12 h. eighty samples from each middleton island (remote location) and the kenai peninsula (urban location) were screened for e. coli and the resultant isolates were tested for antibiotic susceptibility. an aliquot of 100 l from each fecal sample was incubated overnight at 37c in 4 ml of luria - bertani broth (department of clinical microbiology, uppsala university hospital). cotton swabs soaked in the broth were streaked on cystein lactose electrolyte deficient agar plates (department of clinical microbiology, uppsala university hospital) and incubated overnight at 37c. one presumptive e. coli isolate from each sample was collected and confirmed using matrix - assisted laser desorption ionization - time of flight mass spectrometry (maldi - tof ; bruker corporation, germany) as previously described (16). e. coli isolates were spread evenly on muller - hinton agar (department of clinical microbiology, uppsala university hospital) and 10 antibiotic discs ampicillin 10 g, cefadroxil 30 g, chloramphenicol 30 g, nalidixic acid 30 g, nitrofurantoin 100 g, mecillinam 10 g, tetracycline 30 g, tigecycline 15 g, streptomycin 10 g, and sulfamethoxazole trimethoprim 25 g (oxoid ltd, hants, uk) were placed on the plate. inhibition zone diameters were compared to clinical breakpoints for disc diffusion provided by european committee on antibiotic susceptibility (eucast). currently, streptomycin and tetracycline lack eucast clinical breakpoints for disc susceptibility testing, therefore breakpoints for these antibiotics were defined according to the normalized resistance interpretation (nri) method (17). for statistical analyses, samples collected from two urban sites on the kenai peninsula and two remote sites on middleton island the prevalence of antibiotic - resistant isolates and proportion of isolates resistant to three or more antibiotic compounds were compared using fischer 's exact test (graphpad prism version 6). fifty randomly selected samples from each of the kenai peninsula and middleton island collections were screened for extended - spectrum cephalosporin (esbl- and pampc) and carbapenem - resistant e. coli. fecal swabs were placed in tubes with 1 ml phosphate - buffered saline and vortexed. aliquots from each tube were transferred to two vials containing 4 ml luria - bertani broth with either a cefpodoxime 10-g disc (vial 1) or a ertapeneme 10-g disc (vial 2) and incubated overnight at 37c. cotton swabs soaked in the broth from vial 1 were streaked on macconkey agar plates with cefotaxime 5-g disc and ceftazidime 10-g disc and from vial 2 on macconkey agar plates with cefpodoxime 10-g disc and ertapenem 10-g disc (oxoid ltd). colonies growing close to an antibiotic disc were identified as e. coli using maldi - tof. to identify e. coli isolates with esbl- or ampc phenotype, isolates from the previous step were prepared and spread on muller - hinton agar (department of clinical microbiology, uppsala university hospital) according to eucast disc diffusion method for antimicrobial susceptibility testing and five antibiotic discs amoxicillin / clavulanic acid 30/1 g, cefotaxime 5 g, ceftazidime 10 g, cefepime 30 g, and cefoxitin 30 g were placed on the plate according to standard protocol at the department of clinical microbiology in uppsala university hospital. specific inhibition of bacterial growth around the antibiotic discs was used to identify esbl / pampc - phenotypes (18, 19). isolates that displayed esbl or ampc phenotype were subjected to either two multiplex - pcr detecting blactx - m, blashv, blatem, blaoxa-1 or one multiplex - pcr detecting pampc genes and the specific gene variants were determined by sequencing as previously described (20). the esbl / pampc - producing e. coli isolates were further tested for resistance to additional antibiotic compounds. the chosen antibiotics are either frequently used in veterinary and human medicine or are saved for treatment of infections caused by esbl / pampc / carbapenemase - producing e.coli. isolates were spread on muller - hinton agar (department of clinical microbiology, uppsala university hospital) and five antibiotic discs gentamicin 10 g, ciprofloxacin 5 g, meropenem 10 g, tetracycline 30 g, and sulfamethoxazole trimethoprim 25 g (oxoid ltd) were placed on the plate. inhibition zone diameters were compared to clinical breakpoints for disc diffusion provided by eucast and the nri method (16), as described above. in addition, all isolates were tested for colistin susceptibility using etest (biomrieux, marcy letoile, france) according to manufacturer s guidelines. however, due to that the recently described plasmid encoded mcr-1 have been described to show mics below eucast breakpoint and the unreliability of colistin susceptibility results with e - test all esbl - isolates were also tested for the presences of mcr-1 (21, 22). a total of 160 fecal samples were collected in june and july 2014 from large - bodied gulls at two sites from each of two general locations within southcentral alaska 1). middleton island is a remote location in southcentral alaska, approximately 115 km offshore from the mainland in the gulf of alaska. middleton island was once the site of an active military installation (ca., 19581963) but is now uninhabited by humans with the exceptions of small field crews of scientists conducting research activities on the island during summer months and federal aviation administration employees (typically two) that work on the island approximately half - time year round. middleton island is occupied by large numbers of gulls each summer including multiple breeding colonies of glaucous - winged gulls (larus glaucescens). fecal samples were obtained during 2226 june 2014 from glaucous - winged gulls at sites on the northern (59.46n, 146.29w) and southern (59.4159.42n, 146.33146.36w) margins of middleton island, near nesting colonies prior to hatch. individual fecal samples were collected by swirling a sterile swab in fresh feces and placing in bacteria freeze media in cryovials. samples from middleton island were kept cool with icepacks for 48 days until frozen at 80c. map depicting sampling locations for large - bodied gulls (circles) in southcentral alaska. the approximate location of anchorage, the most populous city in alaska, is indicated with a star. the kenai peninsula is a landmass extending south of anchorage, alaska, the most populous city in the state, and supports numerous communities and an extensive road system. communities with the highest human population on the kenai peninsula are soldotna and kenai, both located adjacent to the kenai river below skilak lake. the kenai river is the largest waterway on the kenai peninsula and supports commercial, sport, and personal - use salmon fisheries during summer. in addition to a year - round population of approximately 58,000 residents as of 2015 (united states census bureau), the kenai peninsula also receives a considerable influx of tourists each summer as well as recreational users from the nearby city of anchorage. samples were collected from glaucous - winged gulls, herring gulls (larus argentatus), and potentially, hybrids of these two species on the kenai peninsula at the tidal flats at the mouth of the kenai river (60.54n, 151.26w) and at the soldotna landfill (60.44n, 151.11w) on 24 july 2014. fecal samples were collected as previously described and kept cool until frozen at 80c within 12 h. eighty samples from each middleton island (remote location) and the kenai peninsula (urban location) were screened for e. coli and the resultant isolates were tested for antibiotic susceptibility. an aliquot of 100 l from each fecal sample was incubated overnight at 37c in 4 ml of luria - bertani broth (department of clinical microbiology, uppsala university hospital). cotton swabs soaked in the broth were streaked on cystein lactose electrolyte deficient agar plates (department of clinical microbiology, uppsala university hospital) and incubated overnight at 37c. one presumptive e. coli isolate from each sample was collected and confirmed using matrix - assisted laser desorption ionization - time of flight mass spectrometry (maldi - tof ; bruker corporation, germany) as previously described (16). e. coli isolates were spread evenly on muller - hinton agar (department of clinical microbiology, uppsala university hospital) and 10 antibiotic discs ampicillin 10 g, cefadroxil 30 g, chloramphenicol 30 g, nalidixic acid 30 g, nitrofurantoin 100 g, mecillinam 10 g, tetracycline 30 g, tigecycline 15 g, streptomycin 10 g, and sulfamethoxazole trimethoprim 25 g (oxoid ltd, hants, uk) were placed on the plate. inhibition zone diameters were compared to clinical breakpoints for disc diffusion provided by european committee on antibiotic susceptibility (eucast). currently, streptomycin and tetracycline lack eucast clinical breakpoints for disc susceptibility testing, therefore breakpoints for these antibiotics were defined according to the normalized resistance interpretation (nri) method (17). for statistical analyses, samples collected from two urban sites on the kenai peninsula and two remote sites on middleton island the prevalence of antibiotic - resistant isolates and proportion of isolates resistant to three or more antibiotic compounds were compared using fischer 's exact test (graphpad prism version 6). fifty randomly selected samples from each of the kenai peninsula and middleton island collections were screened for extended - spectrum cephalosporin (esbl- and pampc) and carbapenem - resistant e. coli. fecal swabs were placed in tubes with 1 ml phosphate - buffered saline and vortexed. aliquots from each tube were transferred to two vials containing 4 ml luria - bertani broth with either a cefpodoxime 10-g disc (vial 1) or a ertapeneme 10-g disc (vial 2) and incubated overnight at 37c. cotton swabs soaked in the broth from vial 1 were streaked on macconkey agar plates with cefotaxime 5-g disc and ceftazidime 10-g disc and from vial 2 on macconkey agar plates with cefpodoxime 10-g disc and ertapenem 10-g disc (oxoid ltd). colonies growing close to an antibiotic disc were identified as e. coli using maldi - tof. to identify e. coli isolates with esbl- or ampc phenotype, isolates from the previous step were prepared and spread on muller - hinton agar (department of clinical microbiology, uppsala university hospital) according to eucast disc diffusion method for antimicrobial susceptibility testing and five antibiotic discs amoxicillin / clavulanic acid 30/1 g, cefotaxime 5 g, ceftazidime 10 g, cefepime 30 g, and cefoxitin 30 g were placed on the plate according to standard protocol at the department of clinical microbiology in uppsala university hospital. specific inhibition of bacterial growth around the antibiotic discs was used to identify esbl / pampc - phenotypes (18, 19). isolates that displayed esbl or ampc phenotype were subjected to either two multiplex - pcr detecting blactx - m, blashv, blatem, blaoxa-1 or one multiplex - pcr detecting pampc genes and the specific gene variants were determined by sequencing as previously described (20). the esbl / pampc - producing e. coli isolates were further tested for resistance to additional antibiotic compounds. the chosen antibiotics are either frequently used in veterinary and human medicine or are saved for treatment of infections caused by esbl / pampc / carbapenemase - producing e.coli. isolates were spread on muller - hinton agar (department of clinical microbiology, uppsala university hospital) and five antibiotic discs gentamicin 10 g, ciprofloxacin 5 g, meropenem 10 g, tetracycline 30 g, and sulfamethoxazole trimethoprim 25 g (oxoid ltd) were placed on the plate. inhibition zone diameters were compared to clinical breakpoints for disc diffusion provided by eucast and the nri method (16), as described above. in addition, all isolates were tested for colistin susceptibility using etest (biomrieux, marcy letoile, france) according to manufacturer s guidelines. however, due to that the recently described plasmid encoded mcr-1 have been described to show mics below eucast breakpoint and the unreliability of colistin susceptibility results with e - test all esbl - isolates were also tested for the presences of mcr-1 (21, 22). a total of 55 e. coli isolates were recovered from 80 samples screened from the kenai peninsula and 60 isolates were from 80 samples screened from middleton island, yielding isolation rates of 69 and 75% per location, respectively (table 1). of the e.coli isolates recovered from the urban kenai peninsula, 55% (30/55) were resistant to at least one antibiotic compound, whereas only 8% (5/60) of the isolates from the remote site middleton island displayed antibiotic resistance (table 1). the difference in the prevalence of isolates resistant to at least one antibiotic between the two locations was statistically significant (kenai peninsula vs middleton island, two - tailed p value < 0.0001, fisher 's exact test). additionally, 22% (12/55) of e. coli isolates from the kenai peninsula were resistant to three or more compounds as compared to 2% (1/60) of isolates derived from samples at middleton island, supporting a statistical difference in the prevalence of multi - resistant e. coli between locations (kenai peninsula vs middleton island, two - tailed fisher 's exact test, p value < 0.001 ; table 2). considering antimicrobial activity in all e.coli isolates from the two areas, resistance to eight different antibiotic compounds could be found in kenai peninsula samples as compared to five different antibiotic compounds in middleton island isolates (table 3). isolation of e. coli from large - bodied gull samples collected at urban and remote locations in southcentral alaska, 2014 antibiotic resistance of e. coli from large - bodied gulls sampled in southcentral alaska relative to number of compounds resistance of e. coli isolated from large - bodied gulls in southcentral alaska to specific antibiotic compounds sulfamethoxazole trimethoprim. out of 50 samples screened from the kenai peninsula (urban location), three e. coli isolates that displayed esbl phenotype and five e. coli isolates that displayed ampc phenotype were found. the esbl isolates harbored blactx - m-15 and blatem-1 and the pampc isolates harbored blacmy-2 (table 4). three esbl / pampc - producing e. coli isolates were resistant to at least one other antibiotic compound and all were negative for mcr-1 via pcr (table 4). on the kenai peninsula, all eight esbl / pampc isolates were found among the gull samples from the soldotna landfill site. no esbl / pampc isolates were found at the kenai river site or on middleton island (table 4). phenotype, genotype, and antibiotic susceptibility of eight extended - spectrum cephalosporin - resistant e. coli ; (esbl / pampc) isolates from gulls sampled on the kenai peninsula, alaska. susceptibility (s) and resistance (r) to specific compounds are indicated susceptibility tested by etest sulfamethoxazole trimethoprim. a total of 55 e. coli isolates were recovered from 80 samples screened from the kenai peninsula and 60 isolates were from 80 samples screened from middleton island, yielding isolation rates of 69 and 75% per location, respectively (table 1). of the e.coli isolates recovered from the urban kenai peninsula, 55% (30/55) were resistant to at least one antibiotic compound, whereas only 8% (5/60) of the isolates from the remote site middleton island displayed antibiotic resistance (table 1). the difference in the prevalence of isolates resistant to at least one antibiotic between the two locations was statistically significant (kenai peninsula vs middleton island, two - tailed p value < 0.0001, fisher 's exact test). additionally, 22% (12/55) of e. coli isolates from the kenai peninsula were resistant to three or more compounds as compared to 2% (1/60) of isolates derived from samples at middleton island, supporting a statistical difference in the prevalence of multi - resistant e. coli between locations (kenai peninsula vs middleton island, two - tailed fisher 's exact test, p value < 0.001 ; table 2). considering antimicrobial activity in all e.coli isolates from the two areas, resistance to eight different antibiotic compounds could be found in kenai peninsula samples as compared to five different antibiotic compounds in middleton island isolates (table 3). isolation of e. coli from large - bodied gull samples collected at urban and remote locations in southcentral alaska, 2014 antibiotic resistance of e. coli from large - bodied gulls sampled in southcentral alaska relative to number of compounds resistance of e. coli isolated from large - bodied gulls in southcentral alaska to specific antibiotic compounds sulfamethoxazole trimethoprim. out of 50 samples screened from the kenai peninsula (urban location), three e. coli isolates that displayed esbl phenotype and five e. coli isolates that displayed ampc phenotype were found. the esbl isolates harbored blactx - m-15 and blatem-1 and the pampc isolates harbored blacmy-2 (table 4). three esbl / pampc - producing e. coli isolates were resistant to at least one other antibiotic compound and all were negative for mcr-1 via pcr (table 4). on the kenai peninsula, all eight esbl / pampc isolates were found among the gull samples from the soldotna landfill site. no esbl / pampc isolates were found at the kenai river site or on middleton island (table 4). phenotype, genotype, and antibiotic susceptibility of eight extended - spectrum cephalosporin - resistant e. coli ; (esbl / pampc) isolates from gulls sampled on the kenai peninsula, alaska. susceptibility (s) and resistance (r) to specific compounds are indicated susceptibility tested by etest sulfamethoxazole trimethoprim. through our investigation of antibiotic - resistant e. coli derived from large - bodied gulls at two locations in southcentral alaska with different levels of anthropogenic influence, we observed differences in the prevalence of antibiotic - resistant and esbl / pampc - producing e.coli. thus, although our study design is correlative in nature, our results support the hypothesis that anthropogenic inputs may be a factor in determining the level of antibiotic resistance among e. coli infecting large - bodied gulls in southcentral alaska. the higher incidence of antibiotic resistance detected in e. coli isolated from large - bodied gulls on the kenai peninsula relative to middleton island is likely, at least partially, a function of inputs from inhabitants and visitors of the towns of soldotna and kenai, urban areas in close proximity to sampling sites at the soldotna landfill and mouth of the kenai river. although neither soldotna nor kenai are major metropolitan areas, direct inputs of antibiotic- resistant bacteria and biocides / antibiotic residues, which may lead to the emergence of antibiotic- resistant strains, are likely released into the environment via sewage treatment and the local landfill at higher rates as compared to areas lacking large permanent human settlements. accordingly, the likelihood that gulls may encounter antibiotic - resistant e. coli or plasmids conferring resistant genes from anthropogenic sources via waste management practices may be higher in these more urban environments as compared to more remote sites. food animal production occurs at low levels in alaska and therefore is less likely a major contributor to antibiotic resistance in the area (23). the prevalence of antimicrobial - resistant e. coli detected in gulls at kenai peninsula sites (55%) was similar to that found in yellow - legged gulls in france (47%) (11) and the most recent assessment of gulls at barrow, alaska (48%) (15), areas in close proximity to human settlements providing anthropogenic inputs into the environment. additionally, the finding that all esbl / pampc - harboring e. coli isolated in this study were detected among gull samples collected at kenai peninsula, including globally disseminated blactx - m-15, is similar to the recent detection of blactx - m in barrow, alaska at a sampling site in close proximity to the local landfill (15). as blactx - m-15 and blacmy-2 are relatively common in humans and livestock in the usa (24, 25), it is unsurprising to find these enzymes in gulls inhabiting alaska landfills. further studies to assess the occurrence of antibiotic resistance and antibiotic compounds in the soil and water around the soldotna landfill may help to provide inference regarding the acquisition of antibiotic - resistant bacteria by gulls in this area. the relatively low level of antibiotic resistance detected in e. coli isolated from glaucous - winged gulls at middleton island is likely a reflection of the geographic isolation of this location from mainland north america and a lack of anthropogenic input of antibiotic - resistant bacteria and/or antibiotic compounds. in a survey from the remote commander islands (russia) in the bering sea, hernandez. found 4% (6/145 isolates) of e. coli isolated from seabirds, including glaucous - winged gulls, were resistant to one or more antibiotic compounds (26), comparable to the level found in gulls at middleton island (8%). thus, it appears that glaucous - winged gulls sampled at remote breeding sites have relatively little exposure to antibiotic - resistant bacteria, or compounds driving their emergence, during the breeding season. the detection of some level, albeit low, of antimicrobial resistance among e. coli cultured from glaucous - winged gull samples collected at middleton island suggests that either low levels of resistance may be maintained among bacteria infecting wild birds in the absence of anthropogenic inputs or that resistant bacterial strains arrive at breeding grounds via migrating gulls returning from wintering and staging areas. it should be noted, however, that levels of antibiotic resistance in e. coli among avian populations are not static. the observed prevalence of esbl - producing e. coli in gulls sampled at barrow, alaska has increased between 2005 and 2010 (15). thus, middleton island could be an informative site for future assessments aiming to address the regional and global dissemination of antibiotic - resistant genes through time. although differences of antibiotic resistance levels among e. coli infecting gulls at two southcentral alaska locations are likely directly influenced by bacteria and plasmids from human sources, the role of antibiotic residues in the environment driving the development of resistance in wild birds needs to be further investigated. furthermore, whether birds can maintain antibiotic - resistant e. coli for long durations in the absence of human impact and disperse such bacteria via migration remains unclear and is worthy of further study. finally, given the relatively high prevalence of antibiotic resistance detected in large - bodied gulls on the kenai peninsula (i.e. 55% of isolates tested) and the geographic and temporal proximity of our sampling site at the mouth of the kenai river to a popular personal - use sockeye salmon (oncorhynchus nerka) fishery, additional research may be warranted to understand the risk for human bacterial e. coli infections via environmental transmission at this site during summer. the authors have not received any funding or benefits from industry or elsewhere to conduct this study. | backgroundantibiotic - resistant bacteria pose challenges to healthcare delivery systems globally ; however, limited information is available regarding the prevalence and spread of such bacteria in the environment. the aim of this study was to compare the prevalence of antibiotic - resistant bacteria in large - bodied gulls (larus spp.) at urban and remote locations in southcentral alaska to gain inference into the association between antibiotic resistance in wildlife and anthropogenically influenced habitats.methodsescherichia coli was cultured (n=115 isolates) from fecal samples of gulls (n=160) collected from a remote location, middleton island, and a more urban setting on the kenai peninsula.resultsscreening of e. coli from fecal samples collected from glaucous - winged gulls (larus glaucescens) at middleton island revealed 8% of isolates were resistant to one or more antibiotics and 2% of the isolates were resistant to three or more antibiotics. in contrast, 55% of e. coli isolates derived from fecal samples collected from large - bodied gulls (i.e. glaucous, herring [larus argentatus ], and potentially hybrid gulls) on the kenai peninsula were resistant to one or more antibiotics and 22% were resistant to three or more antibiotics. in addition, total of 16% of the gull samples from locations on the kenai peninsula harbored extended - spectrum cephalosporin - resistant e. coli isolates (extended - spectrum beta - lactamases [esbl ] and plasmid - encoded ampc [pampc ]), in contrast to middleton island where no esbl- or pampc - producing isolates were detected.conclusionour findings indicate that increased prevalence of antibiotic resistance is associated with urban environments in southcentral alaska and presumably influenced by anthropogenic impacts. further investigation is warranted to assess how migratory birds may maintain and spread antimicrobial - resistant bacteria of relevance to human and animal health. |
the extensive use of pesticides harms the soil [14 ], air [5, 6 ], food [710 ] surface and ground waters [1114 ], and quality causing serious impacts on the environment and on human health. in natural waters pesticide residues are present at very low levels and can be degraded when submitted to lower ph levels or exposed to solar radiation. furthermore the complexity of environmental matrices and large variations in physical and chemical properties of the target compounds requires the use of sensitive and selective techniques. several analytical techniques, such as high - performance liquid chromatography (hplc) [16, 17 ], gas chromatography (gc) [18, 19 ], micellar electrokinetic chromatography (mekc) [20, 21 ], enzyme - linked immunosorbent assays (elisa) [2224 ], and gas and liquid chromatography coupled to mass spectrometry (gc / ms, lc - ms) [25, 26 ], have been used for analyses of pesticides in different matrices. the chromatographic techniques combine separation capabilities with sensitivity from the mass systems such as ion trap (it), triple quadrupole (qqq), and time of flight (tof). however, these techniques still remain as challenges related to low detection limits, the variety of pesticides classes, and sample preparation. the analytes extraction in chromatographic analysis is critical to the method 's performance since it enables the elimination of possible array interferences and the preconcentration of analytes. traditional extraction methods such as solid phase extraction (spe) [27, 28 ] and liquid - liquid extraction (lle) are multistage consuming toxic solvents and require a long time to execute. solid phase microextraction (spme) [2931 ] is a technique that is based on the partition between the analyte present in the matrix and the fiber coating over a small fused silica rod. this technique is solvent - free and gathers in a single step extraction and preconcentration. however problems such as low resistance, short lifetime, and high cost remain. recently, several materials have been proposed to increase the strength and durability of spme coatings such as carbon materials. hollow fiber liquid phase microextraction (hf - lpme) [3337 ] and dispersive liquid - liquid microextraction (dllme) [38, 39 ] have been used for the concentration and clean - up step of pesticides analyses in waters. hf - lpme was developed by pedersen - bjergaard and rasmussen and has been used by many researchers in recent years due to its low cost, which enables the rejection of the material after use, eliminating problems of cross - contamination or low reproducibility as well as its decreased consumption of organic solvents. moreover, the process is simple and a clean - up step is not necessary and can be applied to a variety of arrays reaching high enrichment factors. the technique consists of a capillary porous hydrophilic fiber, impregnated with organic solvent and its interior filled with an acceptor phase, so that it does not come into direct contact with the matrices allowing for the application of agitation during extraction. the analyte is extracted from the donor through an organic solvent immiscible in water that fills the membrane pores passing to the acceptor stage, which corresponds to the same solvent. with three phases the analyte is extracted from a donor phase through an organic solvent immiscible in water for an aqueous solution (acceptor phase) inside the fiber. the organic phase acts as a barrier preventing contact between the phases. despite the extensive use of hf - lpme for extraction of pesticides in water [33, 44, 45 ], the reported studies using gc / ms are generally for just one pesticide class. therefore, this study presents the development of a simple and low - cost two - phase hf - lpme methodology for multiresidue microextraction of organophosphorus, phthalimides, organochlorines, and triazoles pesticides from environmental water using gc / ms. the pesticides selected were parathion - methyl (o, o - dimethyl - o - p - nitrophenyl phosphorothioate), chlorpyrifos (o, o - diethyl o-3,5,6-trichloro-2-pyridyl phosphorothioate), captan (n-(trichloromethylthio)cyclohex-4-ene-1,2-dicarboximide), procymidone (n-(3,5-dichlorophenyl)-1,2-dimethylcyclopropane-1,2-dicarboximide), -endosulfan (1,4,5,6,7,7-hexachloro-8,9,10-trinorborn-5-en-2,3-ylenebismethylene sulfite), prothiofos ((rs)-(o-2,4-dichlorophenyl o - ethyl s - propyl phosphorodithioate)), cyproconazole ((2rs,3rs;2rs,3sr)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1h-1,2,4-triazol-1-yl)butan-2-ol), ethion (o, o, o,o-tetraethyl s, s-methylene bis(phosphorodithioate)), triazophos (o, o - diethyl o-1-phenyl-1h-1,2,4-triazol-3-yl phosphorothioate), and phosmet (o, o - dimethyl s - phthalimidomethyl phosphorodithioate). the procedure presented good accuracy and precision and low limits of quantification and detection, besides good recovery. parathion - methyl, chlorpyrifos, captan, procymidone, -endosulfan, prothiofos, cyproconazole, ethion, triazophos, and phosmet of 98% w / w purity grade were purchased from sigma - aldrich (st. louis, mo, usa). the choice of pesticides was based on their use on the region of samples collection. a work solution of 20.00 mg l was prepared by the appropriate dilution in hplc - grade methanol, sigma - aldrich (st. louis, missouri, united states). this work solution was used for the matrix spike in different concentration levels (5.00 to 160.00 g l) to optimize the extraction conditions during the validation study. calibration standards were prepared at 5.00, 10.00, 20.00, 40.00, 80.00, and 160.00 g l concentrations using ultrapure water produced in a purelab uvmk2 purifier from elga (marlow, buckinghamshire, england). 1-octanol hplc grade was purchased from sigma - aldrich (st. louis, missouri, united states), ethyl decanoate, acetonitrile, and ethyl octanoate were purchased from j. t. baker (xalostoc, edo mex, mexico). the analysis was carried out with a shimadzu (kyoto, japan) gc / ms system model gc-2010/qp-2010 high - performance quadrupole. the mass spectrometer operated within the electron impact mode (ei) at 70 ev. a capillary column (30 m 0.25 mm 0.25 m) containing 5% diphenyl and 95% dimethylpolysiloxane hp-5ms from agilent technology, inc. the oven temperature program began at 80c and raised to 200c at a rate of 8c min up to 300c at 30c min and held there for 3 min. helium (99.999%) was the carrier gas at a flow rate of 1.0 ml min. the injector was operated at 280c in splitless mode for 3 min, followed by a 1 : 20 split ratio (rd). the ion source temperature was 200c, and the gc / ms interface temperature was 300c. the collection of raw data was carried out using a labsolution software, shimadzu (kyoto, japan). aqueous standards of pesticides were prepared by spiking an appropriate amount of the working standard. lpme sampling was tested in two and three phases, study of salt addition, stirring speed, and extraction time. the extractions were carried out with propylene hollow fiber of 6.0 cm length, 600 m of internal diameter, and wall thickness of 200 m. before the extraction, then, the u - shaped solvent - filled fiber was connected to two syringe needles and immersed into the vial containing 15.00 ml of aqueous donor solutions spiked with 100.0 g l of standard pesticides solutions for the extraction under magnetic stirring. after the extraction, the acceptor phase was removed with a microsyringe and transferred to a 2.0 ml vial to the injection of 1.0 l in gc / ms. all experiments were performed in replicates (n = 3). real samples of surface water were collected in a rural area of the state of minas gerais, brazil. in this region the amber - glass collection bottles were previously washed with a solution of 5.0% v / v alkaline detergent under ultrasound bath for 15 minutes and rinsed with ultrapure water. water samples collected with these bottles were carefully filled to the brim to avoid trapping air. after filling the bottles they were sealed with teflon lined screw caps, kept in ice, and transported to the laboratory before 24 h where they were stored at 4c until the extraction and analysis. the quality control and the quality assurance method the limits of detection (lod) and limits of quantification (loq) were calculated from mean and standard deviation of 10 blank measurements with 95% confidence. the hartley test using origin 8.0 from originlab co. software (northampton, ma, united states) was used to verify the instrumental response homogeneity of variances. the result of this test indicated heteroscedasticity of variances, so the linear models for the calibration curves were constructed by the least squares method weighted by the experimental variance. the most important factors related to the hf - lpme extraction method such as the extraction mode, solvents, agitation, salt addition, and extraction time were optimized before the validation tests. the pesticides studied were chosen based on the information of their broad use (higher quantity retailed) in the cultures of the region where the samples were collected. the extraction mode with three phases was evaluated using acetonitrile as the acceptor phase ; 1-octanol, ethyl decanoate, and ethyl octanoate were tested as the organic phase. aqueous solutions spiked with standard pesticides were used as donor solutions. a significant loss of the organic phase during the three - phased extraction process significantly affected the recovery of the acceptor phase for further analysis. subsequently, the two - phase mode was tested using 1-octanol or ethyl decanoate and ethyl octanoate as the acceptor phase and the aqueous solutions spiked with standard pesticides as donor solutions. the two - phase extraction method provided significant improvement in the recovery due to good immobilization of the acceptor phase in the fiber. ethyl octanoate was selected as the acceptor phase due to its superior response to most pesticides as shown in figure 1. the results obtained with 1-octanol were relatively lower compared to the other acceptor phases studied. this can be explained by the difference in polarity between them since ethyl decanoate and ethyl octanoate are less polar than 1-octanol. therefore, the studied analytes are best extracted in more hydrophobic solvents because all kow values are greater than 1. this indicates that there is greater solubility in nonpolar solvent increasing the distribution ratio between the organic acceptor solution and the donor solution. the different areas observed for organochlorine pesticides in relation to organophosphorus areas are mainly due to the fact that the electron impact mode used in ms detector show extensive fragmentation. the fiber is depleted of the analytes due to their partitioning in the donor phase ; hence, the agitation diminishes this depletion area by bringing fresh undepleted sample close to the fiber. agitation also reduces the time required to reach thermodynamic equilibrium and induces convection in the membrane phase. to optimize sample agitation, ethyl octanoate was used as the acceptor phase, and aqueous pesticide was used as the donor phase. the stirring rates studied were 0 ; 200 ; 400 ; 800 ; and 1600 rpm using metallic stir bars of 0.5 cm. the results presented in figure 2 show that the largest areas were obtained using 200 rpm of agitation speed. it was also observed that the agitation speed higher than 200 rpm decreases extraction efficiency despite the fact that stirring promotes mass transfer between the donor and acceptor phases. the relative decreased extraction yield was due to the fact that vigorous agitation promotes the formation of air bubbles which adhere to the fiber surface. the efficiency of the extraction at 0 rpm and 400 rpm are similar because in the static stage the diffusion layer close to the fiber is not renewed ; this effect decreases the mass transfer to the donor layer. however, the formation of bubbles on the outer surface of the fiber starts from 400 rpm, which contribute to reducing the mass transfer of the acceptor phase. the effect of salt addition to the lpme extraction of the pesticides was examined in the presence of different concentrations of nacl : 0.0 ; 5.0 ; 10.0 ; and 15.0% w / v. the addition of salt in the aqueous samples generally improves the extraction of analytes in the organic phase. the salt increases the ionic strength decreasing the solubility of hydrophobic analytes in the donor phase ; therefore, it enhances their partitioning into the acceptor phase. however, in this study the addition of salt did not present a positive influence on the extraction process of most analytes (figure 3). phosmet presented a large improvement in the extraction efficiency of 5.0% nacl concentration, although at higher concentrations the peak area decreased drastically. the presence of higher concentrations of salt can change the physical properties of the extraction film reducing the diffusion rates of the analytes into the organic phase. therefore, the increase in ionic strength resulting from the addition of salt increased the salting - in effect. this effect has been observed in other studies regarding environmental water samples [35, 56 ]. lpme sampling is an equilibrium process, in which analytes are partitioned between the donor phase and acceptor phase. the equilibrium time refers to the time after which the amount of extracted analyte remains constant. extraction times of 10.0 ; 20.0 ; 30.0 ; 40.0 ; and 60.0 minutes were tested for the extraction using the best conditions of the variables previously assessed. as seen in figure 4, the extraction efficiency reached its maximum value after 30 min for most of the pesticides evaluated. periods above 30 min reduced extraction efficiency in some analytes, such as parathion - methyl, captan, phosmet, and triazophos. this reduction occurs due to the prolonging of the stirring time, which originates the formation of bubbles in the outer fiber, contributing to increased losses of the donor phase. after evaluating the different parameters that could affect the extraction the following optimized conditions were selected for all experiments : 6 cm polypropylene hollow fiber impregnated and filled with 30 l of ethyl octanoate, immersion in a vial containing 15.0 ml of sample for 30 min under stirring at 200 rpm, and injection of 1.0 l to the acceptor phase in the gc / ms system. figure 5 shows chromatogram of pesticides gc / ms analysis using hf - lpme extraction in aqueous donor solution spiked at 50.0 g l with standards (figure 5(a)) and real environmental water sample (figure 5(b)) under the same extraction conditions. chromatographic separation was satisfactory for all target analytes in a short time. under the optimal extraction conditions the parameters linearity, limits of detection (lod), limits of quantification (loq), recovery, and precision were carefully investigated. the experimental results are presented in table 2. a linear range of 0.14 g the linearity was assessed by the determination coefficient (r) that was in the range of 0.98070.9990. l to 0.44 g l, and loq ranged from 0.14 g l to 1.69 g l. the recovery and precision studies were performed by three replicates of real water samples spiked with concentration of 10.0 the merit parameters values of linearity and precision obtained in this study are consistent with other results reported in the analysis of pesticides in water [37, 5761 ]. on the contrary, the limits of detection and quantification obtained in this study were better than those obtained in other works using hf - lpme [59, 62 ]. besides, comparing other methods described in the literature (table 3) to the multiclass pesticides analysis this hf - lpme method presents better precision and low cost. the application of the hf - lpme - gc / ms method for determination of pesticides of real samples was achieved through the analysis of three real samples of surface water (spring, stream 1, and stream 2) collected in a rural area of the state of minas gerais, brazil. parathion - methyl was quantified in the sample of stream 2 and detected in spring water and stream 1. the value found for this pesticide in the samples analyzed is lower than the limit established by brazilian legislation. these results show the ability of the method for the analysis of ten pesticides of different chemical classes in real samples. this study describes the use of a simple and quick method for the determination of pesticides in environmental water by two - phase hf - lpme - gc / ms. the main factors related to hf - lpme extraction method such as the extraction mode, solvents, agitation, salt addition, and extraction time were investigated and optimized before the validation tests. the proposed method showed good linearity, low detection and quantification limits, high selectivity, and good repeatability for the pesticides selected. this procedure is selective, simple, fast, and low cost ; it has minimal use of solvents and does not require pretreatment of samples. the results obtained from the analyses of three environmental water samples (spring and streams) have demonstrated the ability of the method to measure trace levels of pesticides. | this paper describes a simple and quick method for sampling and also for carrying out the preconcentration of pesticides in environmental water matrices using two - phased hollow fiber liquid phase microextraction (hf - lpme). factors such as extraction mode, time, solvents, agitation, and salt addition were investigated in order to validate the lpme method. the following conditions were selected : 6 cm of polypropylene hollow fiber, ethyl octanoate as an acceptor phase, and extraction during 30 min under stirring at 200 rpm. the optimized method showed good linearity in the range of 0.14 to 200.00 g l1 ; the determination coefficient (r2) was in the range of 0.98070.9990. the lod ranged from 0.04 g l1 to 0.44 g l1, and loq ranged from 0.14 g l1 to 1.69 g l1. the recovery ranged from 85.17% to 114.73%. the method was applied to the analyses of pesticides in three environmental water samples (a spring and few streams) collected in a rural area from the state of minas gerais, brazil. |
medical records from the university of missouri veterinary medical teaching hospital and the university of minnesota veterinary medical center were reviewed to identify dogs that were diagnosed with severe sas between september 1999 and january 2011. information regarding signalment, age at diagnosis, echocardiographic findings, medications, concurrent disease, development of congestive heart failure, endocarditis or other clinical signs related to heart disease, and date and cause of death when applicable was gathered for each patient. when medical records were incomplete, all dogs were diagnosed by or under the supervision of an acvimboard certified cardiologist by using standard transthoracic 2dimensional and doppler echocardiography without sedation.14 three to 5 measurements were averaged for each variable. peak velocity of blood flow in the lvot was measured by continuouswave doppler and used to calculate a pg using the modified bernoulli equation (p = 4v), where p = pg and v = peak velocity. dogs with hemodynamically relevant concurrent congenital cardiac disease such as mitral dysplasia, patent ductus arteriosus, pulmonic stenosis, and valvular or supravalvular aortic stenosis causing clinically relevant pressure or volume overload were excluded from analysis. in addition, dogs that underwent a surgical procedure for palliation of sas or for which there was no followup information were excluded. cardiac death was defined as euthanasia or death after onset of signs of congestive heart failure, or sudden death. sudden death included witnessed sudden death or discovery of deceased animal with no premonitory signs of illness within the past 24 hours. all analyses were performed with a commercial statistical software package.1 normality was analyzed by using the shapirowilk test. baseline descriptive statistics are presented as mean and standard deviation for normally distributed variables whereas nonnormally distributed variables are presented as median and range. betweengroup analyses of baseline variables were performed by using unpaired ttests, or the mannwhitney rank sum test as appropriate for the data distribution. continuous variables were categorized based upon the mean for a given covariate and used in the survival analyses. modelrelative goodnessoffits were analyzed by akaike information criterion and compared by using a chisquare 1 degree of freedom test. tests for proportionality were carried out by visual inspection of schoenfeld residuals, negative log estimated survival distribution function, and formal hypothesis testing of covariates by log (time) interactions followed by wald chisquare statistics and deemed proportional. additional sensitivity analyses were carried out by using parametric accelerated time failure models affirmatively validating the cox proportionality assumptions. fisher 's exact test was used to assess for differences in the proportion of sudden death and congestive heart failure between groups. medical records from the university of missouri veterinary medical teaching hospital and the university of minnesota veterinary medical center were reviewed to identify dogs that were diagnosed with severe sas between september 1999 and january 2011. information regarding signalment, age at diagnosis, echocardiographic findings, medications, concurrent disease, development of congestive heart failure, endocarditis or other clinical signs related to heart disease, and date and cause of death when applicable was gathered for each patient. when medical records were incomplete, owners and referring veterinarians were contacted. all dogs were diagnosed by or under the supervision of an acvimboard certified cardiologist by using standard transthoracic 2dimensional and doppler echocardiography without sedation.14 three to 5 measurements were averaged for each variable. peak velocity of blood flow in the lvot was measured by continuouswave doppler and used to calculate a pg using the modified bernoulli equation (p = 4v), where p = pg and v = peak velocity. dogs with hemodynamically relevant concurrent congenital cardiac disease such as mitral dysplasia, patent ductus arteriosus, pulmonic stenosis, and valvular or supravalvular aortic stenosis causing clinically relevant pressure or volume overload were excluded from analysis. in addition, dogs that underwent a surgical procedure for palliation of sas or for which there was no followup information were excluded. cardiac death was defined as euthanasia or death after onset of signs of congestive heart failure, or sudden death. sudden death included witnessed sudden death or discovery of deceased animal with no premonitory signs of illness within the past 24 hours. all analyses were performed with a commercial statistical software package.1 normality was analyzed by using the shapirowilk test. baseline descriptive statistics are presented as mean and standard deviation for normally distributed variables whereas nonnormally distributed variables are presented as median and range. betweengroup analyses of baseline variables were performed by using unpaired ttests, or the mannwhitney rank sum test as appropriate for the data distribution. continuous variables were categorized based upon the mean for a given covariate and used in the survival analyses. modelrelative goodnessoffits were analyzed by akaike information criterion and compared by using a chisquare 1 degree of freedom test. tests for proportionality were carried out by visual inspection of schoenfeld residuals, negative log estimated survival distribution function, and formal hypothesis testing of covariates by log (time) interactions followed by wald chisquare statistics and deemed proportional. additional sensitivity analyses were carried out by using parametric accelerated time failure models affirmatively validating the cox proportionality assumptions. fisher 's exact test was used to assess for differences in the proportion of sudden death and congestive heart failure between groups. golden retriever (n = 15), newfoundland (n = 9), boxer (n = 6), german shepherd dog (n = 4), irish setter (n = 3), labrador retriever (n = 2), bulldog (n = 2), mixed breed (n = 2), and 1 each : bull mastiff, boston terrier, english springer spaniel, pug, rottweiler, scottish terrier, and siberian husky. twentysix dogs were male (9 castrated) and 24 dogs were female (13 spayed). twentythree dogs received atenolol at a median dosage of 0.55 mg / kg (range, 0.301.2 mg / kg) po q12h, 1 dog received propranolol at a dosage of 0.64 mg / kg po q8h, and 1 dog received sotalol at a dosage of 0.9 mg / kg po q12h. the median age at diagnosis for the treated group was significantly younger than that of the untreated group (0.6 years [range, 0.24.32 years ] versus 1.2 years [range, 0.211.6 years ], respectively ; p =.03). there was no significant difference in the median pg at diagnosis between the treated and untreated groups (127 mmhg [range, 81228 ] versus 121 mmhg [range, 80217 mmhg ], respectively ; p =.20), or the university of minnesota and the university of missouri (116 mmhg [range, 80190 mmhg ] versus 128 mmhg [range, 80227 mmhg ], respectively ; p =.16). twenty cases were contributed by the university of minnesota (treated ; n = 1) and 30 cases were contributed by the university of missouri (treated ; n = 26). no dog had reported episodes of syncope or signs of heart failure at initial presentation. four dogs in the treated group were diagnosed with mitral valve regurgitation (3 mild, 1 moderate), 1 of which also had mild concurrent tricuspid valve regurgitation, and 2 dogs were diagnosed with mild pulmonic stenosis (pg 130 mmhg. in the allcause mortality analyses, 12 dogs that were still alive were censored (130 mmhg, n = 11 ; > 130 mmhg, n = 1). dogs with a pg at diagnosis of 130 mmhg had a median survival of 8.3 years (range, 1.011.8 years), whereas dogs with a pg at diagnosis of > 130 mmhg had a significantly shorter median survival of 2.8 years (range, 0.77.0 years ; p =.03 ; fig 4). in cardiac mortality analyses, 18 dogs were censored from the 130 mmhg group, and 5 were censored from the > 130 mmhg group. the median survival time for cardiacrelated deaths was 11.3 years (range, 1.011.8 years) in the 133 mmhg (p =.01). cox adjusted survival curve of allcause mortality comparing dogs with severe subaortic stenosis with a pg at diagnosis > 130 mmhg (n = 20) and those 130 mmhg (n = 30). the division at 130 mmhg was chosen based on the increased hazard occurring at approximately this pressure based on examination of figure 1. a significant difference in survival between groups at time of analyses, 38 dogs (24 in the treatment group and 14 in the untreated group) had died. ten dogs in the treatment group and 8 dogs in the untreated group were classified as having sudden death. six dogs in the treatment group died or were euthanized because of heart failure, as were 2 in the untreated group. ten dogs died of noncardiaccauses (neoplasia, n = 7 ; and 1 each orthopedic, gastrointestinal, and house fire), and in 2 dogs the cause of death was undetermined. there was no difference in the proportion of sudden death (p =.79) or death because of heart failure (p = 1.00) between groups. the influence of pg at diagnosis, age at diagnosis, and the use of beta blockers on survival was determined by using cox proportional hazards regression analysis. in allcause multivariate mortality analysis, only age at diagnosis (p =.016) and pg at diagnosis (p =.025) influenced survival. the adjusted hazard ratio (ahr) results indicated that as pg at diagnosis increased, there was an associated decrease in survival time (ahr, 1.009 ; confidence limits [cl ], 1.0011.017 ; fig 1). as age at diagnosis increased, there was an associated increase in survival time (ahr, 0.73 ; cl, 0.560.94). two dogs were censored from cardiac mortality analysis because of inability to determine cause of death. in the cardiac mortality analysis, only pg significantly influenced survival time (p =.03 ; ahr, 1.009 ; cl, 1.0011.018). proportional hazards analyses demonstrating an increased risk of mortality as pressure gradient at diagnosis increases. pressure gradient (pg) was analyzed as a continuous variable with 4 degrees of freedom for the spline curve. small vertical lines above the xaxis represents the pgs of individual dogs (n = 50). treatment with a beta blocker did not influence survival time in the allcause (p =.93 ; ahr, 1.034 ; cl, 0.4732.257 ; fig 2) or the cardiaccause mortality analyses (p =.97 ; ahr, 0984 ; cl, 0.4152.333 ; fig 3). median survival time for allcause mortality was 5.9 years (range, 0.711.8 years) in the treated group and 5.1 years (range, 0.810.9 years) in the untreated group. median survival time for cardiacrelated mortality was 6.2 years (range, 0.711.5 years) in the treated group and 6.7 years (range, 0.87.0 years) in the untreated group. cox adjusted survival curve of allcause mortality in dogs with severe subaortic stenosis receiving beta blocker treatment (n = 27) versus untreated (n = 23). there was no significant difference in survival between groups (p =.93). cox adjusted survival curve of cardiacrelated mortality dogs with severe subaortic stenosis receiving beta blocker treatment (n = 27) versus untreated (n = 23). there was no significant difference in survival between groups (p =.97). adjusted survival curves were generated to evaluate the effect of pg at diagnosis on survival in the presence of other covariates in the model. examination of the ahr plot (fig 1) indicated the presence of increased hazard at approximately 130 mmhg, thus this was chosen as the division point for analysis. there were 30 dogs with a pg at diagnosis of 130 mmhg and 20 dogs with a pg > 130 mmhg. in the allcause mortality analyses, 12 dogs that were still alive were censored (130 mmhg, n = 11 ; > 130 mmhg, n = 1). dogs with a pg at diagnosis of 130 mmhg had a median survival of 8.3 years (range, 1.011.8 years), whereas dogs with a pg at diagnosis of > 130 mmhg had a significantly shorter median survival of 2.8 years (range, 0.77.0 years ; p =.03 ; fig 4). in cardiac mortality analyses, 18 dogs were censored from the 130 mmhg group, and 5 were censored from the > 130 mmhg group. the median survival time for cardiacrelated deaths was 11.3 years (range, 1.011.8 years) in the 133 mmhg (p =.01). cox adjusted survival curve of allcause mortality comparing dogs with severe subaortic stenosis with a pg at diagnosis > 130 mmhg (n = 20) and those 130 mmhg (n = 30). the division at 130 mmhg was chosen based on the increased hazard occurring at approximately this pressure based on examination of figure 1. a significant difference in survival between groups to the authors ' knowledge, this is the first study comparing beta blocker treatment in dogs with severe sas against an untreated group. only a high pg at diagnosis was associated with a decrease in survival time in both allcause and cardiac mortality analyses. we speculate that early death of dogs with more severe disease may have influenced this result. however, it is also possible that the difference in age between groups was because of chance resulting from a few dogs in the untreated group that were initially diagnosed with sas at an older age (> 8 years, n = 3) versus no dog being > 4 years of age at diagnosis in the treated group. importantly, the results of this study suggest that a greater percentage of dogs with uncomplicated, untreated severe sas appear to live longer than previously reported. the mortality analysis strongly suggests that within the severe classification of sas, pg at diagnosis may be a useful tool in predicting survival time. the data presented in the log hazard graph (fig 1) showed that an increase in pg in our population was associated with a progressive increase in risk. although the risk leveled out and decreased starting at approximately 180 mmhg, we believe this was secondary to the relatively small number of dogs at the highest pgs, rather than a true decrease in risk. only 4 dogs had pg > 200 mmhg in this study. a pg > 80 mmhg generally has been accepted as severe3, 5 ; however, others have noted up to 100 mmhg before clinically relevant complications are expected.6, 15 redefining mild, moderate, and severe sas would require additional data and was beyond the scope of this study, but does warrant consideration. using the median pg of all dogs in our study, we showed that dogs with a pg at diagnosis 133 mmhg (8.3 versus 2.8 years). the median survival time for allcause mortality in our untreated group was 61.2 months (5.1 years). our data suggest that prognosis with severe untreated sas is better than previously reported,3 but a normal life span in affected dogs is still unlikely. nonetheless, comparing results among studies can be problematic. consideration must be given to the different methods utilized to diagnose sas (cardiac catheterization versus transthoracic doppler echocardiography). however, only a small number of dogs in the previous studies were diagnosed by cardiac catheterization, and catheterization has been shown to have excellent correlation with dopplerderived pgs in sas.16, 17 in addition, the median age at diagnosis in our untreated group was considerably higher than that of the population reported by kienle (1.2 versus 0.5 years, respectively). the association between age at diagnosis and survival time identified in our study may account, at least in part, for the difference in survival times among the studies. in addition, study size and other unknown characteristics of each study population are potential confounders. our study population yielded a total of 50 cases with the majority being large breed dogs. breeds of dogs in which sas has been commonly reported include the newfoundland, golden retriever, german shepherd dog, boxer, rottweiler, and dogue de bordeaux.2, 3, 15, 18, 19 in the golden retriever and newfoundland, strong evidence for a genetic basis of sas has been found, but the exact mode of inheritance does not appear to fit a simple model.15, 18, 19 similar to previous studies, our study population did not show a sex predilection for sas.1, 2 the high prevalence of irish setters (n = 3) within our study population warrants mention because sas has not previously been reported in this breed. these cases were all from a rescue organization near one of the study institutions (missouri). additional investigations would be necessary to determine if irish setters are predisposed to sas in general, or if our results potentially represent a regional founder effect. although the characteristics and progression of the disease are strikingly similar in both humans and dogs,20 a parallel in human medicine to medical management of canine sas is lacking. surgical intervention with removal of the fixed obstruction (fibromyectomy) is the treatment of choice in humans with congenital sas. the timing of the surgery remains a controversial topic because multiple surgeries may be required because of recurrence of stenosis.20 for valvular aortic stenosis in humans, surgical replacement of the valve is the treatment of choice. no medical treatment has been shown to prolong survival in humans once clinical signs of disease begin21, 22 and beta blocker use is not recommended because of the risk of decreased myocardial function and induction of lv failure.23 although no increase in mortality was seen in this study with beta blockade, whether there is a subgroup of dogs at increased risk remains to be shown. veterinary surgical or interventional options have not yielded encouraging results and these studies have intentionally excluded a placebocontrol group because of the reported short survival time in untreated sas.11 surgical resection of the discrete subvalvular ridge successfully decreased the pg across the lvot by 4565% but failed to show a survival benefit versus treatment with atenolol.12 similarly, another study evaluating balloon valvuloplasty for treatment of sas demonstrated a significant reduction in the pg but failed to show a survival benefit versus atenolol alone.11 however, similar to our untreated group, survival times for both studies (70 and 55 months, respectively) were longer than previously reported survival times for untreated severe sas (19 months).3 regardless of treatment, sudden death remains a common outcome in cases of severe sas3, 11, 12 and accounted for 45% of all mortality in this study. the mechanism of sudden death in patients with sas remains unclear, but abnormalities in myocardial perfusion with resulting ischemia are a potential cause of arrhythmia development. alterations to the intramural coronary arteries of the left ventricle in dogs with sas include fibrous intimal proliferation and replacement of medial smooth muscle resulting in a reduction in the lumen of the arteries.24 reversal of coronary artery flow during ventricular systole also has been observed in studies of naturally occurring sas.25 experimental models of canine sas by using aortic banding have demonstrated an increase in myocardial oxygen consumption during exercise suggesting increased vulnerability to ischemic injury compared to normal dogs.26 imbalance of myocardial oxygen supply and demand during exercise results in a functional impairment of the subendocardium. pretreatment with propranolol before exercise appeared to diminish this effect,8 which suggests that beta blocker treatment may help prevent myocardial ischemia, myocardial dysfunction, or both at least during exercise. however, in our study there were no differences in the proportions of sudden death or heart failure between groups. given the retrospective nature of the study, the groups were not randomized and the decision to use beta blocker treatment was solely at the discretion of the clinician managing each individual case. within the study population, animals evaluated at the university of missouri were more likely to be treated with a beta blocker than those evaluated at the university of minnesota. importantly, however, there was no difference in median pg at diagnosis between the 2 institutions or the 2 treatment groups. in addition, when medical records were incomplete, information gained from direct communication with owners and referring veterinarians was subject to recall bias. the limited followup information for some cases made cause of death difficult to determine in those instances. in addition, several dogs within the study were diagnosed with severe sas before reaching maturity and did not have any further echocardiographic evaluation. it is uncertain what influence this may have had on the subjects ' longterm survival. it has been previously shown that sas can increase in severity over time.4, 20 however, progression does not occur in all dogs, nor is there a predictable increase in severity when it does occur, and in fact, some dogs actually will have a decrease in severity over time.27 our wide dosage range, for atenolol also should be considered, and has been previously considered a limitation.11 however, the median dosage used in this study is consistent with what is commonly recommended clinically.7 furthermore, friedman demonstrated that a dosage of 0.7 mg / kg of atenolol in healthy dogs produced a significant decrease in heart rate and cardiac output during exercise compared to control. adequacy of beta blockade also was tested by injection of a standard dosage of isoproterenol, which failed to cause an increase in heart rate. despite these limitations, our results suggest that beta blocker treatment, specifically atenolol at the median dosage used in this study, does not influence survival in dogs with severe sas. whether this was because of a lack of efficacy of the drug, a limitation of insufficient dosing, or failure to select appropriate patients is unknown. a prospective, randomized placebocontrolled clinical trial is necessary to confirm or refute these results. importantly though, the results of this study suggest that dogs with uncomplicated, untreated severe sas (> 80 mmhg) appear to live longer than previously reported. however, a very high pg at diagnosis still confers a poor prognosis for longterm survival. | backgroundsubaortic stenosis (sas) is one of the most common congenital cardiac defects in dogs. severe sas frequently is treated with a beta adrenergic receptor blocker (beta blocker), but this approach largely is empirical.objectiveto determine the influence of beta blocker treatment on survival time in dogs with severe sas.methodsretrospective review of medical records of dogs diagnosed with severe, uncomplicated sas (pressure gradient [pg ] 80 mmhg) between 1999 and 2011.resultsfifty dogs met the inclusion criteria. twentyseven dogs were treated with a beta blocker and 23 received no treatment. median age at diagnosis was significantly greater in the untreated group (1.2 versus 0.6 years, respectively ; p =.03). median pg at diagnosis did not differ between the treated and untreated groups (127 versus 121 mmhg, respectively ; p =.2). cox proportional hazards regression was used to identify the influence of pg at diagnosis, age at diagnosis, and beta blocker treatment on survival. in the allcause multivariate mortality analysis, only age at diagnosis (p =.02) and pg at diagnosis (p =.03) affected survival time. in the cardiac mortality analysis, only pg influenced survival time (p =.03). treatment with a beta blocker did not influence survival time in either the allcause (p =.93) or cardiaccause (p =.97) mortality analyses.conclusionsbeta blocker treatment did not influence survival in dogs with severe sas in our study, and a higher pg at diagnosis was associated with increased risk of death. |
the cannabinoid receptors, endocannabinoids and the proteins for their biosynthesis and degradation constitute the key components of the endocannabinoid system (di marzo., 1998). cb1 receptors and endocannabinoids are highly expressed in the basal ganglia and have close connections with the dopaminergic system, being involved in the central regulation of motor functions. to date, two cannabinoid receptor subtypes have been identified by molecular cloning, cannabinoid receptor type 1 (cb1) (matsuda., 1990) and cannabinoid receptor type 2 (cb2) (munro., 1993). the vast majority of cb1 receptors are located in the central nervous system while cb2 receptors are expressed primarily in cells of the immune system (munro., 1993), microglia, blood vessels and some neurons (van sickle., 2005 ; gong., 2006). both cb1 and cb2 are seven transmembrane gi / o - coupled receptors that activate similar intracellular signaling pathways (mackie, 2008). the discovery of the cannabinoid receptors led to the identification of the so - called natural ligands of the cannabinoid receptors, anandamide (devane., 1992) and 2-arachidonoylglycerol (2-ag) (devane., 1992 ; mechoulam., 1995) which are synthesized on demand in response to elevations of intracellular calcium (di marzo., 1994). although the expression of cb2 receptors has remained controversial (atwood and mackie, 2010), mrna for this receptor has been found in neurons and glial cells from the substantia nigra pars reticulata (snpr) and the striatum (gong., 2006). as for cb1 receptor, high levels are expressed within the basal ganglia (herkenham., 1990 ; mailleux and vanderhaeghen, 1992 ; tsou., 1998 ; mackie, 2005 ; martin., 2008). while mrna for this receptor is found in striatal gabaergic medium spiny neurons (mailleux and vanderhaeghen, 1992) and in the subthalamic nucleus (stn), the expression of the receptor protein is mainly located at the terminal level. thus, cb1 receptors have been observed in subthalamonigral and subthalamopallidal terminals (mailleux and vanderhaeghen, 1992 ; tsou., 1998), glutamatergic corticostriatal afferences (gerdeman and lovinger, 2001 ; kofalvi., 2005), and striatal projections to the globus pallidus (gpi and gpe) and to the snpr (herkenham. distribution of cb1 and trpv1 receptors and their coexpression with dopaminergic d1 and d2 receptors in a simplified diagram of the basal ganglia circuits. cb1, cannabinoid receptor type 1 ; trpv1, transient receptor potential vanilloid type 1 ; d1, dopaminergic receptor type 1 ; d2, dopaminergic receptor type 2 ; gpe, external globus pallidus ; gpi, internal globus pallidus ; stn, subthalamic nucleus ; snpc, substantia nigra pars compacta ; snpr, substantia nigra pars reticulata. neurons expressing d1 receptors form the direct pathway, which projects to the gpi and the snpr, while neurons expressing d2 receptors constitute the indirect pathway, projecting to the gpe (figure 1) (paul., 1992 ; oconnor, 1998 ; nicola., 2000 ; a potential interaction between the d1/d2 and cb1 receptors at the level on the g - protein / adenylyl cyclase signal transduction mechanism has been suggested (giuffrida., 1999 ; combined activation of cb1 and d1 receptors results in a net decrease in adenylyl cyclase, a subsequent decrease in the inhibitory activity of direct striatal projection neurons and finally a decreased motor response due to enhanced activation of snpr neurons. conversely, activation of cb1 and d2 receptors together stimulates adenylyl cyclase (glass and felder, 1997), potentiating the indirect striatal pathway neurons that in turn activate neurons of the stn, also resulting in motor inhibition (brotchie, 2003 ; van der stelt and di marzo, 2003). these data indicate that endocannabinoid system acting on striatal cb1 receptors play a significant role in the regulation of basal ganglia motor circuits. although cb1/d2 receptor heterodimerization has been demonstrated in transfected cells by co - immunoprecipitation and forster resonance energy transfer (fret) techniques (kearn., 2005 ; marcellino., 2008), functionality of those heteromers in striatal glutamatergic terminals is not supported by recent studies (kreitzer and malenka, 2007). in the last years, the transient receptor potential vanilloid type 1 (trpv1) has gained attention for its ability to bind cannabinoids. although neuronal expression and functionality of trpv1 channels are controversial (mezey., 2000 ;, 2006 ; cavanaugh., 2011), this receptor is present in the basal ganglia. indeed, trpv1 is located on nigrostriatal terminals and on tyrosine hydroxylase positive cells in the substantia nigra pars compacta (snpc) (mezey., 2000 ; 2009) which makes it a good candidate for directly modulating dopaminergic neurotransmission (figure 1). on the other hand, the orphan g - protein - coupled receptor 55 (gpr55) has been identified as another possible cannabinoid receptor (ryberg., 2007) that, in contrast to classical cb1 and cb2, is coupled to gq, g12 and g13 proteins (sharir and abood, 2010). despite its high expression in the striatum (sawzdargo., 1999), conflicting pharmacological findings make difficult to consider the gpr55 as a novel cannabinoid receptor (oka. future investigations will clarify the role of trpv1 and gpr55 in modulating basal ganglia circuits. in accordance with its neuroanatomical distribution, functional and behavioral studies have suggested that the endocannabinoid system can act as an indirect modulator of dopaminergic neurotransmission in the basal ganglia. several influences of cannabinoids on motor activity depend on the cannabinoids influence on the dopaminergic system. systemic administration of synthetic and endogenous cannabinoids (-thc, win 55,212 - 2, cp 55,940, or anandamide) characteristically induces inhibition of motor behavior and catalepsy in rodents (prescott., 1992 ; crawley., 1993 ;, 1995 ; romero., 1995 ; de lago., 2004 moreover, the anandamide transport inhibitor, am404, or inhibitors of anandamide hydrolysis produce hypokinesia in rats (compton and martin, 1997 ; gonzalez., 1999). these hypokinetic effects can be reversed by the selective cb1 receptor antagonist, rimonabant, which in itself causes hyperlocomotion in healthy controls (compton., 1996). in agreement with these observations, mice lacking cb1 receptors exhibit several motor anomalies (ledent. although these findings provide evidence for the involvement of cb1-related mechanisms in motor control, other reports demonstrate that also the trpv1 receptors can mediate effects of certain cannabinoids such as anandamide (de lago., 2004). it has been hypothesized that the inhibition of motor behavior mediated by cannabinoids could be related to a reduction in dopaminergic circuitry activity. rotational studies in rats receiving local injections of cannabinoid compounds into the basal ganglia suggest that dopamine - cannabinoid interaction is not a direct mechanism. for instance, cannabinoids increase or decrease motor behavior when locally administered into the direct (sanudo - pena., 1996, 1998) or indirect pathway, respectively (sanudo - pena and walker, 1997 ; miller., 1998). neuroanatomical studies showing that cb1 receptors are not present on dopaminergic neurons or terminals (julian., 2003 ; 2006) suggest that cb1-mediated modifications of nigrostriatal dopaminergic circuits are exerted indirectly by modulation of inhibitory or excitatory inputs to the midbrain dopamine neurons. indeed, cannabinoids are known to dampen both glutamate and gaba transmission in the basal ganglia (szabo., 2000 ; gerdeman and lovinger, 2001 ; wallmichrath and szabo, 2002). in vivo electrophysiological studies have shown that cannabinoid agonists increase the action potential firing rate of snpc neurons (french., 1997 ; melis., 2000 ; since cb1 receptors are poorly expressed in snpc neurons (julian., 2003 ; matyas., 2006), or even absent, the action of cannabinoids is indirectly exerted on dopaminergic neurons through other nuclei. in line with this, in the snpr the cb1 receptors are located on subthalamonigral terminals and their activation inhibits glutamate release (szabo., 2000) resulting in a reduction of gabaergic transmission and, consequently, in an increased activity of snpc cells (morera - herreras., 2008). the increased activity of snpc neurons observed after cb1 receptor activation is in agreement with in vivo microdialysis experiments showing enhanced dopamine release in the striatum after exogenous or endogenous cannabinoid agonists administration (tanda., 1997 ; solinas., 2006). however, this effect is not mediated locally at the terminal level, but rather involves changes in the firing activity of snpc neurons, since in vitro studies in striatal slices have shown that cb1 activation has no effect on dopamine release (kofalvi., 2005). contrary to cb1-mediated mechanisms, the effects of endocannabinoids on dopamine transmission may be mediated by direct mechanisms. indeed, the endocannabinoid anandamide and some analogs (but not classic cannabinoid as -thc), acting via postsynaptic trpv1 receptors, may reduce nigrostriatal dopaminergic cell activity (de lago., however, other authors have reported an increase of dopamine release after activation of trpv1 receptors in the snpc (marinelli., 2003, 2007), although this enhancement may be mediated by trpv1 receptors located in glutamatergic terminals in the snpc rather than by receptors located in dopaminergic terminals. several influences of cannabinoids on motor activity depend on the cannabinoids influence on the dopaminergic system. systemic administration of synthetic and endogenous cannabinoids (-thc, win 55,212 - 2, cp 55,940, or anandamide) characteristically induces inhibition of motor behavior and catalepsy in rodents (prescott., 1992 ; crawley., 1993, 1995 ; romero., 1995 ; de lago., moreover, the anandamide transport inhibitor, am404, or inhibitors of anandamide hydrolysis produce hypokinesia in rats (compton and martin, 1997 ; gonzalez., 1999). these hypokinetic effects can be reversed by the selective cb1 receptor antagonist, rimonabant, which in itself causes hyperlocomotion in healthy controls (compton., 1996). in agreement with these observations, mice lacking cb1 receptors exhibit several motor anomalies (ledent. although these findings provide evidence for the involvement of cb1-related mechanisms in motor control, other reports demonstrate that also the trpv1 receptors can mediate effects of certain cannabinoids such as anandamide (de lago., 2004). it has been hypothesized that the inhibition of motor behavior mediated by cannabinoids could be related to a reduction in dopaminergic circuitry activity. rotational studies in rats receiving local injections of cannabinoid compounds into the basal ganglia suggest that dopamine - cannabinoid interaction is not a direct mechanism. for instance, cannabinoids increase or decrease motor behavior when locally administered into the direct (sanudo - pena., 1996, 1998) or indirect pathway, respectively (sanudo - pena and walker, 1997 ; miller., 1998). neuroanatomical studies showing that cb1 receptors are not present on dopaminergic neurons or terminals (julian., 2003 ; matyas., 2006) suggest that cb1-mediated modifications of nigrostriatal dopaminergic circuits are exerted indirectly by modulation of inhibitory or excitatory inputs to the midbrain dopamine neurons. indeed, cannabinoids are known to dampen both glutamate and gaba transmission in the basal ganglia (szabo., 2000 ; gerdeman and lovinger, 2001 ; wallmichrath and szabo, 2002). in vivo electrophysiological studies have shown that cannabinoid agonists increase the action potential firing rate of snpc neurons (french., 1997 ; melis., 2000 ; since cb1 receptors are poorly expressed in snpc neurons (julian., 2003 ; matyas., 2006), or even absent, the action of cannabinoids is indirectly exerted on dopaminergic neurons through other nuclei. in line with this, in the snpr the cb1 receptors are located on subthalamonigral terminals and their activation inhibits glutamate release (szabo., 2000) resulting in a reduction of gabaergic transmission and, consequently, in an increased activity of snpc cells (morera - herreras., 2008). the increased activity of snpc neurons observed after cb1 receptor activation is in agreement with in vivo microdialysis experiments showing enhanced dopamine release in the striatum after exogenous or endogenous cannabinoid agonists administration (tanda. however, this effect is not mediated locally at the terminal level, but rather involves changes in the firing activity of snpc neurons, since in vitro studies in striatal slices have shown that cb1 activation has no effect on dopamine release (kofalvi., 2005). contrary to cb1-mediated mechanisms, the effects of endocannabinoids on dopamine transmission may be mediated by direct mechanisms. indeed, the endocannabinoid anandamide and some analogs (but not classic cannabinoid as -thc), acting via postsynaptic trpv1 receptors, may reduce nigrostriatal dopaminergic cell activity (de lago., 2004). however, other authors have reported an increase of dopamine release after activation of trpv1 receptors in the snpc (marinelli., 2003, 2007), although this enhancement may be mediated by trpv1 receptors located in glutamatergic terminals in the snpc rather than by receptors located in dopaminergic terminals. as described above, neuroanatomical studies have located cannabinoid receptors in the basal ganglia, and it is widely accepted that the endocannabinoid system influence physiological motor function. these facts predict that pharmacological modulation of the endocannabinoid system may also be beneficial under pathological conditions pertaining to decreased dopamine signaling or the chronic treatment with l - dopa. in parkinson s disease (pd), the progression of the neurodegenerative pathology and the appearance of major motor symptoms are related to increased endocannabinoid levels (pisani., several studies have also found increased cb1 receptor levels in the striatum of parkinsonian monkeys and human patients (lastres - becker., in rat models of pd, publications supporting increased (gubellini., 2002 ; maccarrone., 2003 ; gonzalez.,, 2000 ; kreitzer and malenka, 2007) of endocannabinoid tone have been reported. the heterogeneous results obtained in animal models may depend on methodological differences such as the way of inducing parkinsonism or more importantly on the period of recovery after the lesion before performing experiments (romero., 2000). studies in animal models and patients of pd have indicated that dopaminergic neuronal degeneration produces an imbalance between the direct and the indirect basal ganglia pathways. this imbalance is manifested as reduced activity of striatal gabaergic neurons in the direct pathway and hyperactivity in the indirect pathway striatal neurons. moreover, glutamatergic input from the cortex to the striatum is augmented after dopaminergic denervation (tang., 2001 ; tseng., 2001 ; gubellini., 2002 ; mallet., 2006) cb1 receptors are principally expressed on presynaptic cortical glutamatergic terminals and presynaptic striatal gabaergic terminals (benarroch, 2007). the activation of cb1 receptors reduces the glutamate release from the cortex to the striatum (gerdeman and lovinger, 2001 ; gubellini., 2002 ; brown., 2003) and gaba release to the snpr (wallmichrath and szabo, 2002). in addition, endocannabinoids and cb1 receptors play an important physiological role in the long- and short - term regulation of the synaptic transmission in the basal ganglia shaping the striatal output and therefore modulating motor activities. the two classic forms of long - term synaptic plasticity, long - term potentiation and long - term depression (ltd) are expressed at corticostriatal synapses and abolished in animal models of pd (centonze., 1999 ;, 2005 ; calabresi., 2007 ; kreitzer and malenka, 2007). using different ltd induction paradigms it has been described that this form of plasticity is mostly controlled by endocannabinoids (shen., 2008 ; although probably other mechanisms are also involved in the dopaminergic control of striatal plasticity, pharmacological manipulation of the endocannabinoid system under parkinsonian conditions has been proved not only to rescue ltd in striatum but also to improve the motor deficits evident after dopaminergic denervation (kreitzer and malenka, 2007). in line with this, behavioral studies have shown that modulation of the endocannabinoid system can have a therapeutic impact in animal models of pd. behavioral changes caused by the induction of parkinsonism in rats have been improved by the administration of cb1 receptor antagonists both in unilateral and bilateral pd models in rodents (fernandez - espejo., 2005 ; gonzalez. in mptp - lesioned marmosets, cb1 antagonist administration increased locomotor activity but failed to improve bradykinesia or posture (van der stelt., 2005). on the other hand, co - administration of l - dopa with cb1 antagonists added a positive improvement of the motor symptoms assigned to the antiparkinsonian drug in parkinsonian animals (kelsey., 2009). the latter data suggest that combined therapy with antiparkinsonian drugs and cannabinoid antagonists may permit a reduction of l - dopa dose and therefore, delay the emergence of the motor side effects induced by the chronic treatment with l - dopa., several reports have revealed interesting neuroprotective and anti - inflammatory effects of these drugs in cell cultures and animal models of pd (lastres - becker., 2005 ; garcia - arencibia., 2007 ; fernandez - ruiz., 2011 ; although cb1 receptor - mediated effects can not be excluded, some authors argue that cb1 receptors may have a minimal implication in neuroprotection (lastres - becker., 2005 ; fernandez - ruiz., 2007 ; price., 2009 ; chung., it seems plausible that neuroprotection is principally mediated by the antioxidant effect of cannabinoids and cb1 receptor - independent properties (lastres - becker. 2012), while in parallel, activation of cb2 receptors in astrocytes and microglial cells is responsible for the observed anti - inflammatory effect. although the exact mechanisms should be further investigated, cannabinoid receptor modulation can be potentially useful for protecting dopaminergic neurons from progressive neurodegeneration in pd. the emerging role of the endocannabinoid system as modulator of neurotransmission in the basal ganglia identifies it as a potential pharmacological target for treating motor complications derived from the chronic treatment with l - dopa. l - dopa induced dyskinesia (lid) constitute one of the most disabling complications derived from the long - term therapy with l - dopa affecting up to 40% of pd patients after 5 years of treatment (ahlskog and muenter, 2001). cannabinoid agonists could exert antidyskinetic effect by regulating glutamatergic release in the striatum and/or by re - establishing endocannabinoid - mediated synaptic plasticity affected by dopaminergic denervation. in this sense, pharmacological agents with antidyskinetic properties such as serotonergic 5-ht1b agonists are able to ameliorate the motor complications by depressing the glutamatergic corticostriatal transmission (mathur., 2011). cannabinoid agents have been proposed as promising tools for treating lid, however, different studies in animal models and patients show certain discrepancies. administration of cannabinoid agonists to parkinsonian rats receiving chronic l - dopa treatment attenuated lid via cb1-related mechanisms (ferrer. 2012) and genetic deletion of cb1 receptors prevented the development of severe dyskinetic movements in mice (perez - rial. although the cited studies do not report any reduction of the efficacy of l - dopa to improve the motor performance, a recent publication suggests that the antidyskinetic effect of cannabinoid agonists seem to be based on their general motor suppressant (walsh., 2010a). in mptp - lesioned monkeys and pd patients contradictory results have been found since cb1 receptor activation (silverdale., 2001 ; fox., 2002) or blockage (van der stelt., 2005) other studies in patients have failed to find any correlation between cb1 receptor expression and severity of dyskinesia (van der stelt., 2005) or attribute any positive effect of cannabinoid agent administration in lid (carroll., 2004 ; mesnage., changes in the cannabinoid system are observed after dopaminergic denervation and manipulation of this system has proved to have beneficial effects on parkinsonian symptoms in animal models and pd patients. however, the putative role of cannabinoids in lid is still a matter of controversy. the complex localization of the cannabinoid receptors at different sites in the basal ganglia circuits may contribute to the paradoxical observed effects. further investigations are needed to clarify the role of the cannabinoid system in lid. in conclusion, this system has an important role in dopamine - related movement disorders, as pd, and represents a framework for novel therapeutic approaches in the future. in parkinson s disease (pd), the progression of the neurodegenerative pathology and the appearance of major motor symptoms are related to increased endocannabinoid levels (pisani., 2005, 2010). several studies have also found increased cb1 receptor levels in the striatum of parkinsonian monkeys and human patients (lastres - becker., in rat models of pd, publications supporting increased (gubellini., 2002 ; maccarrone., 2003 ; gonzalez., 2010b), or no modification (romero., 2000 ; kreitzer and malenka, 2007) of endocannabinoid tone have been reported. the heterogeneous results obtained in animal models may depend on methodological differences such as the way of inducing parkinsonism or more importantly on the period of recovery after the lesion before performing experiments (romero., 2000). studies in animal models and patients of pd have indicated that dopaminergic neuronal degeneration produces an imbalance between the direct and the indirect basal ganglia pathways. this imbalance is manifested as reduced activity of striatal gabaergic neurons in the direct pathway and hyperactivity in the indirect pathway striatal neurons. moreover, glutamatergic input from the cortex to the striatum is augmented after dopaminergic denervation (tang., 2001 ; gubellini., 2002 ; mallet., 2006). within the basal ganglia, cb1 receptors are principally expressed on presynaptic cortical glutamatergic terminals and presynaptic striatal gabaergic terminals (benarroch, 2007). the activation of cb1 receptors reduces the glutamate release from the cortex to the striatum (gerdeman and lovinger, 2001 ; gubellini., 2002 ; brown., 2003) and gaba release to the snpr (wallmichrath and szabo, 2002). in addition, endocannabinoids and cb1 receptors play an important physiological role in the long- and short - term regulation of the synaptic transmission in the basal ganglia shaping the striatal output and therefore modulating motor activities. the two classic forms of long - term synaptic plasticity, long - term potentiation and long - term depression (ltd) are expressed at corticostriatal synapses and abolished in animal models of pd (centonze., 1999 ;, 2005 ; calabresi., 2007 ; kreitzer and malenka, 2007). using different ltd induction paradigms it has been described that this form of plasticity is mostly controlled by endocannabinoids (shen., 2008 ; although probably other mechanisms are also involved in the dopaminergic control of striatal plasticity, pharmacological manipulation of the endocannabinoid system under parkinsonian conditions has been proved not only to rescue ltd in striatum but also to improve the motor deficits evident after dopaminergic denervation (kreitzer and malenka, 2007). in line with this, behavioral studies have shown that modulation of the endocannabinoid system can have a therapeutic impact in animal models of pd. behavioral changes caused by the induction of parkinsonism in rats have been improved by the administration of cb1 receptor antagonists both in unilateral and bilateral pd models in rodents (fernandez - espejo., 2005 ; gonzalez., 2006 ; kelsey., 2009). in mptp - lesioned marmosets, cb1 antagonist administration increased locomotor activity but failed to improve bradykinesia or posture (van der stelt., 2005). on the other hand, co - administration of l - dopa with cb1 antagonists added a positive improvement of the motor symptoms assigned to the antiparkinsonian drug in parkinsonian animals (kelsey., 2009). the latter data suggest that combined therapy with antiparkinsonian drugs and cannabinoid antagonists may permit a reduction of l - dopa dose and therefore, delay the emergence of the motor side effects induced by the chronic treatment with l - dopa., several reports have revealed interesting neuroprotective and anti - inflammatory effects of these drugs in cell cultures and animal models of pd (lastres - becker., 2005 ;, 2007 ; fernandez - ruiz., 2011 ; jeon. although cb1 receptor - mediated effects can not be excluded, some authors argue that cb1 receptors may have a minimal implication in neuroprotection (lastres - becker., 2005 ; it seems plausible that neuroprotection is principally mediated by the antioxidant effect of cannabinoids and cb1 receptor - independent properties (lastres - becker., 2012), while in parallel, activation of cb2 receptors in astrocytes and microglial cells is responsible for the observed anti - inflammatory effect. although the exact mechanisms should be further investigated, cannabinoid receptor modulation can be potentially useful for protecting dopaminergic neurons from progressive neurodegeneration in pd. the emerging role of the endocannabinoid system as modulator of neurotransmission in the basal ganglia identifies it as a potential pharmacological target for treating motor complications derived from the chronic treatment with l - dopa. l - dopa induced dyskinesia (lid) constitute one of the most disabling complications derived from the long - term therapy with l - dopa affecting up to 40% of pd patients after 5 years of treatment (ahlskog and muenter, 2001). cannabinoid agonists could exert antidyskinetic effect by regulating glutamatergic release in the striatum and/or by re - establishing endocannabinoid - mediated synaptic plasticity affected by dopaminergic denervation. in this sense, pharmacological agents with antidyskinetic properties such as serotonergic 5-ht1b agonists are able to ameliorate the motor complications by depressing the glutamatergic corticostriatal transmission (mathur., 2011). cannabinoid agents have been proposed as promising tools for treating lid, however, different studies in animal models and patients show certain discrepancies. administration of cannabinoid agonists to parkinsonian rats receiving chronic l - dopa treatment attenuated lid via cb1-related mechanisms (ferrer. 2012) and genetic deletion of cb1 receptors prevented the development of severe dyskinetic movements in mice (perez - rial. although the cited studies do not report any reduction of the efficacy of l - dopa to improve the motor performance, a recent publication suggests that the antidyskinetic effect of cannabinoid agonists seem to be based on their general motor suppressant (walsh., 2010a). in mptp - lesioned monkeys and pd patients contradictory results fox., 2002) or blockage (van der stelt., 2005) ameliorated lid. other studies in patients have failed to find any correlation between cb1 receptor expression and severity of dyskinesia (van der stelt., 2005) or attribute any positive effect of cannabinoid agent administration in lid (carroll., 2004 ; taken together, changes in the cannabinoid system are observed after dopaminergic denervation and manipulation of this system has proved to have beneficial effects on parkinsonian symptoms in animal models and pd patients. however, the putative role of cannabinoids in lid is still a matter of controversy. the complex localization of the cannabinoid receptors at different sites in the basal ganglia circuits may contribute to the paradoxical observed effects. further investigations are needed to clarify the role of the cannabinoid system in lid. in conclusion, this system has an important role in dopamine - related movement disorders, as pd, and represents a framework for novel therapeutic approaches in the future. 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. | there is substantial evidence supporting a role for the endocannabinoid system as a modulator of the dopaminergic activity in the basal ganglia, a forebrain system that integrates cortical information to coordinate motor activity regulating signals. in fact, the administration of plant - derived, synthetic or endogenous cannabinoids produces several effects on motor function. these effects are mediated primarily through the cb1 receptors that are densely located in the dopamine - enriched basal ganglia networks, suggesting that the motor effects of endocannabinoids are due, at least in part, to modulation of dopaminergic transmission. on the other hand, there are profound changes in cb1 receptor cannabinoid signaling in the basal ganglia circuits after dopamine depletion (as happens in parkinson s disease) and following l - dopa replacement therapy. therefore, it has been suggested that endocannabinoid system modulation may constitute an important component in new therapeutic approaches to the treatment of motor disturbances. in this article we will review studies supporting the endocannabinoid modulation of dopaminergic motor circuits. |
lipoma arborescens (la) is a rare, benign intra - articular lesion most commonly found in the knee, characterised by villous proliferation of the synovium. it generally presents as a longstanding, slowly progressive swelling of one or more joints associated which may or may not be associated with pain. mri is the investigation of choice, with images clearest on fat - supressed or stir sequences. we present a 35 year old male patient, who presented with a three year history of bilateral knee pain and swelling. magnetic resonance imaging (mri) scans of his knee showed the characteristic features of lipoma arborescens. lipoma arborescens is a rare, benign intra - articular tumour which may mimic a number of other diagnoses. mri should be considered to exclude this pathology as well as other uncommon intra - articular pathology. lipoma arborescens (la) is a rare, benign intra - articular lesion most commonly found in the knee, characterised by villous proliferation of the synovium. it can be mono-, bi- or polyarticular and can affect patients of all ages (although it is commonest in the fifth decade and above). it often remains undiagnosed for a prolonged period as it mimics the more common arthritis, with secondary degeneration further clouding the picture clinically and radiographically. mri is the investigation of choice, with images clearest on fat - supressed or stir sequences we present a rare case of bilateral lipoma arborescens and undertake a comprehensive review of the literature available. a 35 year old male patient presented with a three year history of bilateral knee pain and swelling. the pain and swelling were spontaneous in onset and there was no documented history of an associated injury. the patient had been investigated elsewhere, where the radiographs had been reported as normal and he had physiotherapy without any benefit. on examination he lacked the last five degrees of extension and flexed to one hundred and twenty degrees. there was no tenderness on palpation and he had an effusion and thickened synovium mainly in the suprapatellar region. plain radiographs were unremarkable. magnetic resonance imaging (mri) scans of his knee showed multiple villous lipomatous synovial proliferations and a frond - like synovial proliferation of fat signal intensity (figs. 1, 2 and 3). a 99technetium bone scan was done to exclude infection, which revealed increased uptake in both knees (fig. nine months following the arthroscopies the patient had no pain or swelling in his knees. mri t2 weighted images showing multiple villous synovi proliferations in the spurapatellar areas mri t2 fat saturated image 99technetium bone scan showing increased uptake in both knees arthroscopic image showing villous appereance medline and embase were searched using the terms lipoma arborescens, villous lipomatous proliferation and arborescent lipoma. references from each paper were cross - referenced and the search was broadened using the related articles function. all abstracts were read by a single author (adl) and relevant papers were retrieved. in total, 72 papers were retrieved, which contained reports of 152 patients. the earliest reports were both in 1952, the most recent paper is from december 2009. lipoma arborescens is undoubtedly rare, but the availability of mri has led to a marked increase in the numbers of reported cases over recent years. the exact incidence is unclear, but vilanova s review of 12,578 consecutive knee mris found 32 patients with la and iovane found 9 out of 6387. this gives an incidence of between 0.14% and 0.25% of scanned knees ; the incidence within the asymptomatic population will be much lower. most cases of la have been described in single - case reports although there are a few larger series. in the 120 cases that we have identified, there was a slight male preponderance (67:53) and patients have a mean age of 45.6 years of age. the youngest case described was 9 years old (although the authors suggest that the patient underwent her first resection at the age of four) and the oldest was 90. lipoma arborescens generally presents as a longstanding, slowly progressive swelling of one or more joints associated which may or may not be associated with pain. a proportion of patients may present with symptoms of secondary degeneration such as crepitus, joint line tenderness and restriction of range of movement. depending on the anatomical site of the disease, some patients may present with exacerbations secondary to interspersion of villi within the joint space ; mechanical symptoms of locking and instability of the knee have been described by the same mechanism. the most common anatomical site by far is the knee, and specifically the pre - patellar pouch, although cases have been described in many other synovial joints including the hip, shoulder, elbow, wrist and ankle. bilateral involvement is uncommon, but when bilateral joints are involved they usually occur at the same time. in very rare cases, examination of the knee reveals a boggy, supra - patellar swelling, occasionally with a palpable mass. aspiration of the joint demonstrates clear, yellow synovial fluid devoid of crystals and cells on microscopy and sterile on culture, although the presence of a haemarthrosis does not exclude the diagnosis. typically, haematological investigations are normal with the exception of a mildly raised esr. aside from soft tissue shadows, mri is the investigation of choice, with images clearest on fat - supressed or stir sequences. these features may occur either separately or together, with a joint effusion present in all cases. other associated findings are degenerative changes with mensical tears, synovial cysts, bony erosion and chondromatosis. ultrasonography is accurate in determining the extent and location of the lesion in the various synovial surfaces of the knee and has the advantage of easy accessibility and low cost. ct is fairly nonspecific, showing a degenerative picture with synovial swelling in affected joints. arthrography was used in the diagnosis of earlier cases but this has been superseded by newer modalities. arthroscopy reveals multiple globular and villous projections of synovial - covered tissue, restricted to the affected area of the joint. microscopy of resected tissue reveals hypertrophic synovial villous proliferation with diffuse replacement of the subsynovial tissue with hyperplastic mature fat cells and an infiltrate of chronic inflammatory cells. it appears that in a subset of patients, there is an antecedent history of local joint trauma or diabetes ; four cases have been described in the context of psoriatic arthritis [25 - 27 ]. it has been postulated that morphologically distinct subtypes of la exist in patients with and without a history of preexisting inflammatory joint disease. one series of 12 patients found that previously normal joints demonstrated synovial fronds alone whilst the more typical villonodular picture were found in patients with a preceding history of joint disease. at cellular level, adipocytes, osteoblasts, chondrocytes and myofibroblasts all have a common origin, and are believed to be derived from multipotent mesenchymal stem cells. it has been suggested that an inverse relationship exists between adipocyte differentiation and the osteogenic activity of bone marrow stromal cells, and that this is reciprocally regulated by bone morphogenetic proteins. from these observations, ikushima have put forward a hypothesis that la is a rare form of a reactive lesion of the synovium in which the mesenchymal stem cells differentiate into adipocytes, whereas osteochondral differentiation of the mesenchymal stem cells results in synovial chondromatosis. one theory put forward to explain osteoarthritis in la suggests that chronic irritation of the synovium and underlying cartilage by the synovial fronds and long standing effusions leads to degenerative changes. the severity of the osteoarthritic changes in the affected knees has been suggested to correlate with the duration of the symptoms. la can mimic nearly any intra - articular pathology, and there are reports of cases being misdiagnosed as acute rheumatic fever and rheumatoid arthritis. the main differential diagnoses are true intra - articular lipoma (which is much rarer), synovial chondromatosis or haemangioma, or pigmented villonodular synovitis (pvns). the diagnosis is made on mri. pvns, atypical synovitis of ra, and synovial chondromatosis can usually be differentiated by mri, since these do not show fat lobules. pvns masses contain haemosiderin and have a low signal intensity on t1 and t2 images. signal intensity in ra masses is low on t1 and intermediate to high on t2 images and in synovial chondromatosis is low on t1 and high on t2 images. chondromatous bodies may contain fat in the centre and have rim - like calcifications which have a low intensity on all sequences and are usually visible on plain radiographs. true intra - articular lipoma does not have the same frond - like appearance as la, and usually occurs in the infrapatellar fat pad. synovectomy is the recommended treatment for la and is usually curative, although recurrence following synovectomy has been reported. open synovectomy has been used in the treatment of this condition though arthroscopic synovectomy is now the treatment of choice. synovectomy has been reported to result in complete and long standing alleviation of symptoms of la in most patients but does not appear to halt the progression of secondary osteoarthritis. non - surgical alternatives to synovectomy appear to be successful, although there are very few reports of their use. erselcan successfully used yttrium-90- radiosynovectomy to treat one patient and chemical synovectomy with osmic acid has also been described with no recurrence of symptoms at one year. in conclusion lipoma arborescens is a rare, benign intra- articular tumour which may mimic a number of other diagnoses. in cases of unexplained chronic joint effusion, mri should be considered to exclude this pathology as well as other uncommon intra - articular pathology. lipoma arborescens is one of the differentials of knee pain and swelling and can present at varied age. | introduction : lipoma arborescens (la) is a rare, benign intra - articular lesion most commonly found in the knee, characterised by villous proliferation of the synovium. it generally presents as a longstanding, slowly progressive swelling of one or more joints associated which may or may not be associated with pain. mri is the investigation of choice, with images clearest on fat - supressed or stir sequences.case report : we present a 35 year old male patient, who presented with a three year history of bilateral knee pain and swelling. magnetic resonance imaging (mri) scans of his knee showed the characteristic features of lipoma arborescens. a 99technetium bone scan revealed increased uptake in both knees. the patient underwent bilateral arthroscopic synovectomies and made an uneventful recovery. the samples sent for histology were reported as being characteristic of lipoma arborescens.conclusions:lipoma arborescens is a rare, benign intra - articular tumour which may mimic a number of other diagnoses. mri should be considered to exclude this pathology as well as other uncommon intra - articular pathology. treatment with synovectomy is frequently curative. |
oxidants are important in the regulation of signal transduction and gene expression. multiple classes of genes are transcriptionally activated by oxidants and are implicated in different phenotypic responses. in the present study, we performed differential mrna display to elucidate genes that are induced or repressed after exposure of rat lung epithelial (rle) cells to h2o2 or crocidolite asbestos, a pathogenic mineral that generates oxidants. after 8 or 24 hr of exposure, rna was extracted, reverse transcribed, and amplified by polymerase chain reaction with degenerate primers to visualize alterations in gene expression. the seven clones obtained were sequenced and encoded the mitochondrial genes, nadh dehydrogenase subunits nd5 and nd6, and 16s ribosomal rna. evaluation of their expression by northern blot analysis revealed increased expression of 16s rrna after 1 or 2 hr of exposure to h2o2. at later time periods (4 and 24 hr), mrna levels of 16s rrna and nadh dehydrogenase were decreased in h2o2-treated rle cells when compared to sham controls. crocidolite asbestos caused increases in 16s rrna levels after 8 hr of exposure, whereas after 24 hr of exposure to asbestos, 16s rrna levels were decreased in comparison to sham controls. in addition to these oxidants, the nitric oxide generator spermine nonoate caused similar decreases in nadh dehydrogenase mrna levels after 4 hr of exposure. the present data and previous studies demonstrated that all oxidants examined resulted in apoptosis in rle cells during the time frame where alterations of mitochondrial gene expression were observed. as the mitochondrion is a major organelle that controls apoptosis, alterations in expression of mitochondrial genes may be involved in the regulation of apoptosis.imagesfigure 1figure 2figure 3figure 4figure 5 |
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in addition to possessing an extraordinary sweeping activity, pulmonary alveolar macrophages (pam) are equipped with the biochemical mechanisms involved in the metabolism of carcinogens, which were found to be inducible in humans by cigarette smoke. moreover, several defense processes were stimulated in rat pam after in vivo administration of the anticarcinogen n - acetylcysteine (nac). benzo[a]pyrene diol epoxide (bpde)-dna adducts, as revealed by synchronous fluorescence spectrophotometry, were selectively detected in pam of smokers and persisted up to 6 months. the amount of adducts was significantly correlated with the number of currently smoked cigarettes but not with the cigarettes smoked in a lifetime (pack - years). nevertheless, deviations from the regression line pointed out the role of interindividual variability factors in adduct formation. probably due to the low mitotic rate of pam in the respiratory lumen, the frequency of micronuclei was not enhanced in smokers. however, parallel assays in rats showed that micronuclei can be enhanced after massive intratracheal administration of benzo[a]pyrene or whole - body exposure to high amounts of mainstream cigarette smoke, which also induced bpde - dna adducts in lung cells and other organs, including the heart. all these adverse effects were markedly inhibited by the oral administration of nac, which provides the premise and rationale for a future study on the protective effects of oral nac in heavy smokers.imagesfigure 1.figure 1. |
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several cases of desmoplastic ameloblastoma (da) of the maxilla that extended into the maxillary sinus have been described,[15 ] but there has been no report of da occupying the entire sinus. here, we report a rare case of a da lesion that filled the entire maxillary sinus. the patient was a 29-year - old japanese female who visited the department of oral and maxillofacial surgery at mie university hospital with a chief complaint of swelling around the upper left premolars. the swelling had persisted for 1 month without subjective symptoms such as pain before she was referred to our hospital by a local dentist. at the initial examination, there was no buccal numbness, tenderness, nasal congestion, or eye manifestation. palpation of the oral cavity indicated the presence of a bone - like and hard, but painless, mass [figure 1 ]. a panoramic x - ray captured an image of a mixture of ill - defined radiolucency and radiopacity that extended from the swollen area to the maxillary sinus, and revealed root divergence between the upper left first premolar and second premolar, but the absence of periapical resorption [figure 2 ]. a computed tomography scan showed that a lesion containing bone - like tissues occupied almost the entire maxillary sinus. the lesion extended from the alveolar process of the premolars to the anterior region of the outer wall of the maxillary sinus, the palatine process, the lateral wall of the nasal cavity, and close to the floor of the orbit. the bone wall was relatively well preserved, but a part of the lesion had entered the nasal cavity [figure 3 ]. the lesion showed heterogeneous isointensity on t1-weighted mri and hypointensity on t2-weighted magnetic resononce imaging [figure 4 ]. the oral cavity at the initial examination showing a painless mass around the upper left premolars a panoramic x - ray captured an image of a mixture of ill - defined radiolucency and radiopacity that extended from the swollen area to the maxillary sinus, and revealed root divergence between the upper left first premolar and second premolar a ct scan showed that a lesion containing bone - like tissues occupied almost the entire maxillary sinus mr image showing heterogeneous isointensity on t1-weighted mri and hypointensity on t2-weighted mri the tumor was pathologically diagnosed as ameloblastoma based on biopsy results. however, based on the patient 's request and her age, removal of the tumor and curettage of the bone around the tumor, and extraction of # 11, # 12, # 13, and # 14 were performed under general anesthesia. the tumor had adhered to the bone and occupied almost the entire maxillary sinus, but did not reach the floor of the orbit or the posterior wall of the maxillary sinus. histopathological findings from isolated tissues showed that the tumor parenchyma had a fiber architecture, which was similar to the enamel organ, with small alveolar or trabecular structures, and spread in the abundant interstitium consisting of scar - like fiber tissues with a few cellular components. the patient was followed up in the outpatient department after discharge, but a ct scan performed at 4 months postoperatively revealed the presence of residual and recurrent tumor in the area of the upper left lateral incisor. therefore, removal and curettage were performed again and the upper left lateral incisor was extracted. the subsequent course has been benign and the patient has been followed up in the outpatient department as of 12 months postoperatively. histopathological view showing that the tumor parenchyma had a fiber architecture with small alveolar or trabecular structures, and spread in the abundant interstitium consisting of scar - like fiber tissues with a few cellular components. ameloblastoma is the most common odontogenic epithelial tumor, accounting for 1118% of all odontogenic tumors, and da accounts for 412% of ameloblastomas. the most common site is from the anterior teeth to the premolars in both the maxilla and mandible, unlike other types of ameloblastoma, which typically occur in the mandibular molars. the size is relatively small and the longest diameter is generally 2 cm or less.[610 ] the majority of ameloblastomas show radiolucency on x - ray, while approximately 50% of da cases have a mixture of radiolucency and radiopacity. da is a histologically solid tumor without capsule formation, and is characterized by abundant collagen fiber formation in the interstitium. yoshimura. reported that da lesions were generally smaller than other ameloblastoma lesions because da growth might be inhibited by collagen fibers. however, the da lesion in our case occupied almost the entire maxillary sinus, which suggests that collagen fibers are unlikely to inhibit da growth. there are several reports of resection and curettage for treatment of da, but the risk of recurrence is higher in these procedures compared to total resection,[79 ] with recurrence in 6 of 19 cases treated with removal and curettage.[379 ] da with extensive spreading to the maxillary sinus is relatively common,[15 ] but a case with the lesion occupying almost the entire sinus, as in our case, is rare. da generally grows slowly, but once it enters the maxillary sinus the growth may be accelerated because there are no tissues to interfere with growth. the prognosis is good with rare recurrence when extended resection is applied in cases of da that spread to the maxillary sinus, but recurrence has been reported after removal and curettage, as in our case. extended resection is the first line therapy for a case in which the tumor has entered and occupied the maxillary sinus. if removal and curettage are requested by the patient or performed for other reasons, wide removal of the lateral wall of the nasal cavity is required to allow detection of recurrence and inspection of the inside of the maxillary sinus from the opening of the nasal cavity using a fiberscope. therapy should be selected based on the quality of life of patients, and the risk of recurrence following removal and curettage should be disclosed to the patient. recurrence may be detected relatively easily based on radiographic characteristics, and therefore follow - up with an x - ray examination such as a ct scan is important. | we report a rare case of desmoplastic ameloblastoma lesion that filled the entire maxillary sinus. the patient visited our hospital with a chief complaint of swelling around the upper left premolars. a panoramic x - ray captured an image of a mixture of ill - defined radiolucency and radiopacity from the swollen area to the maxillary sinus. computed tomography (ct) and magnetic resononce imaging (mri) showed that the lesion occupied almost the entire left maxillary sinus and had entered the nasal cavity. a pathologic diagnosis of ameloblastoma was made after biopsy, and the tumor was removed and the marginal bone curetted under general anesthesia. a ct scan at 4 months postoperatively indicated the presence of residual and recurrent tumor in the area of the upper left lateral incisor, and removal and curettage were performed again. recurrence may be detected relatively easily based on radiographic characteristics, and therefore follow - up with an x - ray examination such as a ct scan is important. |
multiple sclerosis (ms) is a disorder of the central nervous system that affects over 500,000 europeans. the disorder affects three times as many women as men and is more prevalent in northern europe. the social costs associated with ms are high due to its long duration, the early loss of productivity, the need for assistance with activities of daily living, and the use of immunomodulatory treatments. longitudinal population - based studies have found that 50% of patients require assistance with ambulation after 15 years and that over 80% of ms patients reach a level of severe and permanent disability after 30 years. ms is caused by an autoimmune reaction against self - directed myelin antigens in the central nervous system. as a result of the immune deregulation that characterizes ms, clinically, the disease manifests itself as relapses of neurological disability due to dysfunction of the areas in which myelin damage occurs. ms can lead to severe and permanent disability due to the axonal damage and irreversible neurodegeneration. although the causes of ms remain unknown and there is currently no cure, over the last twenty years, a number of treatments have been developed that reduce the number of relapses and slow the progression of the disease. however, advances in our understanding of the underlying pathogenesis of ms will help identify potentially promising avenues of research for novel therapies with unique mechanisms of action. autologous mesenchymal stem cells (mscs) have been shown to induce immunomodulatory and neuroregenerative effects and to have a neuroprotective effect in the animal model of chronic experimental autoimmune encephalomyelitis (eae) [5, 6 ]. studies in animals have shown that mscs can, under the right conditions, mature into myelin - producing cells that counter myelin loss in ms disease models [710 ]. adipose - derived stem cells (ascs) are adult mesenchymal stem cells (mscs) that reside in fat. they can be isolated through enzymatic digestion and have been widely used in clinical trials involving both autologous and allogenic models : over 400 clinical trials that include msc transplantation procedures are currently registered (https://clinicaltrials.gov/). although stem cell therapies for a number of conditions have been previously tested with favorable safety results, few reports of their use in ms patients are available. the goal of this study was to evaluate the safety and tolerability of intrathecal autologous transplantation of ascs over the relatively short period of one year of observation. the secondary endpoints of relapse rate and disability progression were assessed 18 months following randomization. the goal of the study was to achieve local immunomodulation of the patient 's immune system by transplantation of autologous ascs. after the study procedures were approved by the institutional review board, 20 patients with relapsing - remitting (rr) ms (13 patients) and secondary progressive (sp) ms (7 patients) in both groups of patients with relapses were enrolled in the study. the study enrolled patients who were diagnosed with relapsing - remitting ms according to the mcdonald criteria of 2013. patients were diagnosed with secondary progressive ms if they achieved srd (srd : sustained reduction in disability ; srd definition : 1-point decrease in edss (expanded disability status scale) score over 6 months for patients with an edss score 2.0) during the 6 months before the stem cell procedure and did not experience a relapse. the main inclusion criterion was a one - point increase in edss score in the year preceding the asc implantation, with or without new or larger gadolinium- (gd-) enhancing lesions on mri. in addition to the safety endpoints, the investigators tracked several efficacy outcomes, including changes in edss and ms functional scale scores, relapses, and mri lesion burden. clinical and neurologic evaluations were carried out just before the asc procedure (baseline), 3 months after randomization, and then every 3 months for the following 18 months. a neurologic evaluation was completed whenever the patient complained of symptoms or signs suggestive of a relapse. patients whose edss scores were stable over the 24 months who did not relapse were considered responders. adipose tissue was collected from the patients ' abdominal and femoral regions using coleman 's technique and immediately transported to the stem cell laboratory. the fat tissue was mixed with buffered physiological salt solution (pbs) in a 2 : 1 vol / vol ratio and vigorously shaken. following fat phase separation, subsequently, 0.075% collagenase in pbs was added to the adipose tissue (1 : 2 vol / vol), and the mixture was incubated for 1.5 h at 37c. every 15 min, the collagenase / fat mixture was mixed by shaking for 15 sec. after the incubation period, human albumin (20% solution, 2% final concentration) was added, and the mixture was centrifuged (400 g, 10 min). the liquid fat and salt interphases were discarded, and the resulting cell suspension (3 ml) was filtered through a 100 m nylon mesh filter 3x. the cell suspension was washed 3x with pbs, and the final cell suspension was made in 5 ml of physiological salt (clinical grade) supplemented with 2% human albumin. control tests included cell number and viability, sterility control, and flow cytometry characteristics of cell surface markers. all procedures were performed in the stem cells and tissue bank, which is accredited for the processing and storage of ascs for clinical purposes. asc - containing svf was injected intrathecally (12 10 cells / dose) at the time of enrollment, and the injection was repeated at the 3rd and 6th months using cryopreserved ascs. the follow - up observation time varied from 12 to 16 months, and the efficacy parameters (edss and ms functional scale scores, relapse incidents, mri lesion burden, and whole brain and gray matter atrophy rates) were monitored throughout the 12- to 16-month period. the mri analyses included the cumulative number of new t2 lesions that appeared over the first year after therapy as well as the time of appearance of the first new t2 mri lesion. mri examinations were performed in a participating center with mri scanners operating at 3 t using a standardized protocol. contrast agent - enhanced t1-weighted images were obtained for all patients after iv injection of 0.1 mmol / kg body weight gd - based contrast agent. mri of the brain was obtained at screening, at baseline, and after 12 months. the tests were performed in accordance with the declaration of helsinki, and the study protocol was approved by the bioethical committee of the military medical institute in warsaw. all of the patients completed the follow - ups at 12 and 18 months, and some of them also completed a 24-month follow - up. among the recruited patients, 13 were in the rr phase (group i), while 7 were in the sp phase of the disease with relapses (group ii). the average patient age was 37.4 (range : 2758) among the group of patients with rrms and 40.6 (range : 3451) among the patients in the spms group. the baseline edss scores ranged from 3 to 6.5 (mean 4.6) in the first group and from 3.5 to 9 (mean 5.6) in the second group. the average disease duration was 9.5 years in the first group and 15.6 in the second group. all patients clinically deteriorated during the year preceding the study, with at least a 1-point increase in edss score. all of the included patients had failed previous treatments with first - line therapies (glatiramer acetate, interferon - b) or with second- and third - line therapies (fingolimod, natalizumab, azathioprine, and methotrexate) in different combinations. autologous mscs were extracted from adipose tissue at the time of enrollment, and the patients were then observed and examined every 3 months. the number of mscs extracted from a single collection of adipose tissues exceeded the target of 12 10 cells. cell suspensions in a cryoprotectant were divided into portions of 4 10 cells / dose and cryopreserved in the stem cell bank at 170c until transplantation. the patients received a portion of cells intrathecally at the 3- and 6-month follow - ups. clinical data were available for the 20 included patients. within the first 12 months, 18 patients did not exhibit a change in edss score and did not have relapses. two patients from the rrms group had relapses without a change in edss score ; the mri scans of these patients showed new gd+ lesions (in one patient, the lesions were in the brain, and, in the second patient, one lesion was observed in the medulla oblongata and pons). at the 18-month follow - up, edss scores had not changed in any of the patients from either group. within 18 months of follow - up, 3 patients from the rrms group had relapses without exhibiting progression in disability from baseline, and 7 patients showed enticing improvement on some of the exploratory efficacy measures, although no significant benefit of the treatment on edss scores was observed across the entire group. all 7 of these patients showed a slight improvement in terms of msfc scores and certain other efficacy measures. overall, there was no significant change in any of these measures, although the improvements observed in these patients persisted throughout the total observation period. four patients were observed for 24 months, and their clinical status did not change over this period. at 18 months after asc transplantation, the edss scores of two of the patients from the spms group were slightly below the baseline value, but not by 1 point. adverse events after the administration of ascs were not observed in any of the patients at the 12-, 18-, and 24-month follow - ups (table 1). there is a critical unmet need to develop therapies that enable repair in ms patients. preclinical studies using the mouse eae model showed that intrathecal autologous hematopoietic stem cell transplantation (ahct) improved neurological function and that this effect was associated with the suppression of local inflammatory responses and the provision of trophic support for damaged cells at the lesion site. as a result, new ms therapeutic strategies characterized by intense immunosuppression followed by ahct have been proposed in recent years. the suppression of inflammation that occurs after ahct may play a beneficial role in slowing down disease progression and could induce prolonged tolerance to self - antigens. in recent years, over 800 ms cases worldwide have been reported to the registry of the european group for blood and marrow transplantation (ebmt) as having received this treatment procedure. many patients have been treated in phase i / ii studies, and good results have been reported. a beneficial effect of intense immunosuppressive chemotherapy and ahct in the treatment of aggressive ms that is unresponsive to standard therapies later, a major suppressive effect on disease activity was noted based on brain mri, and the procedure was associated with 3% mortality. over the next several years, studies in which ahct was used as a rescue therapy for malignant forms of ms were reported. the most impressive results in patients with the malignant form of ms that were treated with ahct were observed on mri. although this method of therapy appears to be very effective, especially for select ms patients, only one published prospective study has compared ahct with conventional treatments. in this study, aht was shown to be significantly superior to mtx in reducing disease progression on mri and the annual relapse rate (arr) : patients with severe cases of ms in the ahct arm experienced 79% fewer new t2 lesions and a lower arr compared to patients in the mtx arm. another study in which mesenchymal stem cell - neural progenitors, an autologous bone marrow - derived cell population with regenerative potential, were administered intrathecally also showed that this treatment was safe and well tolerated in ms patients and that no adverse events occurred. despite the short observation time, the results of our study suggest that asc intrathecal therapy is safe for use in ms and slows disease progression. as a powerful anti - inflammatory and immunosuppressive treatment, this method may benefit patients with rapidly progressive ms. ascs are an attractive candidate for cell - based therapies aimed at stopping and reversing the loss of myelin in ms, which is ultimately responsible for the progressive disability that is observed over time in the majority of patients. however, the study did not have sufficient power to detect a significant clinical benefit, likely due to the short observation time. nevertheless, there are still many unknowns regarding how to best deliver cell - based therapies in ms, such as the best route of administration, the need for ex vivo manipulations, and the desired dosing levels and intervals. for this reason, we do not view it as a problem that these early studies did not show a dramatic clinical benefit. both in vivo animal experiments and clinical observations suggest that asc treatment causes no adverse effects [10, 13 ] these cells, however, should not be transplanted into cancer patients because msc may induce the formation of blood vessels and secrete cytokines which may stimulate existing tumors, although they do not convert into cancer cells. for that reason, the recruitment of sm patients must exclude these with coexisting cancer disease. our experience with autologous mesenchymal stem cell transplantation in ms raises questions about the acceptable balance between safety, efficacy, and convenience when treating patients with ms. the future importance of these therapies will thus reflect a trade - off between the associated benefits and risks. in our opinion, ascs are an attractive candidate for cell - based therapies aimed at stopping and reversing myelin loss in ms, which is ultimately responsible for the progressive disability that is observed in most patients over time. in our opinion, asc therapy is not recommended for all patients with ms and should be reserved for aggressive cases, for cases still in the inflammatory phase of the disease, and for the malignant form. in conclusion, no difference in 18-month edss score changes was found between the groups of rr and sp ms patients. no progression occurred in any patients from either group at the end of the follow - up, the 18th month of observation. a closely watched one - sided prospective trial of ascs for ms demonstrated that the treatment was safe and suggested that it may have helped some patients. no other treatment - related adverse effects were observed in patients who received intrathecal infusions of autologous ascs, and the study hit safety endpoints. disease progression - free survival (pfs) was 18 months for the majority of patients and did not differ according to disease type, gender, conditioning, or edss score at transplantation. | the clinical outcome of autologous adipose stem cell (asc) treatment of patients with multiple sclerosis (ms) was investigated following one year of observation. methods. the clinical and mri outcomes of 16 asc - treated patients with rrms and spms are reported after a one - year follow - up period. results. at 18 months of follow - up, some patients showed enticing improvements on some exploratory efficacy measures, although a significant benefit was not observed for any measure across the entire group. neither the progression of disability nor relapses were observed in any cases. in four patients, we found new gadolinium+ (gd+) lesions on mri. our results indicate that asc therapy is safe and does not produce any substantial side effects. disease progression - free survival (pfs) of 18 months was seen in all patients with rrms and spms. in these patients, edss scores did not progress above baseline scores. gd - enhancing lesions were observed in two cases with rrms, but these patients did not exhibit changes in edss score. conclusion. intrathecal treatment with ascs is an attractive form of therapy for patients with ms but should be reserved for cases with aggressive disease progression, for cases that are still in the inflammatory phase, and for the malignant form. |
candida is one of the most frequently recovered pathogens in patients with hospital acquired bloodstream infections [13 ], where it is associated with a mortality rate from 30 to more than 60% in case of septic shock [48 ]. major risk factors for candida colonization include length of intensive care unit (icu) stay, use of parenteral nutrition, broad - spectrum and long - term antibiotics, central lines, and abdominal surgery. thus, a colonization index (number of colonized sites / number of sampled sites) of > 0.5, with recovery of the same candida species or genotypes in the colonized sites and bloodstream, has been set up and is associated with an increased risk of candidemia. studies conducted to develop a candida score showed that factors associated with candidemia were surgery, multiple - site candida colonization, severe sepsis, and parenteral nutrition. thus, candida colonization, although not unique, is a reliable independent risk factor for candidemia [1315 ]. therefore, early systemic antifungal therapy (sat) deserves consideration in icu patients in whom they are increasingly used. the diagnosis of candidemia is often difficult and delayed because the sensitivity of blood culture bottles (even in specific milieu) is not higher than 75% and decreased by previous antifungal therapy. non culture - based assays have been developed to improve the diagnosis of invasive candidiasis. (1 - 3)--d - glucan is a cell wall component of candida sp. and other fungi. it becomes detectable early during invasive candidiasis but may also rise in other fungal infections. indeed, (1 - 3)--d - glucan rises in cases of aspergillosis, and pneumocystis jirovecii infections, but also in rare infections, such as infection with fusarium spp., acremonium spp., sporothrix schenkii, coccidioides immitis, histoplasma capsulatum, blastomyces dermatitidis, trichosporon spp. and importantly, (1 - 3)--d - glucan is not found in cryptococcosis or zygomycetes infections (adsidia, mucor, rhizopus). (1 - 3)--d - glucan remains detectable for more than one month after invasive candidiasis. the cut off value proposed for hematological patients is 80 pg / ml but it appears to be higher in icu patients. false positive results have been reported in cases of dialysis with cellulose membrane, albumin perfusions, intravenous immunoglobulin administration, antibiotic therapy with coamoxiclav or piperacillin - tazobactam, and bacteremia. the reported sensitivity and specificity of (1 - 3)--d - glucan is 5797% and 5693%, respectively. given the low prevalence of invasive candidiasis in non - neutropenic patients, a negative test is adequate to rule out a diagnosis of infection. in the recent study of the funginos group, in a selected population of surgical icu patients, two consecutive measurements of (1 - 3)--d - glucan of more than 80 pg / ml were associated with a reasonable diagnostic value (positive predictive value of 72%, negative predictive value of 80%) that need to be confirmed in further study on other non - surgical icu populations. importantly, (1 - 3)--d - glucan positivity preceded the diagnosis of invasive candidiasis by 5 days in mean. mannan, a polysaccharidic antigen from candida cell wall, is another potential diagnostic test. both mannan antigen and anti - mannan antibodies could be measured using one enzyme - linked immunosorbent assay (elisa) kit. mannanemia is more specific than (1 - 3)--d - glucan but showed a lack of sensitivity in a recent study on candidemia. the combination of both tests possesses an acceptable diagnostic value (86% specificity and 83% sensitivity) in a recent meta - analysis. finally, detection of candida dna using polymerase chain reaction (pcr) seems to be both sensitive and specific in the available literature but standardization of tests is lacking. delays in initiating appropriate treatment have been associated with increased mortality in patients with bloodstream infections. importantly, the control of the infected source acts in synergy with the antifungal therapy. in a recent study, both a delay in antifungal treatment of more than 24 hours (odds ratio=5.99, p=0.048) and the absence of source control within the first 48 hours (odds ratio=2.99, p=0.001) were associated with the risk of death in the case of septic shock due to candidemia. the usual risk factors described in the more recent predictive scores included variables that are frequent, and lead us to treat 1020% of the icu patients. more than two - thirds of these treatments are given without definitive proof of invasive fungal infections. as an example, the colonization index 's positive predictive value is less than 9% in the epcan study. in medical icu patients, 39% developed a colonization index of more than 0.5, while, in the same period, no invasive fungal infections were diagnosed. new antifungal agents are well tolerated and overtreatment might be considered safe on an individual basis. however, new data from the us and europe clearly demonstrate that the overuse of antifungal drugs contributes to both the emergence of candida species that are known to be less sensitive to antifungal agents, as well as to the increased occurrence of sensitive candida species with increased minimum inhibitory concentrations (mics). recently, lortholary. reported that azole derivatives and candins pre - exposure increased the risk of fungemia due to species with higher mics to the corresponding antifungal agents. pfaller. found an increase in rates of fluconazole - resistant candida glabrata intermediate or resistant to candins over time, from less than 4% between 2000 and 2002 to more than 12% between 2008 and 2010. reported 20 episodes of fungal infections caused by candin - resistant candida spp. that were harboring diverse and new resistance mutations. for 12 patients, the initial isolates (low mics, wild - type fks gene) and the subsequent isolates (after caspofungin treatment, high mics, fks mutation) were genetically identical. we also recently described a significant relationship between systemic antifungal therapy (sat) consumption and mics of colonizing and infecting fungi in icu patients. finally, two studies clearly showed that the pre - exposure to candins was associated with episodes of c. glabrata septicemia with strains of reduced susceptibility to candins that harbored fks mutation. obviously, sat should be used applying the same rules as for other antimicrobial agents. it must be effective and safe for the patient himself, and also for future patients. regarding which benefits to expect from an empirical or pre - emptive sat in critically ill non - immunocompromized patients, the current literature is inconclusive, and trials demonstrating the efficacy of sat in colonized patients with unresolved sepsis and organ dysfunction are warranted. empiric therapy is usually defined as a therapy instituted in patients with clinical signs suggestive of ongoing invasive infection (new systemic inflammatory response syndrome, organ failures), while pre - emptive therapy is given to patients with risk factors and one or more positive markers such as a rising colonization index or (1 - 3)--d - glucan elevation above a threshold value that remains to be determined in the icu setting. one may question the potential of empirical therapy of icu patients with sepsis and risk factors of invasive fungal infection, and of pre - emptive therapy of icu patients with a positive biomarker such as candida colonization or (1 - 3)--d - glucan. regarding the so - called empirical therapy, no clear demonstration of efficacy has been published. in a randomized controlled double - blind trial, a high dose of fluconazole failed to reduce survival free of invasive fungal infection in medical - surgical icu patients with non - resolving sepsis. in this study, candida colonization was diagnosed at inclusion of patients in only a quarter of the cases. the issue remains uncertain because the diagnosis of invasive fungal infection remains a challenge in icu. in a one - day prevalence study, the authors declared 17% of nosocomial infections to be due to candida spp. the colonization index, validated 20-years ago in long - term surgical icu patients, has been broadly challenged. for instance, its positive predictive value is less than 9% in the epcan study. furthermore, in medical icu patients, 39% developed a colonization index of more than 0.5, while, within the same period, no invasive fungal infections were diagnosed. colonization index remains an important way to characterize the dynamics of the colonization of icu patients, which increases early in patients who will go on to develop invasive candidiasis, but its bedside practicality remains limited. one before - and - after study showed decreased icu - acquired candidemia rate when using a colonization index - based fluconazole therapy, but without any survival benefits. likewise, we are looking forward to having improved diagnostic strategies to increase the sensitivity, specificity and predictive values of the available tools, as well as to reduce diagnostic delays. new tools such as assays to measure (1 - 3)--d - glucan levels provided promising results in icu populations. however, (1 - 3)--d - glucan is not specific to candidiasis, is higher than 80 pg / ml in many icu patients without invasive candidiasis, and decreases slowly under effective treatment. over the last 15 years, several studies have evaluated the potential benefits from sat in icu patients overall [4550 ], and in the subset of icu patients with risk factors for candidemia or sepsis of unknown origin. pre - emptive sat has been suggested for the sickest surgical icu patients, most notably those with peritonitis. more recently, an exploratory study compared the efficacy and safety of micafungin as a pre - emptive treatment of invasive candidiasis vs. placebo in high risk surgical subjects with intra - abdominal infections in a multicenter randomized control trial (intense nct nct01122368). a total of 241 patients were analyzed in the full analysis set. in this study, the rate of independent data reviewing board - confirmed invasive fungal infection after inclusion was similar in the micafungin and placebo arms (8.9 vs. 11.1%). there was no difference in mortality, invasive fungal infection - free survival, and improvement of organ failures between the micafungin and placebo arms. randomized, double - blind, placebo - controlled trial (msg 01) ostrosky - zeichner. tested the use of caspofungin in 222 adults who were in the icu for at least 3 days, were ventilated, received antibiotics, had a central line, and had 1 additional risk factor (parenteral nutrition, dialysis, surgery, pancreatitis, systemic steroids, or other immunosuppressive agents). unfortunately, in terms of trial protocol, patients with sepsis and with two consecutive (1 - 3)--d - glucan samples above 80 pg / ml were classified as probable cases of invasive candidiasis, which allowed the investigators to break the blind and to administer them pre - emptive therapy with caspofungin. the pre - emptive approach analysis included all patients who received the study drug, including those positive at baseline. the incidence of proven / probable invasive candidiasis in the placebo and caspofungin arms was 30.4% (31/102) and 18.8% (22/117) for the pre - emptive approach (p=0.04). there were no significant differences in the secondary endpoints of mortality, antifungal use, or length of stay. the rate of proven invasive candidiasis was low (6.9% and 11.1% in the placebo arms of intense and msg-01 studies) ; it suggests that the targeted population is not fully understood and leads to suggestions that the studies are considerably underpowered. the decrease in the risk of probable infection in the msg-01 study was not associated with an improvement of vital status or duration of icu stay. this may be explained by the fact that (1 - 3)--d - glucan serum level above 80 pg / ml should not be considered an accurate biomarker of invasive candidiasis. patients that may possibly benefit from early (empiric or pre - emptive) antifungal treatment are those with a high risk of invasive candidiasis. given the results of the study from schuster. and the work performed by the epcan groups, we postulated that the combination of multiple organ failure, sepsis of unknown origin and multiple colonization with candida in mechanically ventilated patients for more than four days and receiving board spectrum antibacterial agents, should select a population with a particularly high risk of life - threatening invasive candidiasis. the potential benefits ascribable to sat in this population is currently being tested in the empiricus trial. we consider that definite rules could not be derived for systemic antifungal therapy. we need to strongly encourage and promote studies able to improve diagnostic strategies, and randomized control trials further defining the efficacy of sat in colonized patients with sepsis and multiple organ failures. pre - emptive treatment should be decided at bedside, after sampling at least two separate blood cultures with 10 ml volume of blood preferably on selective milieu, in view of the uncertainty involved. to contain the antifungal selection pressure that is starting to rise, we also propose considering stopping rules after 5 days when no proven invasive candidiasis occurs. until the development of accurate early diagnostic tests or the results from ongoing trials are available, a demonstration of a clinical benefit of treatment of such patients is warranted to solve uncertainties in the issue deciding antifungal treatment in icu setting. jean - franois timsit received educational grants from gilead and lectured in symposia organized by astellas, pfizer and merck. | the potential of the systemic antifungal treatment of non - immunocompromised patients with sepsis, extra - digestive candida colonization and multiple organ failure is unknown, although it represents three out of four antifungal treatments prescribed in intensive care units. it may allow an early treatment of invasive fungal infection at incubation phase, but exposes patients to unnecessary antifungal treatments with subsequent costs and antifungal selection pressure. as early diagnostic tests for invasive candidiasis are still considered insufficient, the potential of this strategy needs to be demonstrated by a randomized controlled trial. such a trial is currently ongoing. |
the first human blood group, that is, the abo system discovered by landsteiner,1 is the most commonly used blood system although many blood systems have been identified so far. the discovery of abo system and findings of red cell agglutination in serum and recognition of blood groups laid the scientific basis for safe practice of blood transfusion.2 the other important blood systems are the rhesus (rh) and the mn system. abo and rh systems have major clinical significance and they are determined by the nature of different proteins present on the surface of red blood cells. the antigens of the abo system are an integral part of the red cell membrane and they are also found in plasma and other body fluids. all human populations share the same blood systems, although they differ in the frequencies of specific types. some variation may even occur in different areas within one small country.3 the blood group distribution also shows variety according to races.4 it was reported that the group a has a wider distribution in eskimos, the group b in chinese and indians, the group o, on the other hand, in american and canadian indians and czechoslovakians and those living in kenya.4 according to statistical distribution of the abo blood types in the turkey, 42.5% had type a and 33.7% had type o and 15.8% had type b, and 8.0% had type ab blood.5 when the rate of rh+ is considered, it was reported to be about 85% in all the population. however, varying percentages were reported in various countries of the world (kenya 96%, india 99%, iran 90%, turkey 87%).4 abo blood groups are the most investigated erythrocyte antigen system, and owing to ease of identifying their phenotypes, they have been used as genetic markers in studies of their associations with various diseases.6,7 studies from the 1950s demonstrated that blood group o is associated with duodenal ulcer disease, while gastric ulcer and gastric carcinoma are associated with blood group a.8 during the last few decades, several reports have suggested that abo blood groups, in particular non - o blood groups, are associated with the risk of ischemic heart disease and of developing severe manifestations of atherosclerosis.912 results from the farmingham study13 and several other reports indicated that the incidence of ischemic heart disease might be higher in subjects of blood group a or its subgroups. stakisaitis10 found that the blood group b might be related to coronary atherosclerosis in lithuanian women. in apparent contradiction, michell14 showed that towns with a higher prevalence of blood group o had higher rates of cardiovascular mortality. although several studies have been carried out to investigate relationships between the abo blood groups and the incidence of certain diseases in medicine, little investigation has been made to explore the relationships between abo blood groups and the incidence of oral and dental diseases. aitchison and carmichael15 studied the distribution of blood groups within two groups, one of whom were the random patients attending the dental hospital and the other consisting of cases with rampant caries. barros and witkop16, on a large group of chileans, found no association between the d.m.f scores for caries and abo blood groups. it is well known that periodontal diseases have high incidence in population.17 although bacterial plaque is considered the primary extrinsic etiologic agent in periodontal diseases,17,18 our purpose in this study was to describe whether there is a relationship between abo blood groups and periodontal diseases in a group of turkish people who have same social and economic conditions. the present investigation was carried out on 1351 subjects, 647 male and 704 female, aged 17 to 63. subjects were selected from among patients who were referred to the clinics of faculty of dentistry at atatrk university for periodontal treatment and for other dental health reasons. certain criteria were followed in order to bring forward abo blood groups of individuals included in the research. it was demanded according to the selection criteria that : all subjects had at least 20 teeth and had received no periodontal treatment or antibiotic - related therapy for medical or dental reasons 3 month prior to the study. they had no history of systemic disease such as diabetes, leukemia, metabolic bone disease or epilepsy. the gingivitis subjects displayed less than 3 mm of attachment loss, periodontal pockets depth less than 3 mm, no radiographic bone loss but displayed gingivitis sign (gingival bleeding, color, gingival contour, position and surface texture in the gingiva). periodontitis subjects exhibited at least one site evidence of radiographic bone loss, attachment loss more than 3 mm, periodontal pockets depth more than 4 mm. healthy subjects displayed less than 3 mm of attachment loss, periodontal pockets depth less than 3 mm, no radiographic bone loss and no gingivitis sign. the clinical oral examination was carried out after the subject had been interviewed on behavior and socioeconomic background. having been informed about the method and purpose of the study, the patients were asked for their consent to participation. all the clinical measurements were made using a manual periodontal probe (williams periodontal probe) on the gingival area adjacent to the teeth in each patient. the subjects were examined clinically for the presence of plaque, gingival bleeding, clinical attachment level, probing pocket depth. according to index scores, subjects were divided into three groups : group i consisted of 356 patients with healthy gingiva (188 females, 168 males) ; group ii consisted of 441 patients with gingivitis (255 females, 186 males) ; and group iii consisted of 554 patients with periodontitis (261 females, 293 males). group i consisted of the subjects selected randomly from patients who applied to our faculty with reasons other than periodontal diseases such as orthodontic reasons and dental caries. then, the subjects in all three groups were referred to the hematology department at the faculty of medicine at atatrk university for determination of abo blood subgroup. in the 1351 blood samples surveyed, a blood group (48.5%) and o blood group (30.3%) were more common, 89.9% had factor rh positive, and 10.1% had factor rh negative. the data of a study4 performed in erzurum, representing the distribution of the blood subgroups among the general population and involving 10493 subjects, was used to control the homogeneity of the study group by taking into consideration the regional changes of abo blood subgroup. when compared with the data obtained in the mentioned study, the blood group distribution in the present study was determined to indicate no significant change. the results were compared with those of random samples obtained from ataturk university medical faculty blood donors.4 the blood group distribution within all the three groups in the present study was determined to indicate no significant change (p >.05). the observation established the homogeneity and unbiased nature of the study group as well as pointing to the natural distribution that is likely to exist in a population. table 1 shows frequency distribution of the abo blood groups in 1351 subjects and the comparative expected frequency distribution of the same blood groups obtained from 10493 controls. table 2 shows frequency distribution of the abo blood groups in 1351 patients with various grades of periodontal involvement. from the data, one finds a higher frequency at periodontal diseases in subjects with group a and o. there is a relatively high percentage of blood group a patients (61.5%) in gingivitis and relatively high percentage of blood group o patients (41.5%) with periodontitis (p.05). it was also determined that there was a relationship between rh (+) factor and gingivitis in border line (p<.05). periodontal diseases, including gingivitis and periodontitis, are serious infections that, if left untreated, may lead to tooth loss.17 the principal cause of periodontal diseases is bacterial plaque. however, a wide range of background factors such as age, sex, education, place of residence, oral hygiene habits, socio - economic status, genetic characteristics and smoking habits have been identified as risk factors for the occurrence of periodontal diseases.1719 it is known that abo blood types indicate differences in terms of their proportion according to races.4 it is also known that periodontal diseases show proportional differences in distribution among races. when this point is taken into consideration, the question arises whether or not the proportion of abo blood subgroup distribution is effective on the proportion of distribution of periodontal disease in various societies. surprisingly, little investigation has been made to explore the abo blood groups and the incidence of oral and dental diseases. in the earliest investigation on this matter, suk20 suggested that particular blood groups and a tendency towards caries might be constitutional characters and they were not particularly related to race, though the o group and good teeth were less common in civilized people than in primitive races. suk s investigation was followed by a study carried out by aitchison and carmichael,15 which revealed a relationship between the patient s susceptibility to caries and his blood group. in a study carried out by arowojolu,21 it was stated that there was a relationship between juvenile and non - juvenile periodontitis and hemoglobin type a. the influence of abo blood types on the risk of developing oral diseases has been the subject of discussion. some authors22,23 claimed that, abo blood types constituted an increased risk for the development of oral diseases whereas a small group of researchers failed to find this increased risk.24 above mentioned studies provided preliminary data concerning the associations between abo blood groups and periodontal diseases. the present study researched the relationship between abo blood subgroups and periodontal diseases in the light of the above data. in this study, it was determined that there was a relatively higher percentage of a blood group in patients with gingivitis and a relatively higher percentage of o blood group in patients with periodontitis. similarly, gawrzewska22 found individuals of blood group o to have greater severity of periodontal diseases, but individuals of blood group a to have greater resistance to periodontal diseases. suk20 found that abo blood types had an increased effect on the risk for the development of oral diseases. pradhan also found significant differences when abo blood groups were related to four grades of periodontal involvement. in one early investigation, orark and lyschon25 found a statistical significance regarding the relation between m.n. witkop16 stated that there were no significant differences between subjects with or without periodontal diseases regarding abo blood group. upon comparison of the rh factor distribution status between the 1351 subjects in the study group and the 10493 subjects in the control group, a significant relationship between rh (+) factor and gingivitis was determined (p<.05). consequently, significant relations were determined between abo blood types and periodontal condition, and rh factor and gingivitis. considering the results of our study, it can be concluded that abo blood subgroups and rh factor could constitute a risk factor on the development of periodontal disease. however, long - term studies are needed to make a more comprehensive assessment of the effects of abo group on periodontal diseases. | objectivesthe purpose of the present study was to determine whether there was a relationship between periodontal diseases and abo blood groups.methodsthis epidemiological study was carried out on 1351 subjects who were randomly selected from individuals referred to the faculty of dentistry clinics for periodontal treatment or for other reasons regarding dental health. the study based on periodontal condition, blood group, and medical history. the subjects were divided into three groups as those with gingivitis, periodontitis, and the healthy ones. the effects of blood subgroups on periodontal health, gingivitis and periodontitis were investigated separately.resultsa relatively higher percentage of a group patients was found in gingivitis group and relatively higher percentage of o group patients was found in periodontitis group. a significant relationship was also determined between rh factor and gingivitis.conclusionsabo blood subgroups and rh factor may constitute a risk factor on the development of periodontal disease. however, long - term studies are needed to make a more comprehensive assessment of the effects of abo group on periodontal diseases. |
emergence of new technologies in food preservation leads to a reduction in the levels of preservatives and promotes the use of naturally derived antimicrobials of animal, plants, and microbial origin. egyptians, chinese, and indians used spices and essential oils since ancient time. some of the spices such as mint, garlic, and ginger are still practiced in alternative health remedies in india [2, 3 ]. about 250 to 500 thousand plant species are estimated to exist on the planet and only between 1 and 10% of them are used as food by humans and other animals. spices and herbs used in foods as flavoring agents, in addition to enhancing flavors, were used as folk medicines and food preservatives. spices and herbs extend the shelf life of foods by restricting rancidity through their antioxidant activity or through their bacteriostatic and bactericidal activity. spices, herbs, and their constituents are generally recogonised as safe (gras) and approved by several regulatory agencies such as us food and drug act, the european union standards, codex alimentarius, and food safety and standards authority of india. in india, the trend of consumption of spices and herbs in food or using them as medicine aims to maintain proper sanitation, health, and hygiene and to increase longevity of life. several spices such as ajowan, clove, ginger, black pepper, cumin, and asafetida are commonly used in the indian diet [3, 6, 7 ]. literature cited the work of several authors on the antimicrobial activity of plants against wide range of bacteria, yeasts, and moulds [2, 3, 820 ]. the general description of spices and herbs used in the present study is tabulated in table 1. spices and herbs, owing to their natural origin, attract more attention of consumers that have doubt regarding the safety of chemical preservatives. several plant extracts have gained momentum in recent years due to their bioactive principals and formed the basis of pharmaceutical and food processing industries [17, 21 ]. unpasteurized fruit juice consumption has increased in the last decades, which is attributed to the contents of antioxidants, vitamins, and minerals. fresh fruit juices are highly vulnerable to spoilage, since fluid components are in contact with air and microorganisms form the environment while handling. fruit juices spoilage bacteria include acid tolerant bacteria such as acetic acid bacteria, lactic acid bacteria, clostridium, bacillus, members of enterobacteriaceae family (klebsiella sp., citrobacter sp., and serratia sp.), and some heat resistant bacteria such as alicyclobacillus acidoterrestris and propionibacterium cyclohexinicum. among yeasts, pichia, candida, saccharomyces, and rhodotorula are commonly encountered genera responsible for spoilage of juices. eurotium, alternaria, cladosporium, paecilomyces, and botrytis have also been reported in spoilage of fruit juices. the presence of pathogenic bacteria and mycotoxin producing mould can not be ruled out in fruit juice which has been responsible for the increase in food borne outbreaks with the consumption of fresh fruit juices during the last two decades [14, 23, 24 ]. therefore, the main objective of this study was to examine the in vitro antimicrobial activity of different spices and herbs extracts and to compare the effect of different solvents in the extraction method for antimicrobial activity. three indian spices, pudina, saunth, and haldi, were procured from local market in yamunanagar, haryana, india. four medicinal plants herbs, amla, brahmi, ashwgandha, arjun, and sarpagandha were collected from ch. b. d. vashishta, plant taxonomist, professor in the department of botany, kurukshetra university, kurukshetra. the scientific name and tested parts of the 8 plants are detailed in table 1. four different solvents, namely, ethanol, methanol, acetone, and aqueous (hot and cold), were used for extraction and plant extracts were prepared according to the methods described by sharma.. in the previous study, microbiological analysis of fruit juices was done by serial dilution agar plate technique. on the basis of percentage of occurrence of microorganisms in juice samples, one gram - positive bacterium, one gram - negative bacterium, one yeast, and two moulds bacterial strains were identified on the basis of gram staining and biochemical and molecular characteristics (16s rrna sequencing). yeast was identified on the basis of staining, morphological, cultural characteristics, and molecular characteristics (28s rrna sequencing). moulds were identified on the basis of morphological and cultural characteristics and further identification was confirmed by cabi international mycological institute, uk. two bacteria, namely, serratia (kc67407) and bacillus cereus krc1 (kc67408), one yeast, rhodotorula mucilaginosa (kc67409), and two moulds, aspergillus flavus (504472) and penicillium citrinum (504473), were identified. the bacterial isolates were subcultured on nutrient agar and r. mucilaginosa, a. flavus, and p. citrinum were subcultured on potato dextrose agar and incubated aerobically at 37c and 25c, respectively. bombay, india (nucleotide sequence of all microorganisms has been submitted to genbank database which provided the genbank accession number, kc67407kc67409 ; international mycological institute reference number). the acetone, methanol, ethanol, and hot and cold aqueous extracts of different plants were used for evaluation of antimicrobial activity by the agar well diffusion method. in this method, a pure isolate of bacteria and yeast was grown on na and pda plates and incubated at 37c and 25c for 24 h and 72 h, respectively. one plate of each microorganism was taken and colonies were transferred into normal saline (0.85%) under aseptic conditions. density of each microbial suspension was adjusted to be equal to that of 10 cfu / ml (standardized by 0.5 mcfarland standard) and to be used as the inoculum for performing an agar well diffusion assay. 100 l of the inoculum of each test organism was spread onto the agar plates so as to achieve a confluent growth. the agar plates were allowed to dry and 8 mm wells were made with a sterile borer in the inoculated agar plates. the dried extracts were reconstituted to 20% in dimethyl sulfoxide (dmso) to the final concentration of 100 mg / ml for the bioassay analysis. a 100 l volume of each extract was propelled directly into the wells (in triplicate) of the inoculated agar plates for each test organism. the plates were allowed to stand for 1 h at room temperature (40c) for diffusion of the extract into agar and incubated at 37c and 25c for 24 h and 72 h, respectively. sodium benzoate (100 mg / ml) was used as positive reference standards to determine the sensitivity of each microbial species tested. the antimicrobial activity, indicated by an inhibition zone surrounding the well containing the extract, was recorded if the zone was greater than 8 mm. the experiments were performed in triplicate and the mean values of the diameter of inhibition zones standard deviations were calculated. the antimould activity of plant extracts in different solvent was accessed by poison food technique. 100 l of plant extract with concentration of 100 mg / ml was poured into sterile petri plate (90 mm diameter) and 15 ml of molten potato dextrose agar (pda) was added to the petri plate and swirled to achieve a uniform mixture and allowed them to solidify at room temperature. the solidified agar plates were inoculated at the centre with fungal disc (6 mm diameter), obtained from the actively growing one - week - old colony of the test fungus, placed with inoculums side down in the centre of each petri plate aseptically and incubated at point 25c for 7 days. dmso was used as negative control and chemical preservative sodium benzoate served as the positive control. the antifungal activity of each extract was evaluated by measuring the radial growth of fungus in terms of diameter and expressed as percentage mycelia inhibition determined by applying the following formula [3537]:(1)inhibition of mycelia growth%=dcdtdc100,where dc is average diameter of fungal colony in negative control plates and dt is average diameter of fungal colony in extract added plates. minimum inhibitory concentration (mic) for each test organism was determined by the broth macrodilution method. the experimental results were repeated thrice in triplicate each time and expressed as mean sd and results were statistically evaluated using spss software version 16 at 5% significant level. the dietary herb and spices are used as food additives in foods not only to improve the sensory characteristics of food but also to increase the shelf life by reducing or eliminating survival of pathogenic bacteria. in the present study, perusal data in table 2 revealed the mean diameters of the inhibition zones of all plant extracts against two bacteria and yeast and percentage mycelia inhibition against two moulds. there was significant variation (p < 0.05) observed between acetone, methanol, ethanol, cold aqueous, and hot aqueous solvents for the antimicrobial activities of each of the tested plant extracts and microorganisms. for b. cereus (kc67408), the diz values of 18 extracts (accounting for 45% of the 40 tested extracts) were between 19.6 mm and 29.3 mm and those of 16 extracts (40%) were between 12.3 mm and 19.3 mm. however, 6 extracts had no inhibitory activity. ethanolic extract of c. asiatica exhibited the strongest antibacterial activity (diz = 29.3 mm), followed by methanolic extract (diz = 26.3 mm) and ethanolic extract of r. serpentina (diz = 25.3 mm) (table 2). for serratia (kc67407), a total of 25 extracts (62.5%) exhibited inhibitory activity with diz values ranging from 11.3 mm to 22.6 mm, and 47 extracts had low activity. the remaining 15 extracts had no inhibitory activity (table 2). the sample with strongest antimicrobial activity was methanolic extract of t. arjuna (diz = 22.6 mm), followed by acetonic extract of r. serpentina (21.3 mm). for r. mucilaginosa (kc67409), the diz values of 9 extracts (22.5%) ranged from 13.3 mm to 25.3 mm (table 2). ethanolic extract of c. asiatica showed the strongest inhibitory activity (diz = 20.6 mm), followed by methanolic extract of t. arjuna (19.3 mm). of the two moulds tested, for a. flavus, the percentage mycelial inhibition of 8 extracts (20%) varied from 20.6 mm to 26.3 mm. nine extracts (22.5%) had low inhibitory activity (percentage mycelial inhibition ranging between 11.6 mm and 19.3 mm). ethanolic extract of c. asiatica exhibited the strongest antimould activity (percentage mycelial inhibition = 26.3 mm), followed by methanolic extract of c. asiatica and t. arjuna (24.6 mm). for p. citrinum, 8 extracts exhibited high inhibitory activity (percentage mycelial inhibition = 19.6 mm24.3 mm) and nine extracts possessed less inhibitory activity (percentage mycelial inhibition ranging between 10.3 mm and 19.3 mm), while the other 23 extracts had no inhibitory activity. methanolic extract of c. asiatica (24.3 mm) showed the strongest inhibitory activity, followed by ethanolic extract of c. asiatica and w. somnifera (23.3 mm). literature search revealed the in vitro and in vivo antimicrobial activities of plant extracts and essential oils against food borne pathogens but they are difficult to compare owing to the use of different methods of extraction, solvents, microbial strains, and antimicrobial test methods [3, 5, 16, 17, 39, 40 ]. in the present analysis, 4 plants, c. asiatica, e. officinalis, m. arvensis, and t. arjuna, exhibited broad spectrum activity against tested microbes. the present results were in line with the observation of earlier workers [57, 1517, 41, 42 ]. methanolic and ethanolic extract of c. asiatica exhibited maximum zone of inhibition against all tested microorganisms in the present investigation. the antimicrobial potential of c. asiatica was observed by arumugam. in a study to check the antimicrobial efficacy of c. asiatica against b. cereus, s. aureus, and pseudomonas aeruginosa in four different solvents such as methanol, chloroform, water, and acetone and they found that methanolic extract of c. asiatica leaves at the concentration of 50 g per ml displayed activity (2427 mm) against all tested microorganisms. acetonic extracts of t. arjuna displayed maximum zone of inhibition (1628 mm) against s. aureus, acinetobacter, p. aeruginosa, and proteus mirabilis in comparison to the other extracts (methanolic, ethanolic, and water extracts) ; however, all the extracts lacked activity against candida albicans. in the present investigation, methanolic and ethanolic extracts of t. arjuna exhibited antimicrobial activity against all tested microbes due to the presence of flavonoids (luteolin) [45, 46 ]. ethanolic extract of e. officinalis leaves showed more activity against p. aeruginosa, proteus mirabilis, s. aureus, and b. cereus in comparison to ethanolic extract of m. arvensis leaves at the concentration of 100 mg / ml. however, in the present investigation, the ethanolic extracts of m. arvensis displayed more activity in comparison to e. officinalis ethanolic extract at the concentration of 100 mg / ml. the antimicrobial potential of e. officinalis and m. arvensis is attributed to the presence of hydrolysable tannins and menthol, respectively. although, in present investigation, r. serpentina and w. somnifera revealed antibacterial and antimould activity but lacked antiyeast activity, several authors confirmed antibacterial and antifungal potential of w. somnifera and r. serpentina [31, 33, 49, 50 ]. sunilson. observed the antimicrobial activity of c. longa and z. officinalis in four different solvents (petroleum ether, chloroform, methanol, and water) against food borne pathogens. the solvent extracts of c. longa and z. officinalis displayed antibacterial and antiyeast activity ; however, in present investigation, c. longa and z. officinalis possessed antibacterial activity. the extraction of biologically active compound from plant material is largely dependent on the type of the solvent used in the extraction procedure. the present study revealed that the organic extracts provided more powerful antimicrobial activity compared to aqueous extracts. among the organic extracts, alcoholic extracts displayed the best antimicrobial activity in comparison to acetonic extracts (table 2), thus substituting the findings of earlier workers who rated methanol and ethanol as best solvent for the extraction of antimicrobial compounds from plants followed by acetone and water [5, 15, 16, 25 ]. several compounds, tannins, flavonoids, coumarins, thiosulfinates, glucosinolates, and saponins, isolated from these plants are secondary metabolites which are responsible for the antimicrobial and medicinal properties of plants [2, 3, 8, 17 ]. b. cereus, serratia, r. mucilaginosa, a. flavus, and p. citrinum revealed varying sensitivities to the 40 tested extracts (table 2). with respect to b. cereus, thirty - four extracts showed inhibitory activity and 16 extracts had no activity. in case of serratia seventeen extracts showed inhibitory activity against two moulds (a. flavus and p. citrinum). based on the inhibitory activity of plant extract, gram - positive bacteria b. cereus demonstrated more sensitivity to the extracts than gram - negative bacteria and fungi. the present findings are consistent with the results obtained by earlier workers who reported that plant extracts are more active against gram - positive bacteria than against gram - negative bacteria [3, 5, 16, 17, 48, 5153 ]. this is attributed to the differences in the outer layers of gram - negative and gram - positive bacteria. gram - negative bacteria possess an outer membrane and a unique periplasmic space not found in gram - positive bacteria [5, 54, 55 ]. the minimum inhibitory concentration was determined for 31 active plant extracts which show antimicrobial activity against microbes associated with juices (table 3). the mic of the 31 active plant extracts showed that mic ranged between 3.12 mg / ml and 50 mg / ml for the organic extracts and between 25 and 50 mg / ml for the aqueous extracts. when comparing the mic values of the tested plants, the ethanolic extract of c. asiatica appeared to be the most effective with the lowest inhibitory concentration being 3.15 mg / ml against b. cereus. methanolic extracts of c. asiatica, m. arvensis, and r. serpentina were the second most effective plant extracts with mic of 6.25 mg / ml. many plant extracts show mic in the range of 2550 mg / ml and were thus shown to be less effective against microbes isolated from juices. it has been established that mic results do not always correlate well with the diz values but, in present investigation, the observation of mic related with the diz value might be due to the adoption of disc diffusion assay for both antimicrobial activity and mic determination of plant extracts. similar results of mic of different plants in different solvents have been observed by several authors against different microbes [3, 5, 913, 16, 56, 57 ]. the results of present work established that all the tested plant extracts possess antimicrobial activity against selected microbes associated with juices. alcoholic extracts of medicinal herbs such as c. asiatica, t. arjuna, and r. serpentina displayed better antimicrobial activity than chemical preservative sodium benzoate ; therefore these plant extracts have the potential to extend the shelf life or they are used as natural preservatives in fruit juices. | in the present investigation, comparison of antimicrobial activities of different spices, curcuma longa, zingiber officinale, and mentha arvensis, and medicinal herbs, such as withania somnifera, rauvolfia serpentina, emblica officinalis, terminalia arjuna, and centella asiatica, was evaluated. different extraction solvents (acetone, methanol, ethanol, and water) were used and extracts were examined against bacillus cereus, serratia sp., rhodotorula mucilaginosa, aspergillus flavus, and penicillium citrinum isolated from juices. extracts from the medicinal herb and spices have significant activity. b. cereus was the most sensitive and r. mucilaginosa was the most resistant among the microorganisms tested. ethanolic and methanolic extract of c. asiatica displayed maximum diameter of inhibition zone against bacteria and yeast and percentage mycelial inhibition against moulds. this study confirmed the potential of selected extracts of spices as effective natural food preservative in juices. |
while these drugs kill tumor cells, they can also cause adverse effects during their use, such as nausea, vomiting, diarrhea, constipation, alopecia, fatigue, skin alterations, and neurotoxicity, among others. chemotherapy - induced peripheral neuropathy (cipn) is a nuisance and a limiting side effect for patients, since the symptoms of pain, dysesthesia, and paresthesia generated by chemotherapy can mean a reduction in the functionality and quality of life for cancer patients. moreover, these side effects may compromise the treatment of the disease and lead to reduced doses of drugs or treatment discontinuation2, 3. the current recommendations of the american society of clinical oncology (asco)4 point out that there are not yet any strongly recommended drugs available to prevent cipn, given the lack of consistent and high - quality research on the issue. chus5 review about the effects of antidepressant drugs and anticonvulsants for the control of cipn confirm asco s data. therefore, the treatment of these disorders could ideally be done without the use of drugs by applying non - pharmacological treatments, which would have the advantage of not compromising the different body systems already burdened by chemotherapy. transcutaneous electrical nerve stimulation (tens) has arisen as an alternative to drug treatment for the control of peripheral neuropathies. the rationale for the use of tens is based on studies showing that electrical stimulation has shown a positive effect on rat s nerve regeneration, revealing a higher number of myelinated fibers, axon density, and blood vessels in the total nerve area compared to control groups6. when using transcutaneous electrical stimulation in mice s peripheral nerve injuries, the density, fiber diameter, and degree of myelination are also similar to animals that have not suffered injuries7. a case - control study evaluated the effect of tens on peripheral neuropathic pain induced by chemotherapy in patients with different types of cancer, pointing out beneficial effects in reducing pain during tens application without generating side effects8. silva9 used tens in the treatment of intercostobrachial pain developed after breast cancer surgery to show that, in addition to pain relief, tens produced changes in somatotopic organization in the parietal cortex after application, causing increased attention to painful processes. other symptoms of cipn, such as paresthesia and decreased quality of life of these patients, have not been evaluated in previous controlled studies. therefore, this study aims to evaluate the effects of tens on symptoms of numbness and tingling and the frequency of symptoms and its impact on the daily life activities of patients with cipn. this two - arm clinical trial was randomized, placebo - controlled, and double - blind. it was conducted in cancer patients who developed symptoms of cipn after the first chemotherapy cycle. the patients were recruited in barretos cancer hospital, institute of cancer of the state of so paulo, and so francisco university hospital. this study was approved by the research ethics committee of the faculty of medicine, university of so paulo, under protocol n 341/13. it was registered at clinicaltrials.gov before the research began, under registration number ntc 02107417. all participants were informed about the objectives of the study and signed a consent form to participate in the study. the study included patients between the ages of 18 and 70 who met the following inclusion criteria : being subjected to chemotherapy treatment with drugs with a high to moderate degree of neurotoxicity, experiencing neuropathic pain and/or peripheral sensory neuropathy of grade i or grade ii on the common terminology criteria for adverse events (ctcae) scale, exhibiting symptoms of peripheral neuropathy after the first cycles of chemotherapy with at least two points on the visual analog scale of paresthesia and/or pain, and having a present performance status of ecog 2 (karnofsky50%). exclusion criteria were : having sensitivity disorders before chemotherapy treatment, having skin lesions on the site of application of the electrodes, exhibiting cognitive difficulties or trouble understanding how to fill out questionnaires, or being cardiac pacemaker users or diabetes mellitus patients. the study sample consisted of 24 patients with diagnoses of breast and colorectal cancer undergoing a chemotherapy treatment containing in its protocol the chemotherapeutic agents paclitaxel or oxaliplatin. patients were divided into 2 groups : active tens (gat) and placebo tens (gpt). the flow diagram of the research the entry of patients in each experimental group was performed randomly by the site www.random.org before the start of treatment. patients were subjected to the following tens (ibramed, neurodyn porttil) parameters : symmetrical biphasic waveform with modulating frequencies ranging from 7 hz to 65 hz, a pulse width of 200 s at the highest tolerable current intensity with an application time of 60 minutes. the stimulation was performed with individual self - adhesive electrodes with an area of 5 cm in the region where the patient had symptoms of neuropathy, typically, the ends of the upper limbs and/or lower limbs. in the application of active tens, the patient was instructed to increase the stimulation intensity each time the intensity reduced during the 60 minutes of application10. in tens placebo application, patients received the same schedule of active tens, but the equipment was produced to apply the current for only 30 seconds and then the intensity began to decrease during the next 15 seconds until reaching zero11. we used the following instruments for data collection : -sociodemographic and clinical data form to characterize the sample profile with the following data : gender, age, race, education, type of cancer, occupational status and disease status. tnm rating (t, refers to the size and extent of the main tumor ; n refers to the number of nearby lymph nodes that have cancer ; m refers to metastasis), treatments conducted (surgical, radiotherapy, hormonal, chemotherapy, other types of medication, acupuncture) and affected segment by neuropathy (upper limb and/or lower limb) were also used to create the profiles. items of the questionnaire used to assess the symptoms of cipn were paresthesia (grade i, ii, and iii), peripheral sensory neuropathy (i, ii, iii, iv, and v), and dysesthesia (grade i, ii, and ii). -performance status scale ecog (eastern cooperative oncology group) to assess the functional capacity of oncologic patients, with the following scores and activity levels : 0) completely active ; able to perform all their activities without restriction. (karnofsky 90100%) ; 1) restriction of rigorous physical activities, but able to perform light work and of a sedentary nature (karnofsky 7080%) ; 2) able to perform all self - care, but unable to carry out any work activity but able to remain standing approximately 50% of waking hours (karnofsky 6050%) ; 3) capable of only limited self - care, confined to bed or chair more than 50% of waking hours karnofsky 3040%) ; 4) completely unable to perform self - care, totally confined to bed (karnofsky 1020%)12. -chemotherapy induced neurotoxicity questionnaire (cinq) for the evaluation of symptoms of pain and dysesthesia and paresthesia of upper and lower limbs. cinq is an instrument containing 20 items to evaluate the symptoms divided into 2 categories : symptoms of acute and chronic neuropathy in the lower extremities (10 items) and symptoms of acute and chronic neuropathy in upper extremities (10 items). this questionnaire evaluates the frequency of symptoms (cinqa) and the symptoms interference with patients daily activities (cinqb). questionnaires were administered before the treatment (assessment 1 : ax1) and after 15 (ax2), 30 (ax3), and 45 (ax4) days of treatment. the data were presented using measures of central tendency (mean and median) and dispersion (standard deviation) for quantitative variables and relative percentages for categorical variables. pearson s test was used to verify the association between the categories of the study and, when necessary, resorted to fisher s exact test. comparisons of the vas scores between groups were performed using the nonparametric statistical mann - whitney u test and comparison of vas along with the assessments by friedman anova. the q - cochran test was used to determine differences in the cinq responses among the evaluations (ax1, ax2, ax3 and ax4). the comparison of the sums of scores of cinq questions among the assessments was done using hierarchical linear models adjusted for restricted maximum likelihood. the mean age in ptg was 46.3 13.7 and 52.7 9 in atg, without significative differences between them. assessing the functional status of the active tens group patients, we found that 10 patients were between 0 and 1 ecog degrees, with no or some functional limitations, while only 1 had more marked limitations related to the inability to work. in the placebo tens group, 8 patients had ecog 01 and 5 ecog 2. the sample characteristics are summarized in table 1table 1.patient characteristicsplacebo tens (n=13)active tens (n=11)total (n=24)%nn%n%genderfemale215.4327.3520.8male1184.6872.71979.2marital statussingle430.800.0416.7married861.51090.91875.0divorced17.719.128.3widowed00.000.000.0race / ethnicitywhite646.2981.81562.5asiatic00.000.000.0black323.100.0312.5admixture430.7218.2625.0educationelementary or less430.8872.71250.0middle school646.219.1729.2high school215.419.1312.5college or higher17.619.128.3worksyes323.1545.5833.3no1076.9654.51666.7stopped workingyes969.2654.51562.5no430.8545.5937.5fisher s exact test. the experimental groups showed no significant differences with regard to gender, marital status, race, education, employment status, or reductions in their occupational activity due to the treatment. table 2table 2.distribution of characteristics of the type and cancer stage according treatment groupstens placebo (n=13)tens ativo (n=11)total (n=24)n%n%n%type of tumorbreast969.2436.41354.2colorectal430.8763.61145.8tt100.019.114.2t2646.2218.2833.3t3646.2763.61354.2t417.619.128.3nn0430.8545.5937.5n1646.2545.51145.8n2323.019.0416.7n300.000.000.0mm0646.2981.81562.5m1538.5218.2729.2m300.000.000.0mx215.300.028.3 test pearson, p>0.01 shows the disease characteristics among groups in relation to cancer type and disease stage. no significant differences were observed between the groups in terms of these characteristics (p>0.05). test pearson, p>0.01 the data relating to the type of chemotherapy performed, different types of therapeutic treatments undertaken by the patient, and the region in which the patient developed neuropathy are presented in table 3table 3.distribution of the type of treatments performed by patients and the body region affected by cipn between groupsplacebo tensactive tenstotaln%n%n%chemotherapyadjuvant1076.9763.61770.8neo - adjuvant215.4436.4625.0paliative17.700.014.2surgeryyes1184.6763.61875.0no215.4436.4625.0radiotherapyyes215.4218.2416.7no1184.6981.82083.3hormone therapyyes215.4218.2416.7no1184.6981.82083.3immunotherapyyes00.000.000.0no13100.011100.024100.0acupunctureyes17.700.014.2no1292.311100.02395.8other medicationsyes753.8763.61458.3no646.2436.41041.7involvementmmss969.2763.61666.7mmii430.8436.4833.3fisher s exact test, p>0.05. no significant differences were observed in the distribution of the characteristics between the groups in any of the variables described (p>0.05). fisher s exact test, p>0.05 table 4table 4.comparison of mean and standard deviation of the frequency of symptoms (cinqa) and interference of symptoms in patients daily activities (cinqb) during treatment with tens in the experimental groupsplacebo tens active tens time groupcinqaax 113.53 (9.29)12.18 (8.28)0.59ax 211.06 (5.02)8.80 (4.87)ax 39.63 (8.46)8.67 (4.33)ax 46.87 (6.40)7.00 (4.31)cinqbax 19.61 (7.73)11.27 (8.66)0.85ax 28.18 (6.61)7.59 (4.67)ax 38.46 (8.28)6.43 (4.19)ax 45.50 (5.93)7.00 (4.58)p - value related to hierarchical linear mixed effect models. ax- (assessment) shows the behavior of cinq scores over the application of tens during the 45 treatment days for the placebo and active groups, indicating that there was no difference between groups during the treatment performed in any assessment carried out, both in terms of the frequency of symptoms (p=0.5906) and daily living activities (0.8565). table 5table 5.comparison of vas of pain and numbness / paresthesia intra- and inter - group during tens treatment in experimental groupsplacebo tensactive tens median (iqr)median (iqr)pain vas ax10.0 (0.00.0)0.0 (0.05.0)pain vas ax20.0 (0.00.0)0.0 (0.01.0)pain vas ax30.0 (0.00.0)0.0 (0.01.0)pain vas ax40.0 (0.02.0)0.0 (0.01.0)p value0.690.632vas numb / pares ax12.0 (2.06.5)4.0 (1.07.0)vas numb / pares ax25.0 (0.08.0)1.0 (1.03.0)vas numb / pares ax31.0 (0.05.0)5.0 (1.07.0)vas numb / pares ax41.5 (0.03.8)2.0 (1.02.0)p value0.240.01iqr interquartile range (25th. 75th percentile);p values from mann - whitney u test for comparison between assessmentp values from friedman test for comparison between groups shows the comparison of vas of pain and paresthesia intra- and inter - group during treatment with tens in the experimental groups. 75th percentile) ; p values from mann - whitney u test for comparison between assessment p values from friedman test for comparison between groups the results of this study show that the use of tens with modulating frequencies between 7 hz to 65 hz indicated no significant difference in the symptoms of paresthesia and the impact of these symptoms on the daily activities among the groups who received applications of active or placebo tens. these symptoms, however, do not increase during the three cycles subsequent to the onset of chemotherapy neuropathy symptoms. these data corroborate the study of wolf13 and pachman14, indicating that numbness and tingling are the most frequent symptoms of cipn. the hands were more affected than the feet by the symptoms of neuropathy, which was corroborated by zanville s study15. review16 states that one of the assumptions for the development of cipn in hands and feet relates to the fact that these neurons have long axons and are highly dependent on transport proteins and other nutrients to work correctly. taxanes can lead to disruption of microtubules and mitochondria, leading to a deficit in cell nutrition and subsequent cell death. platinum - based drugs can also contribute to cell death by inhibiting adn synthesis. the effects of tens on nerve regeneration in rats show a positive effect of low frequency tens in this process, presenting density, fiber diameter, and a degree of myelination of neurons similar to the uninjured rats of the control group7. it has been hypothesized that these results relate to the effect of tens in the modulation of ca2 + levels and of injured neurons through voltage - dependent channels after nerve injury, factors that would contribute to a decrease in the frequency of the firing of these neurons and stimulate production of neurotrophic factors (bdnf, ngf, and vegf) and other transcription factors17. in our study, we use modulating frequencies ranging between 7 and 65 hz. as there are no studies evaluating the symptoms of paresthesia and the impact of symptoms on daily activities, we could not compare the results of our study with others to verify if other tens frequencies would produce better effects on the outcomes. an interesting finding of our study is that there was no significant difference between the active tens and tens placebo groups in the studied variables. it was expected that the symptoms of paresthesia and pain would get worse during the chemotherapy cycles and this may not have happened due to the fact that patients expected to receive treatment for neuropathy symptoms resulting from chemotherapy. this effect was also observed in rakel s study11 for post - surgery pain due to total knee arthroplasty. worsening neuropathy symptoms would be expected since the development of symptoms may occur after the first chemotherapy cycle, worsen after performing subsequent cycles, and even after the end of treatment18. pachman14, 19 points out that the development of neuropathy after the first treatment cycle with oxaloplatin is a risk factor for the worsening of these symptoms in subsequent cycles. patients treated with paclitaxel also have worsening neuropathy symptoms over the course of chemotherapy cycles20. the fact that the patients in this study have not presented worsening symptoms may show a tens placebo effect on cipn. studies show that tens placebo operates effectively in modulating pain11, 21. as the current in placebo tens is applied in the same way as in active stimulation, but for a period of 30 seconds and then decreasing for the next 15 seconds until reaching zero intensity, the fact that the stimulation performed with placebo tens mimics the application of the active tens may have contributed to this placebo effect. the application of tens placebo thus carried out is important since many patients have heard that tens produces a feeling of a small electric shock and the intensity decreases as the application time goes on. some studies, conducting the placebo tens without electrostimulation (patient does nt feel any sensation), show a significant difference between the group of active tens and the placebo stimulation group in the studied parameters, in favor of the active tens22, 23. other studies should be performed with high or low frequency tens to verify these effects. clinical studies show an efficiency of application of transcutaneous electrical stimulation on the quality of life of patients with cipn24, 25 ; however, these studies were not placebo - controlled, not allowing for the analysis of the placebo effect on quality of life. our study found no improvement in the impact of cipn in the daily activities of patients between the active tens groups and placebo group, suggesting that the placebo effect may also have contributed to the fact that there was no impairment in the performance of daily life activities of these patients over the course cycles of chemotherapy. our study has some limitations, such as not accompanying the patients throughout the chemotherapeutic treatment and not evaluating the effect of tens on other types of cancer and neurotoxic drugs. the application of tens with modulating frequencies between 7 hz to 65 hz in patients with breast and colorectal cancer showed no significant difference in terms of the symptoms of paresthesia and the impact on daily life activities between the groups that received application of active tens and those that received a placebo for chemotherapy treatments with oxaloplatin and paclitaxol. there was no worsening of symptoms in subsequent cycles of the onset of cipn symptoms. | [purpose ] the aim of this double - blind, randomized and placebo - controlled study is to investigate the effects of transcutaneous electrical nerve stimulation for reducing the side effects of chemotherapy - induced peripheral neuropathy in cancer patients undergoing chemotherapy with oxaloplatin or paclitaxel. [subjects and methods ] twenty - four patients were randomly allocated into two groups : active or placebo stimulation. all patients were assessed for pain, numbness / tingiling, frequency of symptoms, and quality of life. the transcutaneous electrical nerve stimulation device was applied daily with modulating frequencies ranging between 7 hz and 65 hz in distal limb regions during three cycles of chemotherapy (45 days). the other stimulation parameters were : pulse duration of 200 sec, intensity at the highest tolerable level, and increases in intensity when it diminished. [results ] the data showed no difference between active or placebo groups in terms of pain, numbness / tingling, frequency of symptoms or impact on daily life activities. [conclusion ] these results suggest that transcutaneous electrical nerve stimulation applied in the frequency variation mode was not proven to be effective to improve the symptoms of chemotherapy - induced peripheral neuropathy during chemotherapy cycles. there was no worsening of symptoms in subsequent cycles of the onset of symptoms of the disease. |
endoscopic submucosal dissection (esd) was developed in japan for the treatment of early gastric cancer, and it is now also widely used to treat superficial esophageal cancer. since esd enables the removal of large cancerous lesions from the superficial layer of the esophagus, the indications for endoscopic dissection in guidelines for the diagnosis and treatment of esophageal cancer have been revised : the size limitation for removable lesions (two - thirds of the circumference) by endoscopic techniques has now been lifted. on the other hand, because esophageal stenosis is almost inevitable after the removal of subcircumferential and circumferential lesions, esophageal wall perforation during endoscopy and endoscopic balloon dilatation for the alleviation of stenosis is of particular concern in some cases. here we present a case where conventional endoclip closure of a large perforation of the stenotic esophagus was not possible and non - surgical closure was achieved using the over - the - scope - clip (otsc) system (ovesco endoscopy, gmbh, tuebingen, germany). a 64-year - old woman presented with subcircumferential superficial esophageal carcinoma three - fourths the circumference of the esophagus in the upper thoracic esophagus (fig. the depth of tumor invasion was evaluated as ct1a - mm by white light observation, narrow band imaging with magnifying endoscopy, and endoscopic ultrasound. preoperative diagnosis was 0-iic, ct1a - mm, 40 mm, cn0, cstage 0. according to the japanese guidelines for the diagnosis and treatment of esophageal cancer, the cancer board for esophageal cancer of our hospital decided to perform esd. after obtaining informed consent, esd was performed using an it knife nano (kd-612, olympus medical systems, tokyo, japan). 1b) was necessary and to prevent the likely onset of stenosis, she received a local injection of triamcinolone acetonide (100 mg) into the submucosa underlying the ulcer immediately after esd and was started on oral predonine therapy (30 mg / day) 1 week after esd, which was tapered over a 2-month period. histopathology findings indicated squamous cell carcinoma (0-iic type, t1a - mm, ly1, v0, hm(), and vm()). additional treatment was considered because of the high risk for lymph node metastasis suggested by a positive finding of lymphatic involvement.fig. 1endoscopic submucosal dissection (esd) and an ulcer showing stenosis and perforation. a superficial esophageal carcinoma about three - fourths the circumference of the esophagus located in the upper thoracic esophagus. b immediately after esophageal endoscopic submucosal dissection (esd), circumferential dissection was performed. d large perforation due to the endoscope penetrating the wall in the upper - right side of the stenotic area endoscopic submucosal dissection (esd) and an ulcer showing stenosis and perforation. a superficial esophageal carcinoma about three - fourths the circumference of the esophagus located in the upper thoracic esophagus. b immediately after esophageal endoscopic submucosal dissection (esd), circumferential dissection was performed. d large perforation due to the endoscope penetrating the wall in the upper - right side of the stenotic area two months after esd, upper endoscopy revealed a residual ulcer, but no stenosis. distal end external diameter, 9.8 mm) was inserted to traverse the stenosis but it penetrated the wall through to the mediastinum, leaving a perforation in the upper - right side of the stenotic area (fig.. attempted closure with an endoclip (olympus medical systems, tokyo, japan) was unsuccessful due to the large size of the perforation and fragility of the ulcer floor. the branches of the otsc twin grasper were released to grasp the sides of the lesion. because the perforation hole had gaping edges, i opened one branch and grasped one edge of the perforation first. then i opened the other branch and maneuvered the grasper to the other edge and grasped it, and then retracted into the attachment to place the clip (fig. the patient fasted and received antibiotics, and her only complaint was mild chest discomfort. she did not develop fever, and both her leukocyte count and c reactive protein level were within normal range. contrast radiography performed 5 days after the otsc procedure revealed no leakage (fig. oral intake of food and fluid was resumed and 3 weeks after contrast radiography her condition was sufficiently improved. after discussing the option of surgical treatment which is standard for her with our thoracic surgeon, the patient declined surgery and opted instead for crt. because re - examination by ct revealed no metastasis, preventive crt was considered still effective, despite treatment was delayed by 1 month. months after completing chemotherapy, the endoscope with a conventional - sized distal tip (9.8 mm) could traverse the site despite the presence of minor stenosis (fig. the patient reported no difficulties with nutritional intake. because mild chest discomfort continued due to the otsc a the over - the - scope - clip (otsc) system showing the otsc twin grasper with its branches open outside the endoscope and two clips (image reproduced with the permission of century medical inc., b the perforation was closed by clipping the muscular layer with the otsc systemfig. 3non - contrast radiography showing the otsc system in use (left) and contrast radiography showing no leakage (right)fig. the endoscope could traverse the site despite the presence of minor stenosis suturing devices and perforation closure. a the over - the - scope - clip (otsc) system showing the otsc twin grasper with its branches open outside the endoscope and two clips (image reproduced with the permission of century medical inc., b the perforation was closed by clipping the muscular layer with the otsc system non - contrast radiography showing the otsc system in use (left) and contrast radiography showing no leakage (right) endoscopic image taken 2 months after completing chemotherapy. the number of cases of intraoperative perforation has increased as esophageal esd has becomes more widespread, and perforation is frequently caused by endoscopic balloon dilatation used to alleviate stenosis, a complication of endoscopic dissection of large lesions. conservative therapy, such as perforation closure with an endoclip or placement of a nasogastric tube, is effective when the perforation is relatively small. most perforations can typically be treated conservatively by fasting and administration of antibiotics. with a large perforation, serious mediastinitis and potentially fatal empyema may develop and closure is therefore crucial but often difficult. the otsc system is a new endoscopic tool used for non - surgical treatment of gastrointestinal perforation, fistula, and anastomotic leak. it is the first effective system for closing a fistula endoscopically and it is therefore attracting interest. to our knowledge, this is the first report of the otsc system being used to treat a large perforation of a stenotic lesion that developed after esophageal esd. perforation of large lesions like this, which will likely have hard fibrotic edges and fragile surrounding tissue due to steroid treatment, occasionally need to be treated by surgical drainage or thoracotomy because of the difficulties in closing them with conventional endoclips. the present case demonstrates that the otsc system can be used to close a large perforation of a stenotic site with good postoperative progress. rotation of the otsc twin grasper is not perfect and the target tissue sometimes can not be grasped. also, once clips are placed, they are difficult to remove. special care is required when employing the otsc system in organs with a narrow lumen, such as the esophagus and duodenum, because of the possible risk of stenosis. despite these limitations, the otsc system allows for the closure of large perforations not amenable to closure with conventional methods. as demonstrated in the present study, the otsc system has good utility in difficult cases and thus warrants consideration. | we report here a case of esophageal perforation made by an endoscope while treating cicatrical stenosis that developed after wide circumferential dissection of superficial esophageal carcinoma. perforation closure with a conventional endoclip was difficult as the perforation was large and the surrounding tissue was fragile as a result of steroids administration for stenosis prevention. to avoid surgical intervention, we employed the over - the - scope - clip system and successfully closed the perforation. the favorable outcome suggests the utility of the over - the - scope - clip system for closing perforations when conventional methods are ineffective. |
antimonide semiconductors have potential application in a wide range of electronic and opto - electronic devices due to their unique band - structure alignments, and small effective mass as well as high mobility for electrons [1 - 4 ]. while recent technical advancements have enabled high quality lattice matched gasb epitaxy on native substrates, for many applications gaas substrates are desirable. this is because of the following reasons : gaas is inexpensive, has favorable thermal properties, transparent to more (long wave length) active regions, forms excellent n and p ohmic contacts, and can be semi - insulating compared to gasb. however, the high (7.8%) lattice mismatch between the gasb epilayer and the gaas substrate complicates the growth of sophisticated device structures. currently, this mismatch is accommodated via metamorphic buffer layers and strain - relief superlattices. in metamorphic buffer layer approach, initially the strain within the critical thickness is accommodated by tetragonal distortion followed by defect formation and filtering. while this approach has enabled a number of device demonstrations, it exhibits several deficiencies such as the necessity to grow thick buffer layers (often > 1 m), poor thermal and electrical conductivity, and has resulted in significant material degradation through the presence of threading dislocations (tds). recently, a fundamentally different growth mode, interfacial misfit dislocation (imf) growth mode, has been developed by our group. in this growth, the strain is relieved instantaneously at the mismatched heterointerface unlike the traditional tetragonal distortion approach that relieves the strain after reaching a critical thickness. buffer - free approach to realize monolithic high quality gasb deposited on gaas substrate with exceptionally low threading dislocation (td) densities (~10 cm), despite the high lattice mismatch. the strain created due to the 7.8% lattice mismatch is relieved at the gasb / gaas interface by the formation of a two - dimensional (2d), periodic imf arrays comprised of pure - edge (90) dislocations along both and [1 - 10 ]. to facilitate the growth of buffer - free deposition of gasb on gaas substrate with low td densities, in complex device structures, it is essential to understand the structural properties of imf - grown gasb epitaxial layers. an attempt was made previously to show the proof of existence of the imf array at the gasb / gaas interface along [1 - 10 ] using cross - sectional transmission electron micrograph (xtem) and to calculate the td density using koh etching as shown in ref.. the xtem images look only at one - dimensional sections and hence are not representative of the 2d interface. also, the quantitative analyses like strain relaxation of bulk gasb deposited on gaas substrates, long - range uniformity of the imf array in 2d, and accurate td density calculation for gasb that was not presented earlier, are very important in realizing high quality gasb bulk layers on gaas substrate. in this study, all the issues addressed earlier, namely the material quality of the gasb epitaxial layer is quantified using various analyses like xtem, selective area electron diffraction (saed) double spot pattern, moir fringe patterns, x - ray diffraction (xrd), and plan - view tem. the samples are grown on gaas substrates in a vg v80h molecular beam epitaxy (mbe) reactor equipped with valved crackers for as and sb, and an optical pyrometer for monitoring the substrate temperature. various samples comprising gasb bulk layers are grown on gaas substrates, using imf growth. the thickness of the imf - grown gasb epitaxial layers used for various analyses range from 15 nm to 5 m. for example, thick samples like 5, 0.5 m are used for xrd analyses, and samples with medium thickness, like 120 nm, are used for xtem and saed analyses, respectively. for td density analysis using plan - view tems, very thin 15-nm sample is grown separately for moir fringe analysis to facilitate the transmission of electrons through both the epitaxial layer and the underlying substrate. the sample required for moir fringe analysis is prepared as follows, the substrate is lapped down to ~10 m and ion milled to 30 nm, resulting in a net thickness of 45 nm that includes the 15-nm imf - grown gasb epitaxial layer. another set of gasb bulk samples, which are similar to those of the imf samples are deposited using non - imf growth on gaas substrate for comparison with the former in various analyses as mentioned earlier. if the interface is as - rich instead of ga - rich prior to the deposition of gasb, no imf is observed at the heterointerface and this growth mode is called non - imf growth mode. non - imf growth is also similar to that of the imf growth up to the deposition of gaas smoothing layer. after the smoothing layer, ga source is turned off and the as - overpressure is on while bringing the temperature down to 510 c from 560 c. when the substrate temperature is 510 c, the resulting surface is as - rich. at this point,, imf is not formed at the interface as is explained in the following paragraphs. figure 1shows the high - resolution tem (hr - tem) image of the gasb / gaas interface. the burgers circuit completed around each misfit indicates a pure - edge dislocation along [1 - 10 ]. one of such misfit dislocations are shown in fig.1as a bright spot representing the imf dislocation. hence the dislocation network associated with the imf array formation along both and [1 - 10 ] is characterized as a 2d lomer dislocation network. in general, relaxation kinetics favors the formation of 60 dislocations over 90 dislocations as the former dislocation can glide to the surface from the interface. however, the latter is more preferable as it is more efficient in relieving the strain compared to the 60 dislocations and can be formed under favorable conditions as shown in fig. burgers circuit completed around one misfit dislocation of the imf array at the gasb / gaas interface shown with the help of hr - tem image, where the dislocation is shown as abright spot figure 2a shows the bright - field xtem image of a 120-nm td free imf - grown gasb epitaxial layer on a gaas substrate along zone axis. the imf is seen as dark spots in this figure with a periodicity of 5.6 nm. this periodicity corresponds to exactly one misfit dislocation for every 14 lattice sites of gaas or 13 lattice sites of gasb. this value is in good agreement with the theoretical periodicity for a relaxed gasb deposited on gaas. the strain created by the lattice mismatch is relieved spontaneously by the formation of the imf at the gasb / gaas interface. further proof of spontaneous relaxation of imf - based samples is provided via the saed double spot pattern as shown in fig. the highly resolved diffraction spots in saed demonstrate two separate lattice constants associated with gaas (as = 5.65) and gasb (af = 6.09), respectively. the alignment of the 000 diffraction spot with, for instance, the two 220 spots indicates that there is no lattice rotation. in the imf growth, a sheet of sb atoms are deposited on ga - rich gaas surface before starting the growth of bulk gasb epitaxial layer. if sb is deposited on as - rich gaas surface instead of ga - rich gaas surface, the resulting epitaxial layer will have high defect density as shown in the bright - field xtem of fig. axtem showing a periodic imf array with a periodicity of 5.6 nm, asdark spots, at the gasb / gaas interfacebsaed double diffraction pattern of imf growth mode, andcxtem of non - imf growth mode with high threading dislocation density compared to the imf growth mode the -2 scan of symmetric (004) xrd spectra for a 0.5- m thick gasb epitaxial layers deposited using imf and non - imf growths, and 5- m thick sample deposited using imf growth are shown in fig. in addition to the broad full width at half maximum (fwhm), the non - imf spectrum differs to the imf spectrum due to the presence of additional peak near the gaas substrate as shown in fig. this additional peak in the non - imf sample is attributed to the tetragonally distorted gasb. this means that initially the in - plane lattice constant of the epitaxial layer and of the substrate are equal up to critical thickness, after which the epitaxial layer slowly relaxes to the original lattice constant of gasb by relieving the strain via the formation of misfit and often threading dislocations. in non - imf spectrum, this transition of lattice constant is represented by a negative slope via the transition from additional peak to the epi - peak. similar type of behavior was not observed in the imf samples, and hence no tetragonal distortion is attributed to the imf - grown gasb epitaxial layers. the relaxation of the imf - grown gasb epitaxial layer is determined from the analysis of xrd. the calculation based on the symmetric (004) and asymmetric (115) xrd measurements show approximately 98.5% (complete) relaxation of the gasb epitaxial layer, and similar type of relaxation is observed in gasb grown on gaas with alsb nucleation layer. we believe that the broad fwhm (194 arcsecs) of gasb layers, thinner than 1 m, as shown in fig. 3a is due to the small amount of residual strain (< 2%) in the epitaxial layers after the creation of the imf array. as per our observations, with thicker layers (5 m) the fwhm decreases considerably to ~20 arcsecs in imf - grown gasb epitaxial layers as shown in fig. 3b. xrd (004) scan ofa0.5 m gasb on gaas substrate grown using imf and non - imf growth mode, illustrating highly relaxed gasb for the imf growth, andb5 m gasb on gaas substrate showing a narrow fwhm of ~20 arcsecs for the gasb epitaxial layer figure 4a, b show the bright - field plan - view tems imaged along zone axis for the center and edge of the imf sample, respectively. the average td density was calculated to be 10 cmfrom the plan - view tems. even though, no tds are observed at the center, very few tds are observed at the edge of the imf sample and are attributed to the un - optimized imf growth at sample edges. using the plan - view tem images, the dislocation density has been calculated based on the number of dislocations within the unit area from several wafer surfaces. in the non - imf grown gasb layers, td density is measured to be ~10cmas shown in bright - field plan - view tem shown in fig. this confirms the fact that the td density is reduced in the imf growth compared to the non - imf growth due to spontaneous strain relaxation. also no 60 dislocations were observed in imf - grown gasb, which indicates that the imf dislocations are non - interacting and pure - edge (90) 2d arrays. since the 90 dislocations can relieve strain almost completely at the interface, high quality buffer - free gasb epilayers can be deposited monolithically on gaas substrates in the imf growth. plan - view tem showing tds fromacenter, bedge of the imf sample, andccenter of the non - imf sample for a 5 m gasb epilayer on a gaas substrate figure 5a, b shows the two - beam bright - field plan - view temg.3g[g = (220) and (2 - 20) ] obtained from gasb epitaxial layers deposited on gaas substrates using the imf growth. these tems show moir fringe patterns, which are the interference patterns that are formed when two crystals with different orientations or lattice constants overlap, thus providing an excellent indication of whether the epitaxial layer is strained. the moir fringes shown here are translational moir fringes as the planes and therebygvectors are parallel to each other. moir fringe spacing, which is defined as the spacing between two consecutive white or dark lines is measured to be 2.8 nm from fig. 5a, b. the theoretical spacing for translational moir fringes is given by :, wheredis the inter - planar spacing assuming thatdgasb = 2.155 nm anddgaas = 0.1999 nm for { 220 } reflections and is calculated to be 2.75 nm. the measured value of 2.8 nm is in good agreement with the theoretical spacing, which again indicates that the film is fully relaxed. plan - view tems showing moir fringes of 2d imf arrays alongab[1 - 10 ], andc2d lomer dislocation network along both and [1 - 10 ] measured using diffraction vectors (220), (2 - 20), and both (220) and (2 - 20), respectively. consecutivewhiteanddark linesrepresent moir fringes, and thewhite circlesrepresent the edge dislocations moir fringes are often used to identify dislocations in semiconductors [12 - 14 ] as well as metals. 5a, b, indicated by white circles illustrate the projection of pure - edge dislocations and are similar to the observations made by other groups in various material systems. the pure - edge dislocation density from various areas of the moir fringes averages to 6.62 10 cm. the thls in the moir fringes might also represent tds as shown in ref.. the tds revealed in this way are attributed to the half - period shifts in the moir fringes, which are produced as a result of the interaction between 60 and 90 dislocations. however, no half - period shifts are observed in the moir fringes of imf - grown gasb samples as shown in fig. 5a, b. moreover, no 60 dislocations are observed in the imf sample, which are considered to be the main source for the formation of td when the former interacts with the 90 dislocations. generally, distortions local to the interface, such as stacking faults are revealed as displacements in moir fringes. in this study, displacement of the moir fringes is not observed in the imf samples, hence stacking faults or partial dislocations are not ascribed to the imf growth. the moir fringes are imaged along both and [1 - 10 ] using (220) and (2 - 20) g vectors as shown in fig. the projection of 2d lomer dislocation network is observed to be uniform over a large area that was imaged (0.72 m). in conclusion, high quality buffer - free gasb is grown on gaas substrates with very low td densities (~10 cm) despite the high (7.8%) lattice mismatch. the strain due to lattice mismatch is relieved immediately at the gasb / gaas heterointerface with the help of periodic, pure - edge misfit (imf) arrays of dislocations along both and [1 - 10 ] in the imf - grown gasb. instead, if the gasb is deposited using a non - imf growth, the resulting epitaxial layer has very high td density (10 cm) due to buildup of strain in tetragonal distortion. comparing the imf and non - imf samples using xrd and xtem analyses have shown that the strain is completely (98.5%) relieved in imf sample, whereas it is not the case for non - imf sample. the plan - view tem analysis for both samples also confirmed similar results, where the td density is very low for imf sample (~10 cm) compared to non - imf sample (~10 cm). the long - range uniformity and the strain relief of the imf - grown gasb epitaxial layer measured using the moir fringe patterns have shown a uniform 2d lomer dislocation network over the entire scan area. the moir fringe spacing of 2.8 nm agrees well with the theoretical spacing of 2.75 nm, which proves that the gasb layer is completely relaxed. further proof of strain is also achieved from saed measurements, which shows that gasb and gaas has lattice constants almost similar to the expected lattice constants of the corresponding relaxed materials. we believe that this approach is useful for the deposition of buffer - free high quality gasb on well - studied gaas substrates in complex device structures. the authors gratefully acknowledge the financial support of afosr through fa 9550 - 08 - 1 - 0198. | we report structural analysis of completely relaxed gasb epitaxial layers deposited monolithically on gaas substrates using interfacial misfit (imf) array growth mode. unlike the traditional tetragonal distortion approach, strain due to the lattice mismatch is spontaneously relieved at the heterointerface in this growth. the complete and instantaneous strain relief at the gasb / gaas interface is achieved by the formation of a two - dimensional lomer dislocation network comprising of pure - edge (90) dislocations along both [110 ] and [1 - 10 ]. in the present analysis, structural properties of gasb deposited using both imf and non - imf growths are compared. moir fringe patterns along with x - ray diffraction measure the long - range uniformity and strain relaxation of the imf samples. the proof for the existence of the imf array and low threading dislocation density is provided with the help of transmission electron micrographs for the gasb epitaxial layer. our results indicate that the imf - grown gasb is completely (98.5%) relaxed with very low density of threading dislocations (105 cm2), while gasb deposited using non - imf growth is compressively strained and has a higher average density of threading dislocations (> 109 cm2). |
a 20-year - old female with extensive terminal ileal and colonic crohn 's disease was initially successfully managed on a drug regimen of prednisolone and mesalazine which helped to induce remission. this maintained her disease remission but was associated with neutropenic sepsis, necessitating the withdrawal of the thiopurine therapy with subsequent clinical and endoscopic disease flare. this required an infusion dose and frequency escalation to maintain clinical response. after 58 weeks of infliximab therapy, the patient was requiring 10 mg / kg 6 times weekly and hence was transferred to the fully human anti - tnf- injection adalimumab for loss of response to infliximab. initially this strategy regained disease control, but due to relapse at 40 mg every other week, the dose was increased step - wise to 80 mg weekly which fortunately maintained remission. during her treatment with adalimumab the presence of purple - coloured plaque - like lesions primarily on her abdomen figure 1 demonstrates the presence of psoriasis which is presumed to be a result of treatment with adalimumab. tnf- has been identified as a key cytokine in the pro - inflammatory pathways that drive diseases such as inflammatory bowel disease and rheumatoid arthritis. anti - tnf- agents such as infliximab and adalimumab are widely used in the treatment of these conditions and have been shown to have an impact on pro - inflammatory and apoptotic pathways. they are also effective in the treatment of the dermatological and rheumatological manifestations of psoriasis. however, there have been a number of cases reported where such agents may be responsible for new onset or worsening of psoriasis. a study of 126 patients who underwent treatment with anti - tnf- inhibitors demonstrated evidence of psoriasis in 76 individuals, palmoplantar pustular psoriasis in 37 cases and psoriasis of the nails in 6 cases. there is mounting evidence that a key innate immune pathway for triggering common human autoimmune diseases, including psoriasis, involves plasmacytoid dendritic cell precursors and type 1 interferon (ifn) production. a recent study involving immunohistochemical staining of skin biopsy specimens for myxovirus - resistance protein a (mxa, a surrogate marker for lesional type 1 ifn activity) demonstrated increased staining in tnf- inhibitor - induced psoriasis compared with psoriasis vulgaris. research suggests that a psoriasiform eruption during anti - tnf- treatment seems to be a class effect without any confirmed predisposing factors and does not specifically require treatment discontinuation. further studies are however needed to determine the exact mechanism of and appropriate treatment for anti - tnf--induced psoriasis. | anti - tnf- agents are currently utilised for the treatment of a vast array of autoimmune conditions including inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis and psoriasis. it is however noted that such therapeutic strategies have been linked to the specific induction of cutaneous - based reactions such as dermatitis, erythema multiforme and psoriasis. here we present the case of a young female patient with crohn 's disease who developed psoriasis following treatment with the anti - tnf- drug adalimumab and highlight the possible pathogenetic mechanisms involved in such an occurrence. |
according to classic anatomical textbooks, the suprascapular artery is the first (lower) branch of the thyrocervical trunk and runs transversely in the posterior cervical triangle, parallel, behind, and above the clavicle. the artery first courses laterally, in front of the scalene anterior muscle and the phrenic nerve and then crosses anterior to the third part of the subclavian artery and the primary cords of the brachial plexus. then, it continues backward and skirt the inferior belly of the omohyoid muscle to reach the superior border of the scapula, where it normally passes over the superior transverse scapular ligament (stsl). by this way, it reaches the supraspinous fossa and continues to the infraspinous fossa through the spinoglinoid notch. at the dorsal scapula, it creates an important anastomotic network with the dorsal scapular artery (also known as deep branch of the transverse cervical artery) and the circumflex scapular branch of the subscapular artery. normally, the suprascapular artery joins the suprascapular nerve near the middle half of the superior border of the scapula. the nerve passes under the stsl and through the suprascapular notch, before entering the supraspinous fossa. at the dorsal scapula the suprascapular artery and nerve run together supplying the same targets (supraspinatus and infraspinatus muscles). the suprascapular notch is located at the superior border of the scapula, medial to the base of the coracoid process, and is converted into a foramen by the stsl. variations concerning the origin and course of the suprascapular artery are numerous and well described in the literature. a common trunk providing the suprascapular and the superficial cervical (also known as transverse cervical) artery has been described in 30% of the individuals, whereas in 28% of the cases both arteries arise directly from the thyrocervical trunk (the transverse cervical artery, whenever it exists, provides both the superficial cervical and the dorsal scapular arteries). the origin of the suprascapular artery from different segments of the subclavian artery or from the axillary artery has been reported with a median incidence of 10%. in the most common type, the artery arises from the subclavian artery, with an incidence between 2.8% and 21.3%. the suprascapular artery might also emerge from the internal thoracic artery (1 - 5.1%), from the costocervical trunk (1%), or from the dorsal scapular artery. there has also been a report of two cases of duplicated suprascapular arteries existing bilaterally one rising from the thyrocervical trunk and one rising from the third part of the subclavian artery. in both cases, the artery with normal origin had also a normal course, whereas the other one passed below the transverse scapular ligament. the vein normally drains the same region as the corresponding artery, passes through the suprascapular notch, and finally flows into the external jugular vein (as a rule). this anatomic region (dorsal scapula) may also be drained by the dorsal scapular vein (deep branch of the transverse cervical vein flowing into the jugular vein) and the circumflex scapular branch of the subscapular vein. in the present study, after a thorough examination of the suprascapular artery in a sample of greek (caucasian) origin, we describe one case of anomalous origin of the suprascapular artery from the third segment of the subclavian artery, combined with anomalous course under the transverse scapular ligament. moreover, surprisingly enough, no suprascapular vein was detected, a very rare finding, which has never been mentioned in the available literature. this specific arterial pattern is considered of major clinical importance since it can be related to undiagnosed shoulder pain and neurogenic malfunction of the shoulder 's rotator cuff. the anatomy and course of the suprascapular artery were carefully examined in 31 embalmed adult human cadavers of caucasian (hellenic) origin (16 male and 15 female). the age of the specimens ranged between 39 and 98 years (average age 77.5 years, sd = 11.107, se = 1.385). all the cadavers were derived from body donation with informed consent, written and signed with signature authentication by the donator himself. all donated cadavers were used ; consequently, our specimens may be considered as randomly selected. the proper dissection to reach and enlighten the suprascapular artery, without damaging the structures of the corresponding area, must follow this order : platysma, external jugular vein, sternocleidomastoid and omohyoid muscles, internal jugular vein, vertebral artery, subclavian artery, thyrocervical trunk, suprascapular and superficial (or transverse) cervical arteries, phrenic nerve, anterior scalene muscle, primitive trucks of the brachial plexus, suprascapular nerve and vein, continuation of the subclavian artery, stsl, and the suprascapular notch. all the distances were measured using calipers. to obtain the distances between the vessels, the center of the origin of each vessel the left suprascapular artery presented the usual described course and origin in all cadavers. on the right side of only one female cadaver (deceased at the age of 88 years), we observed at the right posterior cervical triangle an anomalous origin of the right suprascapular artery. the artery emerged from the third segment of the subclavian artery, very close to the first segment of the axillary artery and 4.7 cm distal to the origin of the thyrocervical trunk [figure 1 ]. because of this origin external to the scalene anterior muscle, the suprascapular artery did not cross superficially the phrenic nerve and the muscle, as usual, but it ran immediately in front of the brachial plexus and under the suprascapular nerve. the artery continued parallel to the nerve until it reached the suprascapular notch [figure 1 ]. there, the suprascapular artery and nerve passed under the stsl into the suprascapular notch (converted by the ligament into a foramen). the stsl was found ossified to a great extent, converting the notch into a foramen. the ossification was examined physically : the stsl was harder than a fibrous tissue when pushed and squeezed ; the percussion of the stsl with forceps created a pitched sound as if hitting a rock or bone and not a dull sound. inside this foramen, the artery ran in front of the nerve. the suprascapular artery (spa) originates from the third segment of the subclavian artery (sa), very close to the first segment of the axillary artery (aa) and passes under the superior transverse scapular ligament (stsl), together with the suprascapular nerve (sn) and over the brachial plexus (bp). tca : transverse cervical artery ; om : omohyoid muscle the frequency of the above - described variation was thus 1.6% (1/62 sides). the deviation from the normal pattern is not unusual,[18 ] as the subclavian artery and different types of common arterial trunks had been described at different locations of origin. in the present study the suprascapular artery constituted a branch of the thyrocervical trunk in all but one case (1.6%, 1/62) where it emerged from the third segment of the subclavian artery (right side of a female cadaver), very close to the first segment of the axillary artery. according to the available literature, this type of origin seems to be one of the rarest. mishra and ajmani reported that the suprascapular artery originated from the first part of the axillary artery with an incidence of 1.6% (1/60 sides, variation observed on the left side), whereas adibatti and prasanna observed the same variation in one cadaver (male, left side) out of 30 studied. additionally, mahato recorded a single case of anomalous bilateral origin of the suprascapular artery from the third part of the axillary artery. extensive studies have been carried out on the subligamentous course of the suprascapular artery. according to tubbs., the suprascapular artery, accompanied by the suprascapular nerve, passes under the stsl with a median incidence of 2.5% (3/120). in one (male) cadaver, classified the arrangement of the suprascapular vessels into three types : in type i (59.4%), all suprascapular vessels ran over the stsl ; in type ii (29.7%), the vessels ran over and under the stsl simultaneously (at least one vessel passed under or over the stsl) ; and in type iii (10.9%), all vessels ran under the stsl. furthermore, three cases of the suprascapular artery passing through the suprascapular notch during endoscopic suprascapular nerve release have been reported by reineck and krishnan. anomalous course of the suprascapular artery inside the suprascapular notch very frequently coexists with an ipsilateral variation in the origin of the artery. in particular, this variation was combined with anomalous origin from the subclavian or axillary artery. nevertheless, mishra and ajmani observed three cases where the suprascapular artery coursed inside the notch, with only one of them combined with variation in the origin of the artery (1/60 = 1.6%). in the present study, the sole case of unusual origin of the suprascapular artery was indeed combined with its passage under the stsl (1.6%, 1/62). in our case, variation of the artery 's origin and course is accompanied by total absence of the suprascapular vein. this finding is not described in the available literature. according to the available literature, the incidence of complete ossification of the stsl varied from 3.7% to 13.6%. however, complete ossification of the stsl is exceptionally rare in some populations, such as in alaskan eskimos or indians. reported that stsl appeared calcified and rigid (not necessarily completely ossified) in 26.7% of the cases. the calcified stsl is considered to be a sign of entrapment. on the other hand, simultaneous ossification of the coracoid process and epiphysis, as observed mostly in nigerian infants, might change the shape of the notch. the prevailing conditions lead some vessels to enlarge and form definitive channels and others to regress. during this phase of development, it is possible that different patterns in the vessels may appear, including both the origin and/or the course of either arteries or veins. an origin of the suprascapular artery in the vicinity of the thyrocervical trunk (either from the subclavian artery itself or from a common trunk with the transverse cervical artery) is by far the most common variation and at the same time the least interesting one from an anatomical or clinical viewpoint, since in this case the course and relations of the artery are hardly influenced. on the contrary, an origin of the suprascapular artery from the third part of the subclavian artery or from the axillary artery is rarer and clinically significant. furthermore, this variation is frequently accompanied by the passage of the artery through the suprascapular notch. surgeries in the anterior neck and supraclavicular region, such us radical and modified neck dissections to control the lymphogenous spread of head and neck cancer, may require ligation of the suprascapular artery. thus, knowledge of the possible variations in the origin and the course of the artery are very important. clavicular fractures are fairly common and most often occur in the middle third of the bone.[2224 ] the suprascapular artery supplies with blood the proximal 4/5 of the clavicle and constitutes the exclusive blood supply for the middle 1/3 of the clavicle. on doing so, it runs parallel, above and close to the clavicle, but not in touch with it. a direct origin of the suprascapular artery from the middle of the subclavian artery coursing below the clavicle merely enhances the probability of arterial damage during fracture of this bone. the seemingly illogical origin of the suprascapular artery from the thyrocervical trunk (since it results in its greater length) may be attributed to an effort to minimize the danger of its injury. if the suprascapular artery rises from an anomalous position it is possible that its unusual origin and course could either increase or decrease the danger of being damaged by a broken clavicle, according to the vicinity of this origin with the middle of the bone. in our case, the suprascapular artery emerged very close to the first segment of the axillary artery and almost behind the external third of the clavicle, which means that the usual middleclavicular break would not pose great danger to the artery. however, if the suprascapular artery arises from the internal thoracic, its ascending course behind the middle third of the clavicle could be extremely dangerous not only for the nutrient wigs of the bone but also for the whole suprascapular artery. inside the suprascapular notch, this may lead to friction of the nerve, inflammation, and finally constriction of the nerve, leading to suprascapular neuropathy. factors such as ossification of the stsl and anomalous course of the suprascapular artery under the ligament could lead to a faster and more severe constriction of the nerve, since they reduce the capacity of the notch. chronically, this could lead to atrophy of the supraspinatus and infraspinatus muscles, limited decreasing abduction, and external rotation of the shoulder and chronic deep - seated pain in the shoulder that is aggravated with movement (suprascapular neuropathy symptoms). suprascapular neuropathy may of course occur by other mechanisms as well : for example, by mikroembols in the vasa nervosum of the suprascapular nerve engendered through a damage of the suprascapular artery. unexplained shoulder pain, namely pain not due to arthritis (inflammation of glenobrachial or acromioclavicular joints), or malfunction of the shoulder 's rotator cuff must lead to investigation of an eventual neuropathy. noticeably, in our case, in which the suprascapular vein is absent, the probability of a suprascapular neuropathy was reduced, since the suprascapular notch contained two items (artery and nerve) instead of three. however, the pulsing artery might produce minor annoyance to the nerve, especially when it contacts its sensitive fibers. in a sample of 31 cadavers, we found a suprascapular artery rising in an anomalous way from the third part of the subclavian artery in the right side of only one cadaver (1.6%, 1/62). however, the sample (62 sides) is not big enough to identify the prevalence of the specific variation in the general population. the suprascapular artery exhibited a variation in its course, too, as it passed through the suprascapular foramen together with the suprascapular nerve. both the variations in the origin and the course have already been reported in the past and should be considered as independent variations since they do not always appear together in the same individual. however, the variations in the origin of this artery are numerous and usually coexist with an abnormal course underneath the stsl. surgeons and orthopedics should be familiar with these variations, which may complicate their task. | background : variations concerning the origin and course of the suprascapular artery are numerous and present important clinical implications.aim:in the present study the origin and course of the suprascapular artery are investigated in a sample of greek (caucasian) origin.materials and methods : the anatomy and course of the suprascapular artery were carefully examined in 31 adult human cadavers (16 male and 15 female).results : anomalous origin of the suprascapular artery from the third segment of the subclavian artery was observed in the right side of only one female caucasian specimen (1/62 = 1.6%). the suprascapular artery and the suprascapular nerve passed together under the superior transverse scapular ligament through the suprascapular notch, whereas the suprascapular vein was absent.conclusion:according to the available literature, this type of variation in the origin of the suprascapular artery is considered rare. this variation is clinically important, since it is related to the creation mechanism of suprascapular neuropathy and has also obvious surgical implications. the variation is embryologically enlightened and has an interesting ontogenic aspect. |
a three - month - old male presented with cough, sputum, and intermittent cyanosis that had lasted for one week. a postoperative esophagogram showed a bronchoesophageal fistula, and a secondary operation involving fistulectomy and right lower and middle bilobectomy was performed. nevertheless, he required right pneumonectomy due to recurrent pneumonia in the remaining right upper lobe. on readmission five months after the pneumonectomy, a chest computed tomography (ct) scan revealed post - pneumonectomy changes, with mediastinal shift to the right and collapse of the posterior wall of the trachea compressed by the aortic arch (fig. aortopexy was performed, followed by insertion of a tissue expander into the right pleural cavity. after the insertion of the tissue expander, 45 ml of normal saline was instilled, and the thoracotomy wound was closed. the patient was discharged 29 days after the operation. at a one - year follow - up, he had no symptoms. a 22-month - old female with esophageal atresia (type c) underwent fistula resection and an esophageal anastomosis. a fistulectomy between the right intermediate bronchus and the distal esophagus was performed, as well as right pneumonectomy for persistent collapse of the right lung. twenty - two months after the pneumonectomy, she experienced unspecific respiratory symptoms of cough and sputum for five days. a chest ct scan demonstrated mediastinal shift to the right and a narrowed left main bronchus, which was compressed between the left main pulmonary artery and the descending thoracic aorta (fig. we performed mediastinal repositioning by inserting a tissue expander with 120 ml of saline. during the first two postoperative days she required ventilator therapy, and venovenous extracorporeal membrane oxygenation the patient was discharged on the 77th postoperative day, following successful weaning from extracorporeal membrane oxygenation. at a one - year follow - up, the patient showed no symptoms and a chest x - ray showed no specific findings. a three - month - old male presented with cough, sputum, and intermittent cyanosis that had lasted for one week. a postoperative esophagogram showed a bronchoesophageal fistula, and a secondary operation involving fistulectomy and right lower and middle bilobectomy was performed. nevertheless, he required right pneumonectomy due to recurrent pneumonia in the remaining right upper lobe. on readmission five months after the pneumonectomy, a chest computed tomography (ct) scan revealed post - pneumonectomy changes, with mediastinal shift to the right and collapse of the posterior wall of the trachea compressed by the aortic arch (fig. aortopexy was performed, followed by insertion of a tissue expander into the right pleural cavity. after the insertion of the tissue expander, 45 ml of normal saline was instilled, and the thoracotomy wound was closed. the patient was discharged 29 days after the operation. at a one - year follow - up, he had no symptoms. a 22-month - old female with esophageal atresia (type c) underwent fistula resection and an esophageal anastomosis. a fistulectomy between the right intermediate bronchus and the distal esophagus was performed, as well as right pneumonectomy for persistent collapse of the right lung. twenty - two months after the pneumonectomy, she experienced unspecific respiratory symptoms of cough and sputum for five days. a chest ct scan demonstrated mediastinal shift to the right and a narrowed left main bronchus, which was compressed between the left main pulmonary artery and the descending thoracic aorta (fig. we performed mediastinal repositioning by inserting a tissue expander with 120 ml of saline. during the first two postoperative days she required ventilator therapy, and venovenous extracorporeal membrane oxygenation the patient was discharged on the 77th postoperative day, following successful weaning from extracorporeal membrane oxygenation. at a one - year follow - up, the patient showed no symptoms and a chest x - ray showed no specific findings. postpneumectomy syndrome (pps) is a rare disorder involving the extrinsic compression of the trachea or bronchus due to shifting of the mediastinum and over - inflation of the remaining lung parenchyma. it results in a counterclockwise rotation of the heart and great vessels, along with herniation of the remaining lung into the contralateral hemithorax. pps is more common in children, occurring in up to 15% of patients who undergo right pneumonectomy (or, rarely, left pneumonectomy in patients with a right aortic arch). the frequency with which this syndrome occurs in infants and young children is thought to be related to the increased elasticity and pliability of their lungs and mediastinum, compared with those of adults. they have more pliable and mobile mediastinal tissue than older patients, with a greater tendency to shift that can easily lead to anatomic distortion. pps is characterized by progressive dyspnea, cough, and recurrent pneumonia at least six months after pneumonectomy. because of the rarity and unspecific symptoms of the disease, most patients already have bronchomalacia by the time of diagnosis. various procedures have been proposed, including phrenectomy or crushing of the phrenic nerve, tracheal or bronchial transection with anastomosis, suspension of the aorta to the sternum, and endoscopic bronchial stent insertion [14 ]. more recently, surgical repositioning of the mediastinum with a non - absorbable material has been widely accepted as a standard treatment. many studies have reported that the insertion of an intrathoracic prosthesis can improve clinical symptoms and relieve functional obstructions. shen. reported that placement of a saline - filled tissue expander led to successful mediastinal repositioning. at a median follow - up of 32 months, 10 of 13 patients (77%) reported a significant improvement in their symptoms, with a relatively low operative mortality rate of 27.8%. the early insertion of a tissue expander when pneumonectomy is performed may prevent the possible detrimental consequences that result from the delayed diagnosis and treatment of pps. furthermore, some reports have described an inability to place a tissue expander due to postoperative adhesion when the pps is developed after pneumonectomy. choi. have suggested that prophylactic implantation of a tissue expander may prevent the potentially very serious complication of pps. in our two cases, tissue expanders improved the ventilation of the remaining lung by alleviating the obstruction of the trachea or bronchus and shifting the mediastinum to the midline. although our follow - up duration was short, we observed decreased respiratory compromise and no complications involving leakage or rupture related to the tissue expander. in our opinion, pps can be completely and successfully treated using a tissue expander, which can restore the position of the mediastinum. | postpneumonectomy syndrome (pps) is a rare late complication of pneumonectomy. it occurs more often in children than in adults, and is characterized by respiratory failure resulting from bronchial compression caused by severe mediastinal shift. various methods have been used to treat pps, including aortopexy and the insertion of plastic balls, silastic implants, and saline - filled breast prostheses. we describe two cases of pps corrected with tissue expanders after right pneumonectomy in patients with esophageal atresia. |
treating complex regional pain syndrome (crps) is difficult because it still does not have a recommended therapy. a 29-year - old man was diagnosed with crps after surgery on his 4 and 5 left toes 7 years ago. though he had undergone diverse pain treatment, the symptoms persisted, so he visited dunsan korean medicine hospital of daejeon university. this case report presents results on the effect of bee venom pharmacopuncture in treating patient with crps. bee venom pharmacopuncture (bvp), 0.15 to 0.4 ml dosage, was administered at gb43. the symptoms were evaluated using a numeric rating scale (nrs) and the dosage of pain medicine. on the first visit, he was taking an anticonvulsant, a trycyclic antidepressant, and an analgesic. on the nrs he also complained of severe pain and hypersensitivity when the 4 and the 5 toes were touched just slightly. after treatment, on the nrs, the score for toe pain was 0, and he no longer needed to take pain medication. during the 4-months follow - up period, he has remained without pain ; neither have additional symptoms appeared nor adverse events occurred. complex regional pain syndrome (crps) is a neuropathic pain disease, which is the name now given to group of conditions previously described as reflex sympathetic dystrophy (rsd), algodystrophy, sudeck s atrophy. this chronic pain syndrome appears after big and small tissue damage, such as a sprain, a fracture, neurologic damage, spinal cord injury, cerebral infarction and myocardial infarction. these conditions share a number of clinical features, including spontaneous pain that is mostly on the limbs, associated allodynia, hyperalgesia, autonomic changes, trophic changes, and functional loss. the diagnosis of crps had been based on the criteria of the international association for the study of pain published in 1994. however, due to the lack of specificity and accuracy of diagnosis, the criteria, as revised in 2004, are used instead. the primary medications for treating crps are anti - inflammatory drugs, anticonvulsants, antidepressants, and steroids. for additional pain control, nerve blocks, such as regional nerve blocks and sympathecotomies, and interventional treatment, such as spinal cord stimulation, are used together. though various remedies exist, the therapeutic responses to traditional treatments are relatively poor, and because of the complicated symptoms, a diverse approach is necessary. besides the traditional therapy, patients with crps recently treated in china were reported to have been improved by 80%, and that improvement continued for more than 20 months. also, chronic crps patients treated with acupuncture have been reported to show reduced pain and depression and enhanced quality of life. in additional to international studies on acupuncture, cases using anti - inflammatory pharmacopuncture, harpagophytum radix. pharmacopuncture, and placental extracts have been reported in korea. in this research study, we describe the case of a patient with crps who responded to bee venom pharmacopuncture (bvp) with impressive and sustained results. this patients was a 29-year - old male with a history of congenital polysyndactylia of the 4 and the 5 toes. he had undergone a polydactyly resection, separation of syndactyly, and split thickness skin - graft on march 14, 2006 and surgery on the necrosis site on april 20, 2006. after joining the army in 2006 and complaining of difficulty with military life because of the congenital polysyndactylia, he underwent resection, separation of syndactyly, on his 4 and 5 toes. after the surgery he reported pain and paresthesia on those toes and was diagnosed with crps ; however, according to the operation notes, no neurologic damage had occurred. afterward the diagnosis, he was treated with anti - inflammatory drugs, anticonvulsants and antidepressants and had undergone nerve blocks 10 times before being examined at our clinic. when the pain worsened, he was prescribed a narcotic analgesic, in spite of which he complained of pain of more than 5 on the numeric rating scale (nrs), depression, and anxiety. although he had received treatments such as medications and nerve blocks, the pain had not decreased under a nrs score of 5, even reaching a nrs score of 8 on occasion. also, long - term therapy and medication had led to depression and anxiety. on the first visit, he was taking an anticonvulsant (gabapentin) 2700 mg / day, a trycyclic antidepressant (amitriptyline hydrochloride) 10 mg / day, and an analgesic (tramadol hydrochloride) 50 mg as required. at the first examination in our clinic, he complained of severe pain and hypersensitivity when his 4 and 5 toes were touched just slightly and of dyspepsia, rash, and depression. during the treatment period at our clinic, which ran from january 2, 2014, the patient first visited dunsan korean medicine hospital on december 14, 2013, and started receiving injection of sweet bvp from january 2, 2014. the acupuncture point was gb43, near the tender point, and the dosage of bee venom was gradually increased from 0.15 to 0.4 ml. the initial plan was once a week for 10 weeks, but as the treatment progressed, we added the treatment period was extended to 14 weeks. at the first treatment, the dosage of the bvp was 0.15 ml, at the second treatment, it was 0.2 ml, and at the third treatment, it was 0.3 ml. as the dosage of 0.3 ml induced no side effect, the dosage was kept at 0.3 ml until the 8 treatment. for the remaining treatment after the 8, the irritation decreased, but no change in the nrs score was noted. on the 2 day of treatment, the patient reduced gabapentin from 2700 to 900 mg / day, though the nrs score still persisted. on the 4 day of treatment, the average nrs score declined from 5 to 3, and the sharpest pain appeared just once or twice a week, with rare sudden pain. on the 5 day of treatment after that treatment, the patient experienced pain to a regular degree without sudden severe pain. after the 7 day of treatment, he reduced the dosage of gabapentin from 900 to 600 mg / day for 2 weeks, but upon feeling discomfort, increased it to 900 mg / day again. on the 10 day of treatment, he reduced the dosage to 300 mg / day and experienced no increase in pain after that. while insomnia and anxiety arose without the trycyclic antidepressant, he took additional herbal medicine, ondam - tanggami (wendan - tangjiawei). on the 13 day of treatment, he stopped taking all western medicines. after the 14 treatment, the last injection, the average nrs score was 1 to 2. on the nrs, the patient rated his pain as 0 after the 2-months and the 4-months follow - ups (may 30 and of july 31, 2014). no additional symptoms have occurred (table 1), (fig 2). during the 14 treatments, no side effects due to bee venom especially, at the 11 treatment, the patient noted insomnia and anxiety after discontinuing amitriptyline hydrochloride. he took herbal medicine for 2 weeks, after which those symptoms were relieved, with no further occurrence. after the 14 treatments, the pain had disappeared at the surgical site, and the sensation in the 4 and the 5 left toes was 80% that in the 4 and the 5 right toes (table 1). we administered 0.15 ml of sweet bvp initially, but because the duration of the disease had been long and the pain control had been difficult, the dosage was increased gradually to 0.4 ml (fig 1) we checked for any side effects while increasing the dosage. although the patient felt stiffness at the injection point on the foot, a dosage under 0.3 ml was considered to be appropriate. the initial plan was for one injection to be given each week for 10 weeks, which was based on the experience of the therapist. after 10 treatments and 2 weeks of observation, although his pain level on the nrs was 2 and he felt no inconvenience in daily living, we decided to perform four additional injections until the symptoms disappeared. on the 10 day of treatment, the pain had decreased to an average nrs score of 2, and on the 13 day of treatment, the patient noted pain relief without any medication. especially, on the 14 day of treatment, of the last treatment, pain had disappeared, and just a little paresthesia was noted. we could relieve the symptoms with 10 treatments, but they had completely disappeared after the four additional treatments. thus, we recommend that the treatment plan be based on 10 treatments, with the possibility of about 5 additional treatments, depending on the state of the patient. the patient additionally complained of insomnia and anxiety during the course of treatment, the 11day. he had undergone depression because of persistent pain and failure to control the pain associated with crps. as the insomnia and anxiety were thought to be symptoms caused by discontinuing the nervous system drugs which he had taken for the last 7 years, we administered herbal medicine. he reported that it irritated him, but not strongly, and the insomnia and anxiety disappeared after 2 weeks. a period for discontinuing medication that has been taken for a long time, rather herbal medicine, seems to be needed. no new systemic symptoms appeared following the treatment, and no additional medication was prescribed, except for the herbal medicine ondam - tanggami (wendan - tangjiawei) used for insomnia and anxiety and the intermittent herbal medicine sojukgunbihuan (xiaojijianpiwan) used for dyspepsia. due to the fact that there are few distinct treatments for crps, many studies on anti - inflammatory drugs, nerve blocks, vitamin therapy, and acupuncture for treating patients with crps [11, 12 ]. among those, a study on bee venom therapy is now ongoing, and it has been proven with rodents that bee venom alleviates thermal hyperalgesia. also, a chemotherapy - induced peripheral neuropathic pain model and clinical research [15, 16 ] have already established the effect of bee venom therapy. although these studies are not related to crps, they do show the effectiveness of using bee venom to treat neuropathic disease. thus, using bee venom therapy to treat patients with crps should hold promise. in the present case, bee venom therapy administered to a patient who had been suffering from crps with failure to control its pain for almost 7 years proved to be effective. however, the exact mechanism and therapy methods are beyond the limits of this case report. we particularly suggest further studies on the required length of the treatment and the dosage for bee venom therapy, clinical observations of bee venom therapy, and studies on the mechanisms of bee venom therapy applied to neuropathy. | objectives : treating complex regional pain syndrome (crps) is difficult because it still does not have a recommended therapy. a 29-year - old man was diagnosed with crps after surgery on his 4th and 5th left toes 7 years ago. though he had undergone diverse pain treatment, the symptoms persisted, so he visited dunsan korean medicine hospital of daejeon university. this case report presents results on the effect of bee venom pharmacopuncture in treating patient with crps.methods:bee venom pharmacopuncture (bvp), 0.15 to 0.4 ml dosage, was administered at gb43. the treatment was applied each week for a total 14 times. the symptoms were evaluated using a numeric rating scale (nrs) and the dosage of pain medicine.results:on the first visit, he was taking an anticonvulsant, a trycyclic antidepressant, and an analgesic. on the nrs the worst pain in the toes received a score of 8. he also complained of severe pain and hypersensitivity when the 4th and the 5th toes were touched just slightly. other complaint included dyspepsia, rash, and depression. after treatment, on the nrs, the score for toe pain was 0, and he no longer needed to take pain medication. during the 4-months follow - up period, he has remained without pain ; neither have additional symptoms appeared nor adverse events occurred.conclusion:bvp may have potential benefits for treating patients with crps. |
details of mgs subject recruitment and sample characteristics are provided in the online full methods (section a1). samples (5.3%) were excluded for high missing data rates, outlier proportions of heterozygous genotypes, incorrect sex or genotypic relatedness to other subjects. snps (7% for african american, 25% for european - ancestry and 27% for combined analyses) were excluded for minor allele frequencies less than 1%, high missing data rates, hardy - weinberg deviation (controls), or excessive mendelian errors (trios), discordant genotypes (duplicate samples) or large allele frequency differences among dna plates. principal component scores reflecting continental and within - europe ancestries of each subject were computed and outliers excluded. genomic control values for autosomes after qc were 1.042 for african american and 1.087 for the larger european - ancestry and combined analyses. for mgs, association of single snps to schizophrenia was tested by logistic regression (trend test) using plink10, separately for european - ancestry, african american and combined datasets, correcting for principal component scores that reflected geographical gradients or that differed between cases and controls, and for sex for chromosome x and pseudoautosomal snps. genotypic data were imputed for 192 regions surrounding the best findings, and for additional regions selected for meta - analysis.11 detailed results are available in supplementary datafiles 1 and 2, and complete results from dbgap (www.ncbi.nlm.nih.gov/sites/entrez?db=gap). meta - analysis of the mgs, isc and sgene datasets was carried out by combining p - values for all snps (in the selected regions) for which genotyped or imputed data were available for all datasets, with weights computed from case - control sample sizes. | schizophrenia, a devastating psychiatric disorder, has a prevalence of 0.51%, with high heritability (8085%) and complex transmission.1 recent studies implicate rare, large, high - penetrance copy number variants (cnvs) in some cases2, but it is not known what genes or biological mechanisms underlie susceptibility. here we show that schizophrenia is significantly associated with single nucleotide polymorphisms (snps) in the extended major histocompatibility complex (mhc) region on chromosome 6. we carried out a genome - wide association study (gwas) of common snps in the molecular genetics of schizophrenia (mgs) case - control sample, and then a meta - analysis of data from the mgs, international schizophrenia consortium (isc) and sgene datasets. no mgs finding achieved genome - wide statistical significance. in the meta - analysis of european - ancestry subjects (8,008 cases, 19,077 controls), significant association with schizophrenia was observed in a region of linkage disequilibrium on chromosome 6p22.1 (p = 9.54 109). this region includes a histone gene cluster and several immunity - related genes, possibly implicating etiologic mechanisms involving chromatin modification, transcriptional regulation, auto - immunity and/or infection. these results demonstrate that common schizophrenia susceptibility alleles can be detected. the characterization of these signals will suggest important directions for research on susceptibility mechanisms. |
directed assembly of gold nanorods through the use of dithiolated molecular linkers is one of the most efficient methodologies for the morphologically controlled tip - to - tip assembly of this type of anisotropic nanocrystals. however, in a direct analogy to molecular polymerization synthesis, this process is characterized by difficulties in chain - growth control over nanoparticle oligomers. in particular, it is nearly impossible to favor the formation of one type of oligomer, making the methodology hard to use for actual applications in nanoplasmonics. we propose here a light - controlled synthetic procedure that allows obtaining selected plasmonic oligomers in high yield and with reaction times in the scale of minutes by irradiation with low fluence near - infrared (nir) femtosecond laser pulses. selective inhibition of the formation of gold nanorod n - mers (trimers) with a longitudinal localized surface plasmon in resonance with a 800 nm ti : sapphire laser, allowed efficient trapping of the (n 1)-mers (dimers) by hot spot mediated photothermal decomposition of the interparticle molecular linkers. laser irradiation at higher energies produced near - field enhancement at the interparticle gaps, which is large enough to melt gold nanorod tips, offering a new pathway toward tip - to - tip welding of gold nanorod oligomers with a plasmonic response at the nir. thorough optical and electron microscopy characterization indicates that plasmonic oligomers can be selectively trapped and welded, which has been analyzed in terms of a model that predicts with reasonable accuracy the relative concentrations of the main plasmonic species. |
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appendiceal abscesses represent 2.3% of all the cases of acute appendicitis and usually present a mass in the right lower quadrant. occasionally, abscesses develop in the right subhepatic space, right subdiaphragmatic space and right pararenal space or liver. recently, the ultrasonic or ct guided percutaneous puncture technique has offered an alternative to conventional surgical treatment. we experienced a patient with an epigastric appendiceal abscess, demonstrated by abdominal ct scan and barium enema, which resolved spontaneously. a 49-year - old man was referred to our institution for further evaluation and management of an intraabdominal abscess with the air - fluid level located outside of the greater curvature of the stomach, demonstrated on the abdominal ct scan checked 5 days before the referral (fig 1). he experienced left upper quadrant abdominal pain with fever and chills for 15 days without any preceding trauma and was managed at a private clinic without improvement of symptoms. upon abdominal examination, an adult fist - sized, ill - defined, round, nonmovable and tender mass was palpated at the left epigastrium. no abnormalities were observed in cbc, urinalysis, stool examination, serum amylase and liver chemistry. we treated the patient with intravenous antibiotics and bed rest. on the second day of hospitalization, an gastroscopy was performed which showed a bulging external mass effect with adherent purulent material and mucosal friability around a fistula - like lesion at the posterior wall of the mid - gastric body (fig. 2). we took biopsy specimens which showed necrotic tissues along with an intact gastric mucosa. on the third day of hospitalization, a barium enema was performed and showed a hyperrotated cecum in the left upper quadrant and nonvisualization of the appendix with mucosal irregularity and focal luminal narrowing at the surrounding distal transverse colon on prone position film consistent with a hyperrotated cecum and appendiceal abscess(fig. a follow - up gastroscopy, showed a focal regenerating edematous mucosa without the bulging effect noted in the initial examination (fig. a follow - up ct scan disclosed the abscess pocket had subsided with surrounding inflammatory change only(fig. the patient was discharged on the tenth day of hospitalization and has remained well for over 6 months. the palpable mass associated with acute appendicitis may consist of phlegmon or abscesses of various sizes. 23% of the patients with appendicitis who are admitted to the hospital have abdominal masses and appendiceal abscesses represent 5089% of the patients with an appendiceal mass. complicating abscesses have been reported in 2.3% of all cases of acute appendicitis. patients often have a palpable mass in the right lower quadrant (the appendix mass). rarely does the abscess form in the right subhepatic space, right subdiaphragmatic space, liver or posterior pararenal space distant to the right lower quadrant. therefore, the value of aggressive radiologic work - up and follow - up can not be overemphasized in suspected appendiceal abscesses, especially those present in locations remote from the appendix. jordan has reported that the distinction between an appendiceal mass and an appendiceal abscess could not be made by considering the patient s duration of symptoms, temperature and white blood cell count when the patient was admitted. careful follow - up, routine barium enema study, ultrasonography and abdominal ct scanning would prevent misdiagnosis and delayed treatment. ultrasonography has made it possible to distinguish an abscess from the phlegmon without operation and the abdominal ct scan has proven to be of considerable clinical value in characterizing periappendiceal inflammatory masses and in determining the relative size of the liquefied versus nonliquefied component. despite extensive clinical experiences, the surgical management of appendiceal abscesses remains controversial. however, most authors agree that the initial treatment must be conservative management, including bed rest, nasogastric suction, systemic antibiotics and drainage, rather than early surgery. initial conservative management with intravenous antibiotics, with or without percutaneous drainage of the abscess, is prudent, safe and effective and shows a high success rate (8090%) and low morbidity rate (15%) compared to the high complication rate of early surgery (1550%). guided percutaneous drainage is an effective alternative to surgical drainage nonoperative treatment and, if possible, ultrasonic percutaneous drainage of a verified abscess are safe procedures with few complications and late sequelae. recently, the ultrasonic percutaneous puncture technique has offered an almost atraumatic alternative to conventional surgical treatment. sixty - six percent of the recurrent cases occurred within 2 years of the initial attack. the abscess often spontaneously resolves or drains into the intestine with a low recurrence rate. our case exhibited fever, chills and an epigastric abscess. at first, we suspected a pancreatic abscess because of its location, and managed initially with antibiotics and planned percutaneous drainage. however, the radiological studies, especially the ct scan and the barium enema, revealed an appendiceal abscess in the left upper quadrant of the abdomen due to a hyperrotated ceum. the abscess resolved spontaneously, and we think that the abscess drained into the stomach through a small fistula between the stomach and abscess cavity. the patient has remained well for over 6 months. | the appendiceal abscess is a common complication of acute appendicitis and usually is located in the right lower quadrant of the abdomen. an epigastric appendiceal abscess has never been reported at an unusual location.we experienced an unusual case of a 49-year - old man with an epigastric appendiceal abscess. initially, this abscess was suspected to be a pancreatic abscess. abdominal ct scan and barium enema demonstrated a hyperrotated cecum with an appendiceal abscess in the left upper quadrant of the abdomen. an gastroscopy revealed a small fistula - like lesion with purulent coating at the bulging posterior gastric wall. the abscess resolved spontaneously. we believe that the abscess drained into the stomach through a small fistula between the stomach and abscess cavity. there was no recurrence for over 6 months. |
it is definitely not a matter of dispute that chemotherapy and its constituent cytotoxic agents play a vital role in the clinical management of the vast majority of cancer conditions. chemotherapy measures focus on eradication of tumour presence or (at least) control the degree of tumour progression and metastasis. however, this therapy has its own critical flaws due to two major issues, namely, dose - dependent adverse conditions and the emergence of chemoresistance properties within the tumour. the issue of dose - dependent cumulative adverse effects derives from the pharmacological properties of cytotoxic chemotherapeutic agents, which are not tissue - specific and thus affect all tissues in a widespread manner. in addition, tissues having increased turnover rates, such as the gastro - intestinal system and skin, are more vulnerable to cytotoxic drug activity and are the most prevalent dose - limiting cumulative adverse effects in patients undergoing chemotherapy. table 1 describes in brief the pharmacology and adverse effects of a few of the most commonly prescribed chemotherapeutic agents that are implemented in many cancer chemotherapy strategies. the emergence of chemoresistance within tumour cells of solid tissues is sadly one of the main reasons for treatment failure and relapse in patients suffering from metastatic cancer conditions. resistance of the tumour cell to chemotherapeutic agent exposure may be innate, whereby the genetic characteristics of the tumour cells are naturally resistant to chemotherapeutic drug exposure. alternatively, chemoresistance can be acquired through development of a drug resistant phenotype over a defined time period of exposure of the tumour cell to individual / multiple chemotherapy combinations [1, 2 ] (see figure 1). the biological routes by which the tumour cell is able to escape death by chemotherapy are numerous and complex. however, the major pathways enabling chemoresistance in cancer have been studied in detail and are summarised in table 2. the introduction of nanotechnology in the last few decades has led to an undisputed boom in the conception and development of innovative methods for effective and safe delivery of small - molecule drugs and gene - based therapies to their intended target tissues. the advantages of exploiting nanoparticle delivery systems are many, such as the possibility to protect nuclease - labile drug therapies, such as short interfering rnas (sirnas) and micrornas (mirnas) during transit within the bloodstream [87, 88 ]. in addition, implementation of nanoparticle - based delivery systems has led to improved pharmacokinetic profiles for the specific drug being carried within such a system, together with enhanced targeting of the site of action of the drug [8991 ]. the excellent review by hu and zhang highlighted that nanoparticles also have the capacity to carry combination therapies of two drugs / small molecules and have demonstrated to be particularly effective in circumventing multidrug resistance (mdr) issues in multiple cancer models. the chemical composition of nanoparticles, both from natural occurring compounds (see figure 2) and synthetic ones (see table 3), is varied and the selection of which nanoparticle to utilize for any individual drug delivery system is very much dependent on a multitude of factors such as the chemical nature of the drug to be transported, the loading capacity of the nanoparticle, and resultant pharmacokinetic and pharmacodynamics properties of the nanoparticle following drug loading. it is beyond the scope of this review to delve into the specific technical details regarding each individual type of nanoparticle utilized at present, as this has been already discussed extensively in other technical reviews and research articles within the literature [83, 84, 94, 95 ]. however, a brief summary encompassing the spectrum of varying nanoparticle compositions, key advantages together with toxicity profiles can be viewed in table 3 and figure 3. the study carried out by kang. demonstrated that administration of solid lipid nanoparticles containing doxorubicin (sln - dox) to the adriamycin - resistant breast cancer cell line mcf-7/adr, which also overexpressed p - glycoprotein (p - gp), allowed for chemosensitisation of the cell line. this was induced due to enhanced accumulation of doxorubicin within the cell line, contributed by the nanoparticle - based delivery method, and thus the degree of apoptosis was enhanced. the same principle of exploiting nanoparticle delivery to substantiate chemotherapeutic drug accumulation within the target cancer cell, with the ultimate goal of enhancing tumour chemosensitivity, polymer - cisplatin conjugate nanoparticles were developed and consequently delivered to a2780 human ovarian carcinoma cell line. the added potential of this delivery system relied on the cisplatin analogue prodrug covalently linked to a poly(ethylene glycol)-based polymer, which only released its therapeutic payload in a low ph environment. consequently, clinical administration of such a delivery system would ensure that the drug will remain complexed whilst in transit within the bloodstream due to its neutral ph environment. additionally, rnai therapeutics have come to rely much further on the utilization of nanoparticle delivery systems to exert their biological effects. the study by dickerson. elucidated the efficiency to knock - down genes such as epidermal growth factor receptor (egfr) by the delivery of egfr - specific sirnas contained within core / shell hydrogel nanoparticles (nanogels). the nanogels were also coated with peptides targeting the epha2 receptor to enhance delivery of anti - egfr sirnas within the targeted hey tumour cells. consequently, the knock - down effect on egfr led to enhanced chemosensitivity of cancer cells to taxane chemotherapy. the implementation of nanoparticle technology has also demonstrated to aid the clinical effect of other therapies that were previously unsuccessful due to poor drug delivery issues. developed transferrin conjugated ph - sensitive lipopolyplex nanoparticles with the capacity to bind specific oligodeoxynucleotides (gti-2040 in this case). this delivery system allowed gti-2040 to exert its effect on the r2 subunit of the chemoresistance factor ribonucleotide reductase in acute myeloid leukaemia cell line models. the influence of ultilising such a delivery system was evident in that the 50% inhibitory concentration (ic(50)) for 1 m gti-2040 decreased from 47.69 nm to 9.05 nm. an additional nanoparticle delivery system, adopted against mdr in leukaemic conditions, was investigated by cheng.. this system combined magnetic iron oxide nanoparticles together with daunorubicin and 5-bromotetrandrin, which proved to possess a sustained release pharmacokinetic drug profile when administered to k562/a02 multidrug resistant leukaemic cell lines. the principle behind the utilization of magnetic nanoparticles is due to the effects of magnetic field gradients positioned in a nonparallel manner with respect to flow direction within the tumour vasculature. this allows for physical (magnetic) enhancement of the passive mechanisms implemented for the extravastation and accumulation of such magnetically responsive nanoparticles within the tumour microenvironment, followed by cellular uptake of the nanoparticles within the target tumour cell cytoplasm. the magnetically responsive nanoparticle itself is composed of one or a combination of the three ferromagnetically active elements at physiological temperature, namely, iron, nickel, and cobalt. the delivery system described by cheng. also aided in providing a dose - dependent antiproliferative effect on such cell lines, together with enhanced intracellular accumulation of daunorubicin and downregulated transcript expression of mdr1 gene, the main factor for induction of mdr in most cancer models. these factors all contributed to a reduction in mdr and were directed by the level of endosomal - mediated cellular uptake properties of such nanoparticles. in chronic myelogenous leukaemia (cml), a bcr - abl positive status induces mdr properties through multiple pathways, including resistance to p53 and fas ligand - induced apoptotic pathways. the delivery system devised by singh. consisted of magnetic nanoparticles combined with paclitaxel and was consequently administered to bcr - abl positive k562 leukaemic cell lines. the addition of lectin functional groups to the nanoparticle complex served to aid cellular uptake by the target k562 cell line and also demonstrated a reduction in the ic(50) for paclitaxel within this cell line model. multiple myeloma is an additional tumour model that has seen benefit from the exploitation of nanoparticle technology in its therapeutic avenues. the study by kiziltepe. succeeded in developing a micelle - based nanoparticle delivery system containing doxorubicin and very late antigen-4 (vla-4) antagonist peptides. this delivery method not only accomplished enhanced cytotoxic activity when compared to doxorubicin alone, but also the addition of vla-4 antagonist peptides served well in circumventing the phenomenon of cell - adhesion - mediated drug resistance due to the resultant impaired vla-4 mediated adhesion of multiple myeloma cells to the stroma of bone marrow within cb.17 scid murine multiple myeloma xenograft models. additionally, drug accumulation within the stroma of the multiple myeloma murine xenograft models was also tenfold higher than the control murine model. yet another tumour model that has been investigated for the application of nanoparticle - based chemotherapy, for the purpose of avoidance of chemoresistance, is prostate cancer. gold nanoparticles are an attractive avenue for drug delivery researchers primarily due to their lack of complexity in their synthesis, characterization, and surface functionality. the endosomal - based route for gold nanoparticle cellular uptake can be viewed as the primary advantage for circumventing mdr within the tumour cell, since the drug efflux pump is bypassed and the nanoparticle - held chemotherapeutic agent is released within the acidic environment of the endosome and allowed to penetrate the tumour cell cytoplasm. consequently, tumour progression phenotypes such as cell proliferation and level of apoptosis are affected to direct an amelioration of patient prognosis., with the capacity to selectively bind to two surface receptors which are upregulated in prostate tumour cell surface. thus allowing accumulation of the nanoparticle conjugate specifically within treatment - resistant prostate tumour cells. gold nanoparticles were also exploited in the study conducted by tomuleasa. for the purpose of reducing mdr hepatocellular carcinoma - derived cancer cells. the gold nanoparticles were loaded with doxorubicin, capecitabine, and cisplatin, followed by nanoparticle stabilization by l - aspartate. the resultant cellular proliferation rates of the hepatocellular carcinoma cells treated with this nanoparticle - based therapy were found to be lowered drastically. in the study carried out by punfa., the cytotoxic properties of curcumin on multidrug resistant cervical tumours were maximized through the development of a nanoparticle - curcumin drug delivery system. curcumin was successfully entrapped within poly (dl - lactide - co - glycolide) (plga) nanoparticles, followed by the incorporation of the amino - terminal of anti - p - gp. consequently, the curcumin - nanoparticle conjugates were deployed onto the kb - v1 cervical cancer cell line, having upregulated p - gp expression, together with the kb-3 - 1 cell line that has a reduced p - gp expression level. the results of this study demonstrated that nanoparticle conjugates bearing anti - p - gp surface markers were highly efficient in binding to the mdr - inducing surface protein, allowing enhanced cellular uptake and ultimately aid in the cytotoxic efficacy of curcumin due to increased accumulation of the drug, particularly within the kb - v1 cell line due to its exacerbated p - gp expression status. curcumin / doxorubicin - laden composite polymer nanoparticles were also developed in other studies as a means of enhancing the pharmacokinetic and pharmacodynamics properties of curcumin, thus enhancing its mdr - modulating effect in the target tumour cells. the resultant nanoparticle complex was deployed onto several mdr tumour models such as acute leukaemia, multiple myeloma, and ovarian cancers, both in vitro and in vivo. the results of this study highlighted the possibility of administration of lower doses of doxorubicin due to the circumvention of tumour mdr by efficient curcumin activity, thus enhancing the toxicity profile for doxorubicin in clinical use stemming from the reduction in cardiotoxicity and haematological toxicity dose - dependent adverse effects. retinoblastoma therapeutic avenues have also been increased due to the introduction of nanoparticle drug delivery technology. the study by das and sahoo demonstrated the effectiveness of utilising a nanoparticle delivery system which was dual loaded with curcumin together with nutlin-3a (which has been proven to stimulate the activity of the tumour suppressor protein p53). the results of this particular investigation highlighted an enhanced level of therapeutic efficacy on utilizing the nanoparticle - curcumin - nutlin-3a conjugates on the target retinoblastoma y79 cell lines. in addition, a downregulation of bcl2 and nfb was also observed following cell line exposure to the nanoparticle conjugates. the nanoparticle - based drug delivery system designed by saxena and hussain for its application against multidrug resistant breast tumours was novel in that the actual components of the nanoparticle biomaterials, namely, poloxamer 407 and d--tocopheryl polyethylene glycol 1000 succinate (tpgs), are both known to exert pharmacological activity against p - gp. the drug utilized for nanoparticle loading in this case was gambogic acid, a naturally occurring cytotoxic agent though laden with issues of poor bioavailability and severe dose - limiting adverse effects. similarly to other studies mentioned above, the incorporation of a nanoparticle - based drug delivery system allowed for enhanced cellular uptake by the target breast cancer cell line mcf-7, thus leading to elevated drug accumulation on the intracellular level and ultimately inducing enhanced cytotoxic effects in the target breast cancer cell line. a separate nanoparticle - based drug delivery system for use in circumventing mdr effects in breast cancer is the one developed by li.. in this study, the nanoparticle drug delivery system consisted of a dimethyldidodecylammonium bromide (dmab)-modified poly(lactic - co - glycolic acid) (plga) nanoparticle core that was conjugated to doxorubicin, then consequently coated with a 1,2-dipalmitoyl - sn - glycero-3-phosphocholine (dppc) shell. this system has been described to be specifically effective against mcf-7 breast cancer cell lines overexpressing p - gp. the results obtained from this particular study indicated an elevated accumulation of doxorubicin released from the nanoparticle complex, within the nuclei of the drug resistant mcf-7 cell line. in comparison, the level of accumulation of freely administered (i.e., not utilising a nanoparticle - based drug delivery system) doxorubicin attained lower drug concentration levels within the same cell line. finally, the ic(50) levels for doxorubin on adriamycin - resistant mcf-7 have been observed to be lowered by 30-fold following the incorporation of this nanoparticle delivery system. apart from delivery of conventional chemotherapeutic drugs in drug resistant breast cancer cell line models, researchers also delved into the possibility of adopting sirna therapeutic approaches, using the aid of nanoparticle drug delivery systems. the study conducted by navarro. developed a nanoparticle - based delivery system for sirnas targeting p - gp expression, with the nanoparticle constituent biomaterials being dioleoylphosphatidylethanolamine and polyethylenimine (pei). again, the reduction in p - gp expression led the path to enhanced cytoxic effects brought about by the exposure of the mcf-7 cell line to doxorubicin, thus this nanoparticle - sirna therapy was successful in drastically reducing mdr in this cancer model. quantum dots have also been implemented as novel and effective drug delivery systems for circumventing multidrug resistance in cancer chemotherapy. researchers in this study developed a quantum dot - based drug delivery system that allowed anti - mdr1 sirna and doxorubicin incorporation to two cadmium - selenium / zinc - selenium quantum dots that were eventually functionalized by -cyclodextrin coupling to l - arginine or l - histamine. following deployment of these dual loaded quantum dots in the hela cervical cancer cell line model, elevated accumulation of doxorubicin within the tumour cells was denoted, together with a marked reduction in mdr1 and p - gp expression on analysis by reverse transcription real time quantitative polymerase chain reaction and western blotting. in line with magnetic and gold nanoparticle platforms, quantum dots rely mainly on the endosomal method of tumour cellular uptake and therefore the drug efflux pump system is bypassed, with consequent reduction in mdr properties by the tumour cells. finally, the additional benefit of utilizing quantum dots as a drug delivery system is their capacity to be tracked in real time within specific areas of the target cells, due to their intrinsic fluorescence properties. apart from cell line studies, researchers have also looked into the feasibility of implementing nanoparticle - based drug delivery systems within in vivo models. investigated the efficacy of utilising a egfr - targeting polymer blend nanoparticles, loaded with paclitaxel and the mitochondrial hexokinase 2 inhibitor lonidamine. the nanoparticle polymer blend consisted of 70% polycaprolactone (pcl) incorporating a plga - polyethylene glycol - egfr specific peptide that helped enable nanoparticle active targeting efficiency. following nanoparticle development, four groups of orthotopic mdr breast cancer murine models (mda - mb-231 in nude mice) were treated with free paclitaxel, free lonidamine, free paclitaxel / lonidamine combination, or nanoparticle complexes containing paclitaxel / lonidamine combination. the degree of toxicity of such treatments was also monitored through body weight change measurements, liver enzyme plasma levels, and white blood cell / platelet counts, together with h & e staining of tumour sections was carried out. tumour weight and other clinical parameters such as mdr protein marker (p - gp, hypoxia inducible factor, hexokinase 2, egfr, stem cell factor) were observed over the course of 28 days after - treatment. following this 28-day period, the results demonstrated that only the murine model sample group exposed to the nanoparticle - based paclitaxel / lonidamine combination treatment was the only group to experience statistically significant tumour volume and density reduction, together with overall alteration of the mdr phenotype. toxicity effects due to paclitaxel and lonidamine were also drastically reduced when administered within the nanoparticle - based delivery system, which can ultimately provide enhanced tolerance by the cancer patient. other in vivo studies in this field include the investigations carried out by shen., which focused on the codelivery of paclitaxel and survivin short hairpin rna (shrna) for circumventing chemoresistance in lung cancer. the investigators utilized the pluronic block co - polymer p85 combined with d--tocopheryl polyethylene glycol 1000 succinate (p85-pei / tpgs) for developing the nanoparticles to be implemented in this study. these nanoparticles were based upon triblock structural formation of hydrophilic poly(ethylene oxide) (peo) blocks and hydrophobic poly(propylene oxide) (ppo) blocks, which also gives enhanced capacity to revert chemoresistance due to drug efflux pump inhibition properties, downregulation of atpase activity and p85-induced inhibition of the gluthathione s - transferase compound detoxification enzyme at the subcellular level. paclitaxel and surviving shrna were selected as the ideal drugs for nanoparticle delivery due to the former having poor efficacy due to chemoresistance within the tumour, and survivin was identified as highly expressed within chemoresistant tumours. the in vivo activity of such nanoparticle systems (with / without paclitaxel and survivin shrna) was evaluated on balb / c nude mice injected with viable, paclitaxel - resistant, a549/t lung adenocarcinoma epithelial cells. the results of this study demonstrated that deployment of the nanoparticle - based chemotherapeutic drug proved to have distinct enhancement of antitumour efficacy, when compared to deployment of the drug / s alone. chemoresistance to the aromatase inhibitor letrozole in postmenopausal breast cancer is another major therapeutic hurdle which was investigated in vivo. biodegradable plga - polyethylene glycol copolymer nanoparticles were developed by nanoprecipitation and designed to incorporate hyaluronic acid - bound letrozole (ha - letr - nps). the addition of hyaluronic acid served to enhance letrozole binding specificity to cd44 on the target tumour cell surface, with the expected consequences of enhanced drug accumulation within the target tumour cell cytoplasm and resultant re - sensitization of the target tumour cells to letrozole activity. such ha - letr - nps, once produced at a size of less than 100 nm diameter, were deployed within a letrozole - resistant murine xenograft tumour model. the results of this study demonstrated a highly efficient nanoparticle - based drug delivery system, with the ic(50) for ha - letr - nps within the murine xenograft model being only 5 m when compared to the control groups, thus enhancing the in vivo aromatase enzyme activity within the xenograft and ultimately inducing a prolonged resensitising of the breast cancer tumour to letrozole activity. the naturally occurring compound chitosan was also utilized for the development of in vivo nanoparticle - based therapies to circumvent ovarian cancer chemoresistance properties induced by overexpression of the jagged1 notch ligand. murine orthotopic models, utilising female athymic nude mice, were injected with skov3trip2 taxane - resistant ovarian cancer cell line and consequently, following one week, subjected to anti - jagged1 sirna / chitosan nanoparticle complexes (5 g dose of sirna) with / without taxane, applied via intraperitoneal route twice weekly for a total period of five weeks. the results of this study indicated that such nanoparticle - based complexes had the capacity to reduce tumour weight by over 70% within such murine models and also induced taxane sensitization within the tumour. in a similar study, cationic liposome - polycation - dna (lpd) and anionic liposome - polycation - dna (lpd ii) nanoparticle systems were developed to incorporate doxorubicin and vegf sirna within a murine ovarian cancer animal model. female, athymic nude mice were treated with 5 10 cells of the mdr ovarian cancer cell line nci / adr - res. once the murine tumours reached a size of approximately 1625 mm, the mice were consequently injected with individual nanoparticle complexes bearing either sirna or doxorubicin at a dose of 1.2 mg / kg in both cases, once daily for three consecutive days. the results of this study demonstrated the effectiveness of such nanoparticle complexes for inhibiting tumour progression within the treated murine model groups, mainly due to impaired vegf expression - related mdr. other human cancer conditions which were investigated for circumvention of tumour mdr properties through nanoparticle delivery include uterine sarcomas. in the study carried out by huang., ph - sensitive mesoporous silica nanoparticles incorporating hydrazine and doxorubicin were developed for in vivo testing on murine models of doxorubicin - resistant uterine sarcoma. since the composition of such nanoparticles specifically allow for cellular uptake through endocytosis, bypassing of the p - gp efflux pump induced a marked reduction in p - gp dependent mdr properties. consequently, the murine mdr tumour model treated with such nanoparticles demonstrated enhanced tumour apoptotic effects which were clearly confirmed by active caspase-3 immunohistochemical validation analysis. the latest studies described above undoubtedly serve as a testament to the immense clinical value represented by nanoparticle technology. the ability of such nanoparticles, irrelevant of biomaterial composition to efficiently load individual or combinations of chemotherapeutic drugs and/or chemosensitising agents (such as curcumin) and novel rna interference - based therapies has been clearly demonstrated above. this property provides an excellent escape mechanism for circumventing target tumour cell multidrug resistance properties based on drug efflux pump activity on the tumour cell surface, such as that exerted by p - gp. the overall advantage of deploying nanoparticles includes the drastic reduction in the ic(50) parameter for most of the carried chemotherapy agents, due to marked intracellular accumulation pharmacodynamics. this in turn would lead to a reduction in the clinical doses of the conventional cytotoxic agents required for chemotherapy, ultimately demonstrating a striking reduction in dose - dependent adverse effects in the oncology patient. presently, this does not mean that nanotechnology - based translational therapies are not fraught with challenges, such as biocompatibility issues of the nanoparticle components and the level of complexity required for cost - effectively translating these novel therapies to the patient bedside. however, it is the firm belief of the authors that through constant accumulation of marginal gains in knowledge, derived from persistent and motivated researchers on a global scale, will ultimately overcome such scientific hurdles, thus nanoparticle - based drug delivery aided therapies will eventually become commonplace in the oncology clinic in the near future. | the implementation of cytotoxic chemotherapeutic drugs in the fight against cancer has played an invariably essential role for minimizing the extent of tumour progression and/or metastases in the patient and thus allowing for longer event free survival periods following chemotherapy. however, such therapeutics are nonspecific and bring with them dose - dependent cumulative adverse effects which can severely exacerbate patient suffering. in addition, the emergence of innate and/or acquired chemoresistance to the exposed cytotoxic agents undoubtedly serves to thwart effective clinical efficacy of chemotherapy in the cancer patient. the advent of nanotechnology has led to the development of a myriad of nanoparticle - based strategies with the specific goal to overcome such therapeutic hurdles in multiple cancer conditions. this paper aims to provide a brief overview and recollection of all the latest advances in the last few years concerning the application of nanoparticle technology to enhance the safe and effective delivery of chemotherapeutic agents to the tumour site, together with providing possible solutions to circumvent cancer chemoresistance in the clinical setting. |
prior to use of ophthalmic non - steroidal anti - inflammatory suspensions, topical ophthalmic steroids were the mainstay treatment of post - operative, surgically induced ocular inflammation. although considered very effective, the use of topical corticosteroids is limited by well known side effects which in some serious cases can precipitate vision loss, and limits such therapy to short, intermittent use. topical non - steroidal anti - inflammatory drugs (nsaids) are notable for a definitive lack of corticosteroid - defined toxicity and have secured an important role, albeit in some cases an undefined role, in the treatment of ocular inflammatory disease. acute inflammation can be the result of exogenous injury either iatrogenic or due to accidents as well as of endogenous origin such as occurs in autoimmune disease. the use of the term inflammation in the context of the present report, however, will be limited to that of inflammation due to exogenous origin secondary to post - operative ophthalmic surgery related to the production of various eicosanoids. eicosanoids are defined as prostaglandins, leukotrienes, and other compounds that are products of the action of phospholipase a2 on the cellular phospholipid membrane and are, in general, derived from the production of arachidonic acid (figure 1). it has been shown that cyclooxygenase enzymes play a key role in maintaining cellular integrity and preventing apoptosis in eukaryotes (xiaojun 1995). the eicosanoid products of cyclooxygenase play a vital role in cellular homeostasis such as modulation of platelet function as well as renal regulation of salt and water balance (morrow and roberts 2002). however, up - regulation of the cyclooxygenase enzyme system has been documented secondary to injury and results in overproduction of eicosanoids normally required for cellular homeostasis. an overabundance of eicosanoids converts these otherwise essential compounds to rampant mediators of inflammation. unlike corticosteroids, which exhibit a broader effect on suppression of inflammation through inhibition of phospholipase a2 and through various cellular effects, nsaids act primarily through the inhibition of the cyclooxygenase (cox) enzyme isoforms. interestingly, nsaids also have been demonstrated to exert anti - inflammatory activity by mechanisms unrelated to cox inhibition through suppression of polymorphonuclear (pmn) locomotion and chemotaxis as well as by decreasing expression of inflammatory cytokines and mast cell degranulation (periann 1985 ; leonardi 2000). there is also evidence that nsaids exert activity as free radical scavengers, a finding that may also contribute to lessen the inflammatory response (flach 1992). it is now well established that two forms of cox exist. a constitutive isoform, cox1, is equally expressed upon the endoplasmic reticulum of all cells including platelets, cellular elements of the small and large bowel mucosa, vascular endothelium renal medullary collecting ducts, interstitium, pulmonary and hepatic sites, as well as the spleen (needleman and isakson 1997). cox2 is the induced isoform formed in part by various cytokines and various inflammatory mediators secondary to inflammation (crofford 1997). it is noteworthy that in the mammalian cornea, arachadonate may also be routed into a distinct metabolic pathway involving cytochrome p 450 (cyp) enzyme systems in addition to cox and lipoxygenase pathways (figure 2) (schwartzman and abraham 1990). in the corneal epithelium, arachidonate is metabolized by cyp systems that results in the biologically active species, 12(r) hydroxyl 5,8,10,14 eicosatetraenoic acid [12(r) hete ] and 12(r) hydroxyl -5,8,14-eicosatetraenoic acid [12(r) hetre ] (schwartzman and abraham 1990 ; mieyal 2000). 12(r) hete is a potent inhibitor of na - k atpase as well as mediating pmn aggregation (masferrer 1990 ; mieyal 2000, 2001). 12(r) hetere also plays a role in inflammation through mediation of conjunctival vasodilatation, and increasing permeability of the blood aqueous barrier in addition to acting as an aggregate for pmn leukocytes (davis 1990). descriptive clinical ramifications of corneal inflammation derived from activity of the cyp systems remain to be further elucidated. classification of nsaids is often noted by their chemical constituents as well as their activity on cox1 and cox2, respectively. nsaids available commercially for topical ophthalmic use are considered organic acids, with the exception of nepafenac, which is a benzoylbenzeneacetamide prodrug metabolized in vivo to its corresponding acid. currently available ophthalmic nsaid products in the us are considered phenylacetic or phenylalkanoic acids, including nepafenac which is a substituted phenylacetic acid. other chemical entities considered as nsaids such as salicylates, fenamates and pyrazolone derivatives an outline of currently used topical ophthalmic nsaids, their approved use in the us, as well as their chemical structure and class are outlined in table 1 and figure 3, respectively. commercially available topical ophthalmic nsaids are in large part solutions, with the exception of nepafenac which is a suspension, and vary based on the inclusion of inactive components utilized in embodiments as preservatives, surfactants, tonicity agents, viscosity enhancers, and buffers. as multi - use ophthalmic preparations require the presence of a preservative, the inclusion of anti - microbial compounds in the ophthalmic solution is imperative in the prevention of contamination of the solution. as cox inhibition is the major outcome parameter of anti - inflammatory potential of nsaid application in vitro, the comparative effect of topical ophthalmic nsaids on cox1 and cox2 activities is also of interest, at least from an experimental standpoint. classical nsaids with arylacetic acid structure are known to be potent inhibitors of the cox isoforms that promote a time - dependent inactivation of enzyme activity with increasing duration of drug exposure (flach 1990). the inhibitory action against cox is markedly reduced, however, when arylacetic nsaids are esterified. indeed, as an amide analog, nepafenac demonstrates weak cox activity ; however its free acid, amfenac, is a potent inhibitor of both cox1 and cox2 isoforms (gamache 2000). topical nsaids differ based on their affinity for the cox enzyme and activity in terms of enzyme inhibition. however, studies examining relative inhibitory cox activity of nsaids often utilize differing protocols and enzyme sources ; therefore the comparative assessment of nsaid ic50 is not directly correlative. it is important to exert caution when interpreting ic50 values for various nsaids in terms of clinical effectiveness. for example, cox activity can vary based solely on the species used to derive enzyme (warner 2001). furthermore, one must differentiate between in vitro testing at the cellular level or use of isolated enzymes. if one uses cells, a further caveat includes the inclusion of cofactors, for example glutathione or epinephrine that may affect enzyme rate (warner 2001). finally, the choice of an index of activity, eg, prostaglandin e2, thromboxane, or other markers of cox enzyme activity may affect determination of nsaid inhibitory potential (warner 2001). despite the intuitive impulse to equate clinical effectiveness with nsaid potency, the non - parallel nature of inhibitor curves makes interpretation of ic50 levels clinically quite difficult (warner 2001). indeed potency is relatively unimportant in the overall scheme of pharmacotherapy as long as the chemical entity in and of itself is devoid of marked dose - dependent toxicity as is the generally the case with ophthalmic application of nsaids (nies 2001 ; lekhanont 2007). although all topical ophthalmic nsaids may be considered non - selective cox inhibitors, the relative ratio of cox1:cox2 inhibitory potential of each agent differs markedly. in terms of cox isoforms demonstrable in corneal epithelium subsequent to experimentally induced hypoxia - derived inflammation, cox2 protein, but not cox1, was found to dramatically increase rabbit corneal epithelium after cellular hypoxia (schwartzman 2003). cox2 protein has also been noted in the canine model of corneal inflammation as well (schwartzman 2003 ; sellars 2004). however, corneal inflammation as result of hypoxia was found unrelated to the presence of the inflammatory mediator pge2 in the rabbit model. the lack of a concomitant increase in prostanoid pge2 despite elevation in cox2 protein thereby suggests an alternative source of hypoxia - defined corneal inflammation aside from prostanoid compounds. of further interest, several studies have demonstrated that prostaglandins may in some cases have frank anti - inflammatory properties and thus may be beneficial in combating ocular inflammation. for example, pge2 has been shown to inhibit ltb4-induced pmn infiltration into the anterior chamber of the rabbit eye (kulkarni 1991). furthermore, in the rabbit hypoxia model of corneal injury, inhibition of cox by indomethacin or aspirin resulted in potentiation of the release of pmns into the tear film (srinivasan and kulkarni 1989). although the role of prostaglandins in benefiting human corneal inflammation has not been tested, it is widely known that in general topical ophthalmic nsaids are only partially effective in reducing corneal inflammation in the human. nsaids may also potentiate peroximase proliferator - activated receptors with subsequent induction of cyp4b1 activity and production of the pro - inflammatory compounds 12-hete and 12-hetre. if such is the case, topical nsaids may not only be ineffective in treating corneal inflammation in the human eye, but actually contribute to the inflammatory response. these findings back the general notion that topical nsaids are in general inferior to corticosteroids in treatment of ocular surface inflammation secondary to hypoxia (srinivasan and kulkarni 1989). since the introduction of topical indomethacin for use in ophthalmic disease, several generations of nsaid have been brought to market. one of the more recent products commercialized for topical ophthalmic use is nepafenac, a prodrug of amfenac approved for use in the us for the treatment of post - operative inflammation after cataract surgery. nepafenac is described chemically as 2-amino-3-benzoylbenzeneacetamide manufactured as a 0.1% suspension for topical ophthalmic use (gamache 2000). unlike other topical nsaids nepafenac is not a free acid and unique in that it is the first prodrug nsaid formulation. until recently, all ophthalmic nsaid preparations, including flurbiprofen, diclofenac, ketorolac, and bromfenac, were relatively water - soluble phenylalkanoic and phenylacetic acids. thus, due to their inherent water solubility, phenylalkanoic and phenylacetic acids would be predicted to have limited ability to penetrate corneal epithelium. nepafenac, an amide prodrug analog of amfenac, is less polar and therefore ionic influences associated with corneal epithelial absorption are reduced. nepafenac as an amide prodrug that requires hydrolysis to the more active amfenac entity, as noted in figure 4. nepafenac as a parent compound exhibits weak intrinsic cox inhibitory activity (cox1 inhibitory activity ic50 64.3 m) (hargrave 2002) ; however, it readily penetrates the rabbit cornea and distributes in all ocular tissues including aqueous humor, iris, ciliary body, retina, and choroid (gamache 2000). amfenac is an arylacetic acid derivative (2-amino-3-benzoylbenzene acetic acid), which exhibits potent antipyretic and analgesic properties. amfenac is currently marketed in japan as a systemic formulation for treatment of rheumatoid arthritis and for pain and/or inflammation after surgery (fenazox) (needleman and isakson 1997 ; gamache 2000). a series of structural analogs of amfenac have been prepared to enhance the compound s therapeutic index (walsh 1990 ; ruiz 1993). interestingly, oral use of nepafenac, the amide analog of 2-amino-3-benzoylbenzeneacetic acid, exhibited potent ant - inflammatory activity but greatly reduced intestinal toxicity (walsh 1990). studies examining the in vitro comparative pharmacodynamic properties of nepafenac, amfenac, ketorolac, or bromfenac on cox activity demonstrated that ketorolac displays the greatest cox1 inhibitory activity while amfenac showed the greatest cox2 inhibitory potential (walters 2007). additional studies using pge2 as a surrogate marker of cox activity have noted that ketorolac demonstrated greater effect in lowering aqueous levels of pge2 levels compared to nepafenac (bucci 2007). it should also be noted that nepafenac, as well as other topical ophthalmic nsaids, do not demonstrate activity on the 12(r) hete and 12(r) hetre inflammatory mediators generated within the cornea by the action of cyp450 enzymes on arachidonic acid. topical ophthalmic nepafenac targets the anterior segment of the eye, intraocular and vascularized tissues. following topical application the onset of nepafenac activity is around 15 minutes and duration of action is greater than 8 hours (stewart 2005) after topical administration (nevanac). small quantifiable plasma concentrations of nepafenac and amfenac have been observed in subjects 23 hours after topical administration. after ocular administration the mean - steady state cmax of nepafenac and amfenac in serum were 0.310 0.014 ng / ml and 0.422 0.121 ng / ml, respectively (gamache 2000 ; nevanac). after oral administration nepafenac in rats was eliminated in urine (57%) and by the fecal route (40%) over a 7-day period (bucci 2007). nepafenac in the us was approved by the fda (food and drug administration) on august 19, 2005 as a priority drug approval (lane 2006). nepafenac is currently approved in the us only for treatment of pain and inflammation associated with cataract surgery (nevanac). it has been studied for the relief of pain and photophobia associated with photorefractive surgery and retinal edema secondary to diabetic retinopathy but is currently not approved for these indications in the us (colin and paquette 2006). drug formulation is an important factor affecting drug absorption and takes into consideration the ph of the drug, preservatives, and vehicle type. the corneal absorption of a drug depends on its lipid solubility and inversely on its polarity or degree of ionization. an important factor in the design of drugs for topical ophthalmic use that are commonly weak acids or bases involves the degree of penetration of a drug through the corneal epithelium. the more drug in its unionized form, the more likely it is to be lipid soluble and transferred by passive diffusion across the epithelial membrane. the ph of normal tears, which acts as a buffering system for many substances, varies between 6.5 and 7.6. altering the ph solution of a slightly basic drug, for example from 6.2 to 7.5, renders a drug to a more unionized state and therefore more lipid soluble, which therefore increases corneal penetration and, notably, systemic absorption as well. hence ophthalmic nepafenac being a base and maintained as an ophthalmic solution at ph 7.4 would exist more as a unionized drug and is therefore absorbed readily across the cornea at higher levels of tear ph. the marked advantage of using prodrugs to enhance corneal absorption of ophthalmic drugs has been demonstrated using isolated rabbit corneal tissue (ke 2000). in a rabbit model, corneal permeability of nepafenac was demonstrated by a permeability coefficient that was approximately 4, 19, and 28 times greater than diclofenac, bromfenac, and ketorolac respectively (ke 2000 ; linstrom and kim 2006). however, it should be pointed out that significant differences exist between rabbit and human cornea both anatomically and in terms of pharmacokinetics of drug absorption (owen 2007). in general, penetration of drugs applied to the rabbit cornea far exceeds that seen in the human due to the relative thinness of the rabbit cornea (owen 2007). it is also notable that bioactivation of nepafenac into the active amfenac entity is negligible in the cornea (ke 2000). however, as noted previously nepafenac does appear to penetrate the rabbit cornea in amounts relatively higher than other nsaids, but once again a caveat must be noted concerning differences between the rabbit and human cornea. the tissue that appears to demonstrate the greatest hydrolytic activity for the nepafenac molecule has been shown in the rabbit to be the retina / choroid followed by the iris / ciliary body (ke 2000). however, it is known that enzyme - protein concentration in the human aqueous is negligible with albumin being the largest contributor of aqueous protein concentration. enzyme activity is also poorly discernable in the aqueous with exception of plasminogen and plasminogen activator (blakemore 1995). as an amide, nepafenac (benzoylbenzeneacetamide) may be considered chemically analogous to n - methylacetamide and may undergo similar water hydrolysis once reaching the aqueous humor (zahn 2004). it is quite possible however that metabolism of nepafenac in the aqueous occurs by amide hydrolysis induced by water. at neutral ph, nucleophilic attack by the mechanism of water assisted hydrolysis has been investigated with use of n - methylacetamide using car - parrinello molecular dynamic (cpmd) simulation, which suggests a proton transfer and attack of the amide by the hydroxyl ion (zahn 2004). in vivo study in humans has shown that when applied topically to the cornea, nepafenac demonstrates both a faster as well as a higher aqueous humor concentration time to cmax than either bromfenac or ketorolac (walters 2007). the mean area under the time - concentration curve of aqueous drug concentration was found to be significantly higher for nepafenac than amfenac, ketorolac, or bromfenac (walters 2007). however, it should be noted that the mean area under the curve for aqueous amfenac was found comparable to that of ketorolac (180.7 vs 176.9 ng h / ml, respectively) (walters 2007). in contrast, additional studies have noted that levels of aqueous ketorolac following topical ophthalmic administration in humans far exceeds that of amfenac or nepafenac following 2 days of 4-times - a - day drug administration (bucci 2007). this finding is curious in view of the marked penetration potential of the prodrug nepafenac entity. it should be noted that benzalkonium chloride and other cationic surfactants increase ocular absorption of drugs by increasing corneal permeability by compromise of corneal integrity (forrester 2002). indeed alternative ophthalmic nsaids such as ketorolac, flurbiprofen, and bromfenac include benzalkonium chloride as a preservative, which may contribute to corneal permeability variances seen among these products. cataract surgery is an invasive procedure that requires incision and cutting of ocular tissue as well as considerable intraocular tissue manipulation. iatrogenic injury to the eye leads to disruption of the blood aqueous barrier with cellular infiltration leading to intraocular inflammation. fibrin and protein accumulation in the anterior chamber may lead to complications of intraocular inflammation such as increased intraocular pressure as well as corneal endothelial precipitates. adhesions of the iris to the angle and/or lens implant as well as opacification of posterior capsule may also be seen. cellular debris on the lens implant and posterior capsule may also lead to poor visual recovery. in severe cases post - operative inflammation of the anterior segment secondary to surgical trauma interestingly, topical nsaid treatment appears more effective than topical steroids in re - establishing the blood aqueous barrier as quantitatively measured with anterior ocular flurophotometry (jampol 1994). additional uses of nsaids in regard to cataract surgery include prevention of surgically induced miosis that may hamper surgical outcome and treatment of complicating cystoid macular edema after lens removal. there is evidence that prophylactic use of nsaids prior to cataract surgery may in fact lessen post - operative inflammation avoiding intraocular pressure - related complications incurred with frequent administration of high dose corticosteroids postoperatively (jampol 1994). a total of 5 unpublished studies in humans have examined the use of nepafenac after cataract surgery (nevanac ; lane 2006 ; national pbm drug monograph 2006). these studies included 1 pivotal trial (c-03 - 32), 1 efficacy and safety study (c-02 - 53), 2 dose response studies (c-95 - 93 and c-97 - 30), as well as 1 trial comparing safety and tolerability of nepafenac with diclofenac c-95 - 91. in studies c-02 - 53 and c-03 - 32 patients were randomized to receive nepafenac 0.1% at different dosing schedules, 1 drop daily, twice daily, 3 times a day, or placebo given with the above dosing regimen. all of the studies (except c-95 - 91) used aqueous cells and flare (signs of inflammation) as basis for evaluating the efficacy of the drug product. using slit - lamp biomicroscopy is a standard practice in ophthalmology to evaluate inflammation, using aqueous cells and flare ; the lower the score the lower the inflammation. the results of study c-02 - 53 demonstrated that 3-times - a - day regimen proved to be more efficacious than nepafenac given daily or twice daily, based upon the percentage of treatment failures (3 times a day 19.6% vs daily 25% vs twice daily 30%). as one would expect, nepafenac was superior to placebo in all three dose regimens. both studies achieved statistical significance in terms of efficacy with more patients receiving nepafenac vs placebo pain free on days 1, 3, 7, and 14 (national pbm drug monograph 2006). in a randomized double - blinded study of 476 patients evaluating safety and effectiveness of nepafenac in preventing and treating post - cataract surgery inflammation and pain, a higher percentage of patients in the nepafenac group were pain - free at all visits (83.1%93%) compared with vehicle - treated patients (41.6%46.4%) (p 4 weeks) resulting in epithelial breakdown, corneal thinning, corneal erosion, corneal ulceration or corneal perforation (gaynes and fiscella 2002 ; nevanac). it should be pointed out, however, that nsaids - related corneal toxicity often occurs inconsistently with variable dose relationships, thereby suggesting etiologies perhaps more related to susceptibility rather than frank chemical toxicity as the defining cause of corneal injury (flach 2001). in addition to local toxicity, isolated reports of systemic toxicity have been associated with use of topical nsaids (estes 1993 ; chan 1995 ; sharir 1997). reports of asthma exacerbation, gastrointestinal erosions, and bleeding have been associated with use of systemic as well as topical ophthalmic nsaids (estes 1993 ; chan 1995 ; sharir 1997). according to studies in rabbits, 74% of the administered topical ophthalmic dose of ketorolac reaches the systemic circulation through nasolacrimal drainage (ling and combs 1987). nepafenac is not an inhibitor of the in vitro metabolism of the major cytochrome (cyp) isozymes (cyp1a2, cyp2c9, cyp2c19, cyp2d6, cyp2e1, and cyp3a4) at therapeutic concentrations used in ophthalmic indications (nevanac). therefore, interactions involving concurrently used systemic drugs that are metabolized by cyp are unlikely. drug interactions with nepafenac mediated by protein binding are also unlikely according to the opinion of the manufacturer (nevanac). concurrent use of nepafenac with medications that prolong bleeding time may increase the risk of hemorrhage due to interference by nepafenac with thrombocyte aggregation. moreover, there have been reports that ophthalmic non - steroidal anti - inflammatory drugs may increase bleeding in ocular tissues (including hyphema) in conjunction with ocular surgery (gaynes and fiscella 2002). one must be particularly cautious in initiating topical nsaid use particularly for long - term usage in the presence of concomitant risk factors such as with those who use concurrent systemic nsaids, use tobacco and/or alcohol, and/or are included in geriatric, pediatric, or other vulnerable populations. nepafenac warning labels indicate there is cross - sensitivity to acetylsalicylic acid, phenylacetic acid derivatives, and other non - steroidal anti - inflammatory agents (nevanac). therefore caution should be used when treating individuals who have previously exhibited sensitivity to these drugs. as a rule the inhibition of cox2 determines the clinical efficacy of a topical nsaid. in systemic use nsaid selectivity for one cox isoform drugs with more selectivity for cox2 than cox1 tend to have fewer gastrointestinal side effects but have been associated with increased risk for hepatotoxicity, edema, hypertension, and cardiovascular problems. although uncommon, the occurrence of systemic adverse events related to topical ophthalmic nsaids should not be discounted (jampol 1994 ; malhotra 2004 ; warner 2004). the most frequent adverse events reported in clinical trials with nepafenac to date have been ocular in nature ; however, the definitive causality is obscure. approximately 5%10% of the patients demonstrated post - operative capsular opacity, decreased visual acuity, foreign body sensation, increased intraocular pressure, and a sticky sensation (nevanac). fewer frequent ocular adverse events (1%5%) included conjunctival edema, corneal edema, dry eye, lid margin crusting, ocular discomfort, ocular hyperemia, ocular pain, ocular pruritis, photophobia, tearing, and vitreous detachment. non - ocular adverse (1%4%) events included hypertension, headache, nausea / vomiting, and sinusitis (nevanac). concentrations of this drug have been evaluated at 15 times the commercial marketed dose without any significant complications in animal studies (mcgee 2005 ; walker 2005). in the randomized vehicle - controlled clinical trial by lane and colleagues evaluating nepafenac for treatment of pain and inflammation after cataract surgery, no ocular adverse events of definitive relationship to nepafenac therapy were reported (lane 2007). ocular adverse events reported by lane occurring at an incidence of 1% or greater included decreased visual acuity, photophobia, capsule opacity, foreign body sensation, ocular hyperemia, ocular pruritis, ocular discomfort, dry eye, increased intraocular pressure, and blurred vision (lane 2007). none of the events were considered serious and in large part of mild intensity and resolved without complication. the incidence of ocular adverse events such as decreased visual acuity rates and capsular opacity were indeed similar in patients receiving nepafenac 0.1% and those receiving vehicle (3.2% vs 3.8% and 2.0% vs 2.5% respectively (lane 2007). the use of nsaids after refractive surgery for control of pain and inflammation is a growing indication for this class of drug. in addition to typical lasik procedures, refractive surgery also involves surface ablation photorefractive keratectomy such as laser - assisted sub - epithelial keratectomy (lasek) and epi - lasek. after surface ablation procedures, a contact lens is placed in the eye to promote epithelial regeneration. in one randomized double masked study of ketorolac 0.4% vs nepafenac 0.1% for control of pain after surface ablation, eyes treated with nepafenac tended to demonstrate greater corneal haze as well as slower epithelial healing compared with ketorolac, resulting in discontinuation of the study due to safety concerns (trattler 2005). it appears that use of nepafenac after surface ablation greatly augments the risk of corneal toxicity, notably corneal haze and delayed healing, particularly if used for longer than a 3-day post - operative interval (durrie 2006 ; reilly and cadwell 2006 ; william trattler pers comm march 3, 2008). the rationale for nepafanac - related corneal injury following prk is unclear as bioactivation of nepafenac within the cornea is reported to be very low (colin and paquette 2006). persistent corneal haze after use of nepafenac after surface ablation procedures may, although unlikely, be related to direct chemical toxicity of the prodrug entity rather than a consequence of the pharmacodynamic actions of amfenac. more compelling is the notion of water - based hydrolysis of nepafenac within the matrix of the hydrophilic soft contact lens leading to nepafenac bioactivation. with repeated dosing, nepafenac may be adsorbed and accumulate within silicon hydrophilic bandage lenses which provide a substrate for water - based amide hydrolysis resulting in a steady source of amfenac leaching from the lens matrix. it should be noted, however, that analysis of nepafenac in terms of corneal healing and analgesia following prk as studied by caldwell and reily found that while analgesia was significantly enhanced in the nepafenac group, a concomitant delay in corneal wound healing was not evident. corneal injury following prk was indeed fully healed equally in both the nepafenac and placebo groups by post - operative day 5 (caldwell and reilly 2008). further analysis by donnefeld found that neither nepafenac nor ketorolac reduced corneal healing time following prk and both drugs demonstrated a beneficial effect in terms of analgesia (donnenfeld 2007). colin and paquette described corneal epithelial healing rate with use of 0.03% or 0.1% nepafenac or 0.1% diclofenac. furthermore, colin and paquette found no difference in corneal epithelial healing rate among the three study formulations ; however, a small corneal infiltrate was noted in the 0.03% nepafenac group that was deemed possibly related to the study medication (colin and paquette 2006). it should be noted, however, that in all studies mentioned describing prk and corneal epithelial healing rate with various topical nsaids, subjects were followed for a maximum of 57 days with no comment on the appearance of the cornea following re - epithelization. interestingly, corneal haze following prk can occur weeks, months, or even years following prk and is related to abnormalities in healing of the corneal stroma rather than epithelium (majmudar 2000). as a corollary to issues pertaining to refractive surgery, it is interesting to note that nepafenac is considered to demonstrate greater corneal analgesic effect compared with drugs such as diclofenac or ketorolac (acosta 2007). this finding is largely due to the ability of nepafenac to partition into corneal epithelium quickly and efficiently compared more polar analogs. it has also been shown that unlike diclofenac, nepafenac does not exhibit local anesthetic qualities (acosta 2007). reproduction studies performed with nepafenac in rabbits and rats at oral doses up to 10 mg / kg / day have revealed no evidence of teratogenicity due nepafenac, despite induction of maternal toxicity (nevanac). at this dose, the animal plasma exposure of nepafanac and amfenac was 80 and 680 times human plasma exposure for rabbits, respectively, when nepafanac was used at manufacturer recommended ophthalmic doses. in rats maternal toxic doses 10 mg / kg were associated with increased post - implantation loss, reduced fetal weights and growth, and reduced fetal survival. there have been no adequate, well - controlled trials for the use of nepafenac in pregnant women ; therefore, its use should be avoided in the late stages of pregnancy (third trimester) due to the known effects of prostaglandin biosynthesis inhibition on the fetal cardiovascular system (closure of the ductus arteriosus). nepafenac has been shown to cross placental barrier in rats. because animal reproduction studies are not always predictive of human response, nepafenac should be used during pregnancy only if the potential benefit justifies the potential risk to the fetus (nevanac). nepafenac was excreted in the milk of pregnant rats. because it is not known whether this drug is excreted in human milk, caution should be exercised when nepafenac is administered to a nursing woman (nevanac). increased chromosomal aberrations were observed in chinese hamster ovary cells exposed in vitro to nepafenac suspension. nepafenac did not impair fertility when administered orally to male and female rats at 3 mg / kg (approximately 90 and 380 times the plasma exposure to the parent drug, nepafenac, and active metabolite, amfenac, respectively, at the recommended human topical ophthalmic dose) (nevanac). the approved fda labeling for nepafenac states that 1 drop of nepafenac is to be applied to the affected eye(s) 3 times a day. therapy begins 1 day prior to cataract surgery and continues the day of surgery and through the first 2 weeks of the post - operative period. the use of nepafenac beyond the recommended therapeutic time frame may increase the risk of adverse corneal events as noted in the fda - approved drug labeling (nevanac). safety of nepafenac has not been determined in pediatric patients below the age 10 years (nevanac). each milliliter of nepafenac suspension contains 1 mg of nepafenac and should be shaken well prior to instillation. topical ophthalmic nsaids such as ketorolac have been administered without incident in combination with other topical ophthalmic products including antibiotics, beta blockers, carbonic anhydrase inhibitors, cycloplegics, and mydriatics (acular 2003). nepafenac would also be expected to be safely administered in combination with other topical ophthalmic products ; however, dosage should be spaced at least in 5-minute intervals to prevent drug washout and dilution and to maximize drug absorption. the concomitant use of topical nsaids, however, in conjunction with topical and/or enteral corticosteroids must be carefully monitored, particularly among patients at high risk for corneal injury such as those with rheumatoid arthritis or fulminate collagen vascular disorders (gaynes and fiscella 2002). nepafenac is the first prodrug ophthalmic nsaid formulation approved for use in the us for the treatment of post - operative pain and inflammation after cataract surgery. nepafenac itself has little activity on cox enzyme activity and requires deamination to the more active congener amfenac for therapeutic action. although amfenac demonstrates marked cox inhibitory potential, differences in nsaid potency as described in comparative reviews of cox ic50 activity should not be the sole determinant of effective ophthalmic nsaid utilization clinically. however, the expected therapeutic advantage of nepafenac based on its corneal permeability profile and absorption is not fully recognizable in comparative assessment of clinical anti - inflammatory efficacy. the issue of bioactivation as it pertains to nepafenac is a key point in pharmacodynamics pertaining to the active amfenac entity. although nepafenac is metabolized in the mammalian iris and retina, the rate of amide hydrolysis demonstrable in human aqueous has not been fully elucidated ; therefore, comparative prediction regarding peak nepafenac concentration in aqueous as an implied indicator of effectiveness in the human is at best equivocal. all commercially available topical ophthalmic nsaids are effective inhibitors of cox activity ; whether or not one drug has greater or lower ic50 value is therefore not directly correlated with clinical indicators of therapeutic effectiveness. in the case of topical ophthalmic nsaids, practitioners should carefully weigh the cost - benefit of implementing highly potent new drug products because perturbations in pharmacodynamic response due to the inherent novelty in terms of chemical designs may outweigh the demonstrated replicative pharmacologic action of all topical ophthalmic nsaids. | the removal of diclofenac sodium ophthalmic solution as a viable pharmaceutical entity in september 1999 from the us market spurred considerable interest in the general safety and effectiveness of topical ophthalmic nsaids for treatment of anterior segment inflammation. in late 1999 the use of topical ocular nsaids declined in the us as a result of incidents involving corneal melts and toxicity surrounding use of generic diclofenac. however, since the removal of diclofenac sodium ophthalmic solution from the marketplace, ophthalmic nsaids have regained use as viable pharmacotherapeutic entities. moreover, several new ophthalmic nsaid products have recently been introduced for commercial use in the us including the novel chemical entity nepafenac. the purpose of this report is to revisit the use of topical ophthalmic nsaids for the treatment of surgically induced anterior segment inflammation with a particular focus on nepafenac. nepafenac is unique among ophthalmic nsaids in that it is a prodrug deaminated to amfenac, a highly effective non - selective cyclooxygenase inhibitor. in the case of topical ophthalmic nsaids, practitioners should carefully weigh the cost - benefit of implementing highly potent new drug products because perturbations in pharmacodynamic response due to the inherent novelty in terms of chemical designs may outweigh the demonstrated replicative pharmacologic action of all topical ophthalmic nsaids. |
lab courses have been a staple of the undergraduate biology curriculum since its inception, but too often the labs have taken on a cookbook form where students follow a protocol, much like a recipe, to obtain a known answer (24, 34). bio2010 (26) and vision and change (6) advocate shifting how we teach undergraduate laboratory courses from traditional cookbook labs to research - focused labs (which have been described a number of ways, including : inquiry - based ; discovery ; investigative ; project - based ; research - based ; and course - based research experiences). these efforts seek to make labs more representative of authentic research, so that students spend more time thinking like a biologist rather than following a set of directions that often fail to engage deeper thinking. in response to the call for reform, a significant number of research - based curricula have been developed, many of which have been formally evaluated and published. research on these curricula report significant gains in student confidence in their abilities (4, 9, 10, 11, 14), their attitudes towards science and authentic research (13), and content knowledge using an array of pre-/post- surveys (3, 5) and restricted response summative assessment tests (e.g., biology field test) (13). based on these data, reformed biology lab courses are measurably better. while the biology education community has made progress in the development of novel research - based lab curricula, it is important that we interpret the effectiveness of these curricula with caution and remain mindful of inherent limitations to our study designs that may impact internal validity. what research design allows us to claim that these research - based labs are better ? looking at pre - course and post - course scores within one course ? comparing reformed courses to cookbook labs ? working with 10 students or 100 students ? furthermore, on what basis could we claim that what we find in a local context generalizes to other contexts diverse populations of students, different types of institutions, or variations in instructional methods ? unfortunately, the specific context of many recent studies is often not addressed, which can lead to biased generalizations threatening external validity. by citing the results of the study without limiting the conclusions to the context in which it was implemented, these potentially inaccurate generalizations can weaken the evidence - based foundation of undergraduate biology education reform. in addition, these generalizations can create the false assumption that continued investigation of research - based lab courses is unnecessary because research has already supported their efficacy. although standards for reporting education research have been well documented and provide a framework of expectations for both quantitative and qualitative research in education (1, 2), some of these guidelines, particularly discussing the limits of a study s conclusions, seem to be underemphasized in the biology education research community. in this paper, we highlight some of the common limitations of educational research. as an example of the challenges of implementing one of these research - based lab courses, we will discuss our own data from a research - based lab course that highlights how very different conclusions can be drawn about the effectiveness of a study depending on the type of data used. as the biology education research community continues to use social science methodology to explore better ways to teach undergraduate biology, it is paramount that we acknowledge how the specific context of our research design influences our data and conclusions. logistical and institutional barriers that influence a study s design remain major challenges to conducting biology education research on research - based lab courses. since randomized experimental design may not always be possible, the biology education research community must take extra precautions to address factors that can threaten the validity of reported conclusions (12, 28). this includes, but is not limited to, the bias of volunteer populations of students, small sample sizes, and not using a comparison group. double - blind randomized trials have long been considered the gold standard of scientific research, including scientific educational research (8, 12, 23, 31). randomization minimizes the chances of unobserved, systematic differences between two conditions biasing the results. however, some studies can not avoid using volunteers who have some information about the intervention. among other problems, this can result in researchers finding a significant impact resulting from an intervention even if none exists the so - called hawthorne effect, where participants show changes because they are being studied. in other scenarios, the bias might arise from self - selection if volunteers are aware of what will occur in the treatment condition (27). volunteers may also produce a sample that differs from the comparison condition in terms of gender, level of self - confidence, willingness to take risks, or previous experience doing experiments (27). when recruiting volunteers to pilot a new classroom intervention, the study will almost certainly attract individuals more pre - disposed to the treatment than students who do not volunteer. for ethical or logistical reasons, the use of volunteers with some knowledge of the treatment sometimes can not be avoided, thus making the careful interpretation of results extremely important. often, equipment, personnel, scheduling, and space are limiting factors that dictate how many students can participate in a reformed lab course, often resulting in reforms that focus on upper - level elective courses with a small number of students. moreover, reforming courses requires extensive time from faculty, teaching assistants, and external evaluators to change the nature of teaching and learning and respond flexibly to obstacles that may arise. thus, initial pilots might involve small numbers of students before scaling - up for hundreds of students. clearly, advantages accrue for piloting new courses with small samples, including reducing cost and minimizing logistical problems. reporting data from these pilot studies is important, as results from these studies provide an indicator of potential directions for improving teaching and learning. however, our reporting of results should make clear the ways in which the data might be impacted by the small sample size and what added affordances or obstacles will likely exist if the class is scaled up. ideally, researchers should also publish data from the larger, scaled - up version of the course. traditional institutional structures and logistics make randomizing students into different versions of a science course very difficult, although all too often such designs are simply eliminated from consideration due to the perceived amount of effort it would take. quasi - experimental methods without randomization provide an alternative for comparing student outcomes (8, 30). publishing student achievement gains on a pre- and posttest in a pilot course provides helpful data about the likelihood that the course fosters student learning in this content area. while useful, if learning gains are not compared to the traditional course s outcomes, conclusions can not be made about whether the new approach is just as beneficial as the traditional option. a comparison to the traditional course is critical to making the most informed decisions about whether the differences in performance are worth the costs of scaling - up new innovations. generalizations made from the results of a study without a comparison group might wrongly attribute the learning gains to the specific reform intervention when, in fact, statistically similar gains are seen in other courses that have the same time on task, thus presenting a threat to external validity (23). students received detailed protocols that they followed step by step to achieve a known answer. the revised course was a research - based laboratory that was aligned with the goals of bio2010 and vision and change students worked collaboratively on experiments with unknown answers and emphasis was placed on data interpretation and analysis, as opposed to getting the right answer. the revised lab course s content was rooted in ecology ; students investigated the relationships of biotic and abiotic factors on yeast and bacteria that reside in a flowering plant called the monkeyflower. students collected data, compiled it into a large database shared by all students in the class, posed novel hypotheses, and used statistical analysis to investigate correlational relationships. for more details about the course structure and design, see kloser. data reported here include a combination of novel and previously published data ; a description of research methodologies and statistical analyses can be found in brownell. volunteers were recruited because the department did not believe it fair to require participation in an untested lab course. we compared the volunteers in the pilot lab to matched non - volunteers in the traditional lab. we checked that there were no differences in the two populations in gender distribution, class year, previous research experience, and self - reported gpa. using likert - scale surveys, we found that students in the pilot research - based lab course showed significant positive changes in attitudes towards authentic research, confidence in their abilities to do lab - related tasks, and interest in pursuing future research compared to students in the traditional lab (7). based on these encouraging results, the department agreed that no ethical problems would result from randomizing students into either course for a larger implementation study. the next year, we randomized students into the research - based lab or the traditional lab. we scaled up to 34 students in the research - based lab, while keeping the section size the same (approximately 10 students). for the evaluation, we first focused on pre- to post - course gains that students made in the research - based lab. using the same set of likert - scale surveys, we found that students in the research - based course showed significant gains in their attitudes towards authentic research, confidence in their ability to do lab - related tasks, and interest in future research (20). students in the research - based lab also showed improvement on a performance assessment focused on data interpretation and designing an experiment. however, when we compared the responses of students in the research - based lab to those of students in the traditional lab, we did not see the same differences observed previously (fig. students in both conditions had similar gender distribution, class year, previous research experience, and self - reported gpa there were no differences in these self - reported characteristics. although we saw significant gains in student attitudes towards authentic research in the research - based course, there were no differences between the two groups in student confidence in their ability to do lab - related tasks or student interest in pursuing future research. so why would the students in the research - based course in the randomized year not show higher gains in interest or confidence in ability compared with students in the traditional lab ? likert - scale survey data from a three - year study of a research - based biology lab course. students were asked a series of questions about (a) their interest in future research (2 questions), (b) their confidence in their ability to do lab - based tasks (6 questions), and (c) their attitudes towards authentic research (4 questions). student scores on each question on the pre - course survey were subtracted from their scores on the post - course survey and averaged for that block of questions to get the main gain per question. data shown are from three years that the course was offered to : volunteer students (n = 20 for the cookbook, n = 20 for research - based course), randomized students (n = 33 for cookbook, n = 33 for research - based course), and scaled - up research - based course students (n = 128). p < 0.05 (note : data from cookbook and research - based volunteers (7) and research - based randomized students (20) have previously been published.) the notable difference between the pilot and randomized experiment was that students in the pilot course were volunteers. might it be that students partial to authentic research and willing to take risks in a new course volunteered for the original pilot, thus skewing the study s results ? while the initial study was important for our own internal evaluation and the results were interesting to the field as an initial indication of the impact of the treatment, the difference between the volunteer and non - volunteer group outcomes in our own work highlights the need to qualify findings and explicitly state limitations (7). we, along with the biology education research community, must carefully consider the limits of generalizing results from a volunteer population to all students in all universities because it could potentially have a negative impact on knowledge - building around issues of undergraduate lab reform. in the third year, we scaled up from approximately 34 to 132 students. all students were enrolled in the research - based lab with a section size of approximately 20 students. we compared this larger group of non - volunteer students to the non - volunteer students in the traditional lab from the previous year and found results similar to the second year study (fig. students in the research - based course compared with students in the traditional lab showed significant gains in their attitudes towards authentic research, but no differences in their interest in research or confidence in their ability to do lab - based tasks. thus the difference in sample sizes (34 compared to 132) did not seem to have an impact, even though it was a potential limitation of our initial studies (7, 20). we have compiled some of our previously published (and some unpublished) data to illustrate how important the context of a research study can be, particularly how the specific population of students and the inclusion of a comparison group can affect one s interpretations of the effectiveness of a course. to be clear, these are not new methodological findings, but rather, this case serves as an example from our own work that, in order for the field to move forward, we must be explicit about the limitations of our findings. in our initial studies, if we had only reported the data from the volunteers in our research - based lab, we would have concluded that students in the research - based lab show increased interest in future research and confidence in their ability to do lab - related tasks (fig. if we had compared these volunteers to matched non - volunteer students in the traditional lab, we would have concluded that students in the research - based course have greater increases in interest and confidence in their ability to do lab - related tasks than students taking a cookbook lab (fig. conclusions about the effectiveness of the course differ based on which data are used. (a) the conclusion from only examining the data from the volunteers in the research - based course is that students show gains in both confidence in lab - based tasks and interest in pursuing future research. (b) the conclusion from comparing the volunteers in the research - based course with non - volunteers in the cookbook course is that students in the research - based course show higher gains than those in the cookbook course. (c) the conclusion from assessing non - volunteer students in the research - based course is that students show gains in confidence but no gains in interest. (d) the conclusion from comparing non - volunteers in the research - based course to non - volunteers in the cookbook course is that there are no differences between the students in interest or confidence in their ability to do lab - based tasks. however, if we used the data from our second year of evaluation and reported data only from non - volunteers in the research - based lab, we would conclude that students only show gains in their confidence in their ability to do lab - related tasks, but not in their interest to pursue future research (fig. these different conclusions likely stem from the participant population recruited did these students volunteer or were they randomized without choice into the lab ? if we added in the comparison data from randomized non - volunteers in the cookbook lab, we would conclude that students show gains in confidence in their ability to do lab - related tasks in both labs and do not show gains in their interest in doing future research in both labs (fig. using the comparison group demonstrates that students, regardless of what lab they are enrolled in, believe that their ability to do lab - based tasks improves there is no difference between students in the two lab conditions. thus, the conclusions based on these likert - scale self - report surveys differ when using randomized non - volunteers with a comparison group from conclusions drawn from volunteers enrolled in a research - based course with no comparison group. although we do not see a difference in self - report interest or confidence in their ability to do lab - based tasks between the two groups when using randomized non - volunteers, we do see differences in students in the two labs in other measures : we consistently saw higher gains in students in the research - based course in their attitudes towards authentic research, regardless of whether students were volunteers or non - volunteers. we interpret this to mean that our authentic research - based course is effective at changing student attitudes about research in a positive way, independent of whether they chose to be in the course as volunteers or whether they were randomly assigned to it. we also saw that students in the research - based course showed gains in their experimental design and data interpretation skills. while we developed our own assessment of students ' ability to design an experiment and interpret data, this assessment is limited to the ecological content studied in the research - based course and could not be used in the cookbook course. recently published content - independent assessments for skills such as the experimental design ability tool (33) and scientific literacy test (18) could now be used to assess skills addressed or not addressed in the differently structured courses. logistical and institutional barriers that influence a study s design remain major challenges to conducting biology education research on research - based lab courses. since randomized experimental design may not always be possible, the biology education research community must take extra precautions to address factors that can threaten the validity of reported conclusions (12, 28). this includes, but is not limited to, the bias of volunteer populations of students, small sample sizes, and not using a comparison group. double - blind randomized trials have long been considered the gold standard of scientific research, including scientific educational research (8, 12, 23, 31). randomization minimizes the chances of unobserved, systematic differences between two conditions biasing the results. however, some studies can not avoid using volunteers who have some information about the intervention. among other problems, this can result in researchers finding a significant impact resulting from an intervention even if none exists the so - called hawthorne effect, where participants show changes because they are being studied. in other scenarios, the bias might arise from self - selection if volunteers are aware of what will occur in the treatment condition (27). volunteers may also produce a sample that differs from the comparison condition in terms of gender, level of self - confidence, willingness to take risks, or previous experience doing experiments (27). when recruiting volunteers to pilot a new classroom intervention, the study will almost certainly attract individuals more pre - disposed to the treatment than students who do not volunteer. for ethical or logistical reasons, the use of volunteers with some knowledge of the treatment sometimes can not be avoided, thus making the careful interpretation of results extremely important. often, equipment, personnel, scheduling, and space are limiting factors that dictate how many students can participate in a reformed lab course, often resulting in reforms that focus on upper - level elective courses with a small number of students. moreover, reforming courses requires extensive time from faculty, teaching assistants, and external evaluators to change the nature of teaching and learning and respond flexibly to obstacles that may arise. thus, initial pilots might involve small numbers of students before scaling - up for hundreds of students. clearly, advantages accrue for piloting new courses with small samples, including reducing cost and minimizing logistical problems. reporting data from these pilot studies is important, as results from these studies provide an indicator of potential directions for improving teaching and learning. however, our reporting of results should make clear the ways in which the data might be impacted by the small sample size and what added affordances or obstacles will likely exist if the class is scaled up. ideally, researchers should also publish data from the larger, scaled - up version of the course. traditional institutional structures and logistics make randomizing students into different versions of a science course very difficult, although all too often such designs are simply eliminated from consideration due to the perceived amount of effort it would take. quasi - experimental methods without randomization provide an alternative for comparing student outcomes (8, 30). publishing student achievement gains on a pre- and posttest in a pilot course provides helpful data about the likelihood that the course fosters student learning in this content area. while useful, if learning gains are not compared to the traditional course s outcomes, conclusions can not be made about whether the new approach is just as beneficial as the traditional option. a comparison to the traditional course is critical to making the most informed decisions about whether the differences in performance are worth the costs of scaling - up new innovations. generalizations made from the results of a study without a comparison group might wrongly attribute the learning gains to the specific reform intervention when, in fact, statistically similar gains are seen in other courses that have the same time on task, thus presenting a threat to external validity (23). double - blind randomized trials have long been considered the gold standard of scientific research, including scientific educational research (8, 12, 23, 31). randomization minimizes the chances of unobserved, systematic differences between two conditions biasing the results. however, some studies can not avoid using volunteers who have some information about the intervention. among other problems, this can result in researchers finding a significant impact resulting from an intervention even if none exists the so - called hawthorne effect, where participants show changes because they are being studied. in other scenarios, the bias might arise from self - selection if volunteers are aware of what will occur in the treatment condition (27). volunteers may also produce a sample that differs from the comparison condition in terms of gender, level of self - confidence, willingness to take risks, or previous experience doing experiments (27). when recruiting volunteers to pilot a new classroom intervention, the study will almost certainly attract individuals more pre - disposed to the treatment than students who do not volunteer. for ethical or logistical reasons, the use of volunteers with some knowledge of the treatment sometimes can not be avoided, thus making the careful interpretation of results extremely important. often, equipment, personnel, scheduling, and space are limiting factors that dictate how many students can participate in a reformed lab course, often resulting in reforms that focus on upper - level elective courses with a small number of students. moreover, reforming courses requires extensive time from faculty, teaching assistants, and external evaluators to change the nature of teaching and learning and respond flexibly to obstacles that may arise. thus, initial pilots might involve small numbers of students before scaling - up for hundreds of students. clearly, advantages accrue for piloting new courses with small samples, including reducing cost and minimizing logistical problems. reporting data from these pilot studies is important, as results from these studies provide an indicator of potential directions for improving teaching and learning. however, our reporting of results should make clear the ways in which the data might be impacted by the small sample size and what added affordances or obstacles will likely exist if the class is scaled up. ideally, researchers should also publish data from the larger, scaled - up version of the course. traditional institutional structures and logistics make randomizing students into different versions of a science course very difficult, although all too often such designs are simply eliminated from consideration due to the perceived amount of effort it would take. quasi - experimental methods without randomization provide an alternative for comparing student outcomes (8, 30). publishing student achievement gains on a pre- and posttest in a pilot course provides helpful data about the likelihood that the course fosters student learning in this content area. while useful, if learning gains are not compared to the traditional course s outcomes, conclusions can not be made about whether the new approach is just as beneficial as the traditional option. a comparison to the traditional course is critical to making the most informed decisions about whether the differences in performance are worth the costs of scaling - up new innovations. generalizations made from the results of a study without a comparison group might wrongly attribute the learning gains to the specific reform intervention when, in fact, statistically similar gains are seen in other courses that have the same time on task, thus presenting a threat to external validity (23). the department of biology at stanford university recently redesigned its introductory biology laboratory course. the traditional version of the course was a standard students received detailed protocols that they followed step by step to achieve a known answer. the revised course was a research - based laboratory that was aligned with the goals of bio2010 and vision and change students worked collaboratively on experiments with unknown answers and emphasis was placed on data interpretation and analysis, as opposed to getting the right answer. the revised lab course s content was rooted in ecology ; students investigated the relationships of biotic and abiotic factors on yeast and bacteria that reside in a flowering plant called the monkeyflower. students collected data, compiled it into a large database shared by all students in the class, posed novel hypotheses, and used statistical analysis to investigate correlational relationships. for more details about the course structure and design, see kloser. (21) and fukami (16). data reported here include a combination of novel and previously published data ; a description of research methodologies and statistical analyses can be found in brownell. volunteers were recruited because the department did not believe it fair to require participation in an untested lab course. we compared the volunteers in the pilot lab to matched non - volunteers in the traditional lab. we checked that there were no differences in the two populations in gender distribution, class year, previous research experience, and self - reported gpa. using likert - scale surveys, we found that students in the pilot research - based lab course showed significant positive changes in attitudes towards authentic research, confidence in their abilities to do lab - related tasks, and interest in pursuing future research compared to students in the traditional lab (7). based on these encouraging results, the department agreed that no ethical problems would result from randomizing students into either course for a larger implementation study. the next year, we randomized students into the research - based lab or the traditional lab. we scaled up to 34 students in the research - based lab, while keeping the section size the same (approximately 10 students). for the evaluation, we first focused on pre- to post - course gains that students made in the research - based lab. using the same set of likert - scale surveys, we found that students in the research - based course showed significant gains in their attitudes towards authentic research, confidence in their ability to do lab - related tasks, and interest in future research (20). students in the research - based lab also showed improvement on a performance assessment focused on data interpretation and designing an experiment. however, when we compared the responses of students in the research - based lab to those of students in the traditional lab, we did not see the same differences observed previously (fig. students in both conditions had similar gender distribution, class year, previous research experience, and self - reported gpa there were no differences in these self - reported characteristics. although we saw significant gains in student attitudes towards authentic research in the research - based course, there were no differences between the two groups in student confidence in their ability to do lab - related tasks or student interest in pursuing future research. so why would the students in the research - based course in the randomized year not show higher gains in interest or confidence in ability compared with students in the traditional lab ? likert - scale survey data from a three - year study of a research - based biology lab course. students were asked a series of questions about (a) their interest in future research (2 questions), (b) their confidence in their ability to do lab - based tasks (6 questions), and (c) their attitudes towards authentic research (4 questions). student scores on each question on the pre - course survey were subtracted from their scores on the post - course survey and averaged for that block of questions to get the main gain per question. data shown are from three years that the course was offered to : volunteer students (n = 20 for the cookbook, n = 20 for research - based course), randomized students (n = 33 for cookbook, n = 33 for research - based course), and scaled - up research - based course students (n = 128). p < 0.05 (note : data from cookbook and research - based volunteers (7) and research - based randomized students (20) have previously been published.) the notable difference between the pilot and randomized experiment was that students in the pilot course were volunteers. might it be that students partial to authentic research and willing to take risks in a new course volunteered for the original pilot, thus skewing the study s results ? while the initial study was important for our own internal evaluation and the results were interesting to the field as an initial indication of the impact of the treatment, the difference between the volunteer and non - volunteer group outcomes in our own work highlights the need to qualify findings and explicitly state limitations (7). we, along with the biology education research community, must carefully consider the limits of generalizing results from a volunteer population to all students in all universities because it could potentially have a negative impact on knowledge - building around issues of undergraduate lab reform. in the third year, we scaled up from approximately 34 to 132 students. all students were enrolled in the research - based lab with a section size of approximately 20 students. we compared this larger group of non - volunteer students to the non - volunteer students in the traditional lab from the previous year and found results similar to the second year study (fig. students in the research - based course compared with students in the traditional lab showed significant gains in their attitudes towards authentic research, but no differences in their interest in research or confidence in their ability to do lab - based tasks. thus the difference in sample sizes (34 compared to 132) did not seem to have an impact, even though it was a potential limitation of our initial studies (7, 20). we have compiled some of our previously published (and some unpublished) data to illustrate how important the context of a research study can be, particularly how the specific population of students and the inclusion of a comparison group can affect one s interpretations of the effectiveness of a course. to be clear, these are not new methodological findings, but rather, this case serves as an example from our own work that, in order for the field to move forward, we must be explicit about the limitations of our findings. in our initial studies, if we had only reported the data from the volunteers in our research - based lab, we would have concluded that students in the research - based lab show increased interest in future research and confidence in their ability to do lab - related tasks (fig. if we had compared these volunteers to matched non - volunteer students in the traditional lab, we would have concluded that students in the research - based course have greater increases in interest and confidence in their ability to do lab - related tasks than students taking a cookbook lab (fig. conclusions about the effectiveness of the course differ based on which data are used. (a) the conclusion from only examining the data from the volunteers in the research - based course is that students show gains in both confidence in lab - based tasks and interest in pursuing future research. (b) the conclusion from comparing the volunteers in the research - based course with non - volunteers in the cookbook course is that students in the research - based course show higher gains than those in the cookbook course. (c) the conclusion from assessing non - volunteer students in the research - based course is that students show gains in confidence but no gains in interest. (d) the conclusion from comparing non - volunteers in the research - based course to non - volunteers in the cookbook course is that there are no differences between the students in interest or confidence in their ability to do lab - based tasks. however, if we used the data from our second year of evaluation and reported data only from non - volunteers in the research - based lab, we would conclude that students only show gains in their confidence in their ability to do lab - related tasks, but not in their interest to pursue future research (fig. these different conclusions likely stem from the participant population recruited did these students volunteer or were they randomized without choice into the lab ? if we added in the comparison data from randomized non - volunteers in the cookbook lab, we would conclude that students show gains in confidence in their ability to do lab - related tasks in both labs and do not show gains in their interest in doing future research in both labs (fig. using the comparison group demonstrates that students, regardless of what lab they are enrolled in, believe that their ability to do lab - based tasks improves there is no difference between students in the two lab conditions. thus, the conclusions based on these likert - scale self - report surveys differ when using randomized non - volunteers with a comparison group from conclusions drawn from volunteers enrolled in a research - based course with no comparison group. although we do not see a difference in self - report interest or confidence in their ability to do lab - based tasks between the two groups when using randomized non - volunteers, we do see differences in students in the two labs in other measures : we consistently saw higher gains in students in the research - based course in their attitudes towards authentic research, regardless of whether students were volunteers or non - volunteers. we interpret this to mean that our authentic research - based course is effective at changing student attitudes about research in a positive way, independent of whether they chose to be in the course as volunteers or whether they were randomly assigned to it. we also saw that students in the research - based course showed gains in their experimental design and data interpretation skills. while we developed our own assessment of students ' ability to design an experiment and interpret data, this assessment is limited to the ecological content studied in the research - based course and could not be used in the cookbook course. recently published content - independent assessments for skills such as the experimental design ability tool (33) and scientific literacy test (18) could now be used to assess skills addressed or not addressed in the differently structured courses. how biology education research addresses volunteer bias, sample size issues, and the presence or lack of comparison groups is important for determining the generalizability of research findings (12, 28). for example, even with a structured curriculum, the level and type of instruction is significantly impacted by an instructor s content knowledge (19, 36), pedagogical knowledge (25), pedagogical content knowledge (17, 22, 32), or personality (15, 29). similarly, students range of general academic ability, extent of their prior knowledge and experiences, socioeconomic status, ethnicity, first generation status, and career aspirations can vary significantly within and among institutions (35). for example, conclusions drawn from data collected from a high - achieving student population may not apply to other student populations that have different learning needs. making the problem more complicated is that some of these factors may appear to be at odds with each other. for example, if a specific number of students take a biology lab course each year, then taking volunteers or randomizing students into a treatment and comparison group will reduce the sample size within each condition, making any effect more difficult to statistically detect. however, without the comparison group, interpreting the impact of the new intervention will be limited. when dealing with a finite number of possible participants, it may be necessary to sacrifice a large sample size and qualify the interpreted data in light of this limitation. such is the difficulty of conducting educational research, yet we must face these challenges and acknowledge these limitations in order to move the field forward. while we focused on educational research, overextending conclusions based on limited or biased datasets for example, cost and labor constraints often limit the number of samples or subjects that are used in animal studies, large - scale metabolomic or transcriptomic experiments, or clinical trials. we should likely be asking about the appropriateness of generalizing from these conclusions as well. the impetus for writing this article stemmed from a series of discussions at recent biology education conferences. on several occasions, when questions were raised regarding possible limitations of studies done on research - based lab courses, presenters dismissed these questions as irrelevant to the interpretation of the results. perhaps these encounters are anecdotes, limited to only a few isolated public comments, and do not reflect the general consensus of the biology education community. but whether anecdote or common occurrence, it is important for the field to remind itself about the need for qualifying our claims and generalizations so that we may better construct new knowledge in this important area of research. for the field to move forward, the biology education research community needs to be more aware of the potential limitations of its research and be cautious when generalizing specific work to other contexts. at the most basic level, all peer - reviewed biology education publications and presentations could be required to include an explicit limitations section. we must be more mindful of our specific recruitment methods, the demographics of our student population, and the caveats of comparing our data to other student groups. given the complexities of in situ educational research we must continue to value exploratory studies that may not meet the rigors required in other scientific research, but in doing so we must also recognize the results as warrants for larger - scaled randomized trials and not final justification for a course design that works. then, we must convince members of our departments and institutions that larger - scaled, randomized trials are both possible and necessary to truly promote an evidence - based scholarship of teaching and learning. we hope that our case study exemplifies the importance of recognizing limitations in the interpretation of results and, perhaps more importantly, the generalization of conclusions. we want our foundation of research to be secure, even if that means less flashy conclusions in the short - term, but conclusions that in the long run will better prepare the next generation of biologists. our research community and | the shift from cookbook to authentic research - based lab courses in undergraduate biology necessitates the need for evaluation and assessment of these novel courses. although the biology education community has made progress in this area, it is important that we interpret the effectiveness of these courses with caution and remain mindful of inherent limitations to our study designs that may impact internal and external validity. the specific context of a research study can have a dramatic impact on the conclusions. we present a case study of our own three - year investigation of the impact of a research - based introductory lab course, highlighting how volunteer students, a lack of a comparison group, and small sample sizes can be limitations of a study design that can affect the interpretation of the effectiveness of a course. |
overweight and obesity in childhood are recognised as a major health problem worldwide and while considerable data have been published relating to influences such as sedentary behaviour and nutritional habits on weight status, there is a dearth of information pertaining to the structural and functional limitations of excess weight in adults [2, 3 ], with even less data in children. this is despite authors noting that children display alterations to their functional movement as a consequence of excessive weight and suboptimal movement patterns found in overweight and obesity can seriously impede daily physical activity level and limit functional performance [3, 4 ]. the concern here is that an inability to complete fundamental daily tasks coupled with excessive and prolonged loading of tissues in childhood may lead to orthopaedic abnormality in later life. therefore, minimizing joint deterioration from excessive joint loading or impaired movement patterns evident in obese and overweight children should be treated at the earliest opportunity. therefore, the aim of this study was to examine relations between habitual physical activity, functional movement patterns, and weight status in children. following institutional ethics approval, fifty - eight children (29 boys and 29 girls, 86% caucasian) from a primary school in central england volunteered and returned signed parental informed consent forms to participate in the study. children were from school year 6 (aged 10 - 11years) and were predominantly caucasian (81%). the mean age (sd) of the children was 10.7 (0.4) years. the school the children was drawn from was in an urban area of the city of coventry and was located in the least deprived ward in the city with an average household income of 43,842 in comparison to 31,695 for the city average. body mass (kg) and height (m) were measured using a stadiometer and weighing scales (seca instruments, germany, ltd) prior to the monitoring period. from this children were classified as normal weight (68.4%) and overweight / obese (31.6%) according to iotf criteria. physical activity was assessed using a sealed, piezoelectric pedometer (new lifestyles, nl2000, montana, usa) worn over four days (2 weekdays and 2 weekend days) in accordance with recommendations for the assessment of physical activity in children and using protocols previously described. across the measurement period, the children completed a brief survey to verify that the pedometers were worn for the entire time of the study. survey results were used to identify participants who reported removing the pedometer for 1 h, resulting in three exclusions from the data set, similar to other studies. the functional movement screen (fms) is a preparticipation screening tool which evaluates the fundamental movement patterns that underpin performance of all movement [9, 10 ]. the fms consists of seven tests which challenge an individual 's ability to perform basic movement patterns that reflect combinations of muscle strength, flexibility, range of motion, coordination, balance, and proprioception [9, 10 ]. the fms was administered by a trained rater using standardised procedures, instructions, and scoring processes [9, 10 ]. each participant was given 3 trials on each of the seven tests (deep squat, hurdle step, in - line lunge, shoulder mobility, active straight leg raise, trunk - stability push - up, and rotary stability) in accordance with recommended guidelines [9, 10 ]. each trial was scored from 0 to 3 with higher scores reflecting better functional movement. for each test, the highest score from the three trials was recorded and used to generate an overall composite fms score with a maximum value of 21 and in accordance with recommended protocols [9, 11 ]. relationships between total fms score, bmi and pa (average steps / day) were analysed using pearson 's product moment correlations. a 2 (gender) 2 (weight status : normal weight versus overweight / obese) 2 (pa : children meeting / not meeting the health related steps / day cut off (8)) analysis of variance (anova), using backwards elimination to achieve a parsimonious solution, was employed to examine any differences in functional movement (fms score) according to gender, weight status, and physical activity. where any significant differences were detected, bonferroni post - hoc multiple comparisons were used to detect where these differences lay. multiple linear regression was also employed to predict functional movement (fms score) from bmi and physical activity. total fms score was significantly, negatively correlated with bmi (r =.806, p =.0001, see figure 1(a)) and positively related to pa (r =.301, p =.029, see figure 1(b)). furthermore, anova found a significant main effect for weight status (f 1, 53 = 50.4, p =.0001) for total fms score with normal weight children scoring significantly better than their overweight and obese peers (mean diff = 4.941, p =.0001, see figure 2). mean s.d. of fms scores were 15.5 2.2 and 10.6 2.1 in normal weight and overweight / obese children, respectively. multiple linear regression also revealed a significant predictor model (f 2, 53 = 40.369, p =.0001, adjusted r =.602) whereby bmi and physical activity (average steps / day) predicted 60.2% of the variance in functional movement score. however, bmi was a stronger predictor (=.712, p =.0001) of functional movement predicting 52.9% of the variance in total fms score in comparison to physical activity (= 1200.0, p =.03) where average steps / day predicted 7.3% of the variance in fms score. of total fms score, pa (average steps / day), and bmi between gender and weight status groups are presented in table 1. results of this study suggest that functional movement is related to weight status and habitual physical activity, with weight status having a stronger association with functional movement in this population. in this case although bmi and physical activity were both significant predictors of functional movement predicting 60.2% of the variance in fms score, bmi alone predicted 52.9% of this variance in comparison to physical activity, where average steps / day predicted 7.3% of the variance in fms score. this study supports prior research in adults which has suggested that excess weight results in functional limitations [2, 3 ] and assertions that children display altered functional movement as a consequence of excess weight. the positive association seen between fms score and habitual physical activity in the present study supports prior assertions that the lack of physical activity is associated with the lack of functional movement skills in adults. however, in the context of the present study and population examined, it is possible that functional limitation may have existed prior to overweight / obesity. as such, excess weight and/or functional prowess are the result of natural selection since children who are functionally limited will remain inactive and will not develop the fundamental movement patterns that underpin performance to the same level of mastery as children who do not have a functional limitation. in the same way, children who are not functionally limited may more likely enjoy physical activity and thus, engage in more regular practise of the fundamental movement patterns that underpin performance. these children will consequently become more proficient in performance of fundamental movement patterns resulting in greater physical self efficacy and increased likelihood of participation in physical activity. to the authors knowledge this is the first study to examine associations between weight status, physical activity, and fms in a pediatric population. the data presented here are however, important as they highlight those children who are overweight / obese present maladaptive movement patterns needed to accomplish tasks of daily life. over time, these movement patterns coupled with the effect of excess weight on joint loading are likely to lead to orthopaedic abnormality in later life. moreover, such suboptimal movement patterns can prevent individuals from undertaking health enhancing physical activity. there are several proposed mechanisms for the impact of overweight and obesity on functional movement. however, these suggestions have tended to be based on studies in adult populations with few studies examining this issue in children and adolescents. one suggestion is that overweight and obesity lead to alterations in the musculoskeletal system that place overweight individuals at higher risk of musculoskeletal pain [4, 12 ]. such pain may then result in restricted range of movement and/or lower levels of physical activity. one study by messier. showed that a weight loss of approximately 5% following a combined diet and exercise programme improved physical function, mobility, and reduced pain in overweight and obese adults with osteoarthritis. however, musculoskeletal pain is considered a chronic condition that is less likely to affect children. some studies in children and adolescents [13, 14 ] have evidenced changes in foot structure and plantar pressure distribution, as a result of structural adaptation to excess weight, which was suggested to lead to functional movement complications. likewise, research by zoico., with older adults, has concluded that a bmi of 30 kg m or greater is significantly associated with functional limitation. however, stenholm. has further suggested that it is the ratio of fat free mass (ffm) to body mass which is important as individuals with higher ffm are more likely to be functionally proficient than those with lower ffm. however, while these studies were conducted with older adults, there is a lack of data examining the association between bmi, ffm, muscle strength, and functional movement in children. similarly, only one other study to date has reported specifically on the use of the functional movement screen in a pediatric population. burton. reported that, in a sample of 39 middle school children, there were significantly higher total fms scores in girls compared to boys, principally due to better performance in the deep squat, in line lunge, straight leg raise, and shoulder rotation. they concluded that girls exhibit superior quality in functional movement compared to boys but suggested that it was important to develop musculoskeletal interventions to improve functional movement in children and adolescents. however, although burton. did not account or control for variation in weight status in their study making it difficult to draw parallels with the current study, the range of total fms scores reported in their study is similar to those documented in this study. however, there was no gender difference found for fms score in the present study but as burton. did not assess or control for weight status it is difficult to draw parallels between the two studies. irrespective of whether it is functional limitation that results in lack of physical activity and increases in overweight / obesity or whether lack of physical activity and overweight / obesity leads to suboptimal movement, the data present here support the need for interventions to increase habitual physical activity and improve functional movement in british children generally but those who are overweight and obese specifically. it is also important to note that there may be other factors which influence performance of the functional movement screen in children such as motivation to perform the movement patterns and particularly motor learning. although the children in the present study had been familiarized with the movements involved in the fms, it may be that there are practice effects in the fms. no studies to date have examined this issue and the recommended protocols were followed when administering the fms into the current study. however, future research examining the effects of motor learning on performance of the fms would be useful in elucidating this area further. in respect to the clinical significance of the current study, the results present here do not necessarily suggest a clinical need for those children exhibiting poor functional movement at their present age. the worry, however, is that if suboptimal movement patterns, present in children who are overweight / obese, persist into adolescence and adulthood, this will lead to a range of musculoskeletal problems of clinical significance. this includes a range of factors including knee osteoarthritis, early need for hip replacement and chronic pain, in addition to limitations in tasks of daily living (see for a review). thus, in overweight / obese children who exhibit suboptimal functional movement, interventions aimed at improving the quality of functional movement via weight loss and/or increases in physical activity energy expenditure may offset more severe clinical implications in later life. considering the association between physical activity and fms found in this study, it may be that the relationship between physical activity and weight status is magnified as children become older if their weight is not managed, resulting in further reduction in physical activity, functional movement, or both. this suggestion is, however, speculative and further research is needed to verify this claim. this exploratory study is also limited by a small sample size and larger scale studies would be welcomed to verify the claims made here. in addition, cause and effect in relation to physical activity and functional movement could not be determined in the present sample. the results of this study do, however, highlight that ambulatory physical activity and weight status are significant predictors of functional movement in british children. in the current study weight status had a greater influence on functional movement compared to physical activity and children who were classified as overweight / obese demonstrated significantly poorer functional movement than children classified as normal weight. | although prior studies have suggested that overweight and obesity in childhood are associated with poorer functional movement performance, no study appears to have examined this issue in a pediatric population. the relations between bmi, ambulatory physical activity and functional movement screen (fms) performance were compared in 58, 10 - 11-year - old children. total fms score was significantly, negatively correlated with bmi (p =.0001) and positively related to pa (p =.029). normal weight children scored significantly better for total fms score compared to children classified as overweight / obese (p =.0001). mean s.d. of fms scores were 15.5 2.2 and 10.6 2.1 in normal weight and overweight / obese children, respectively. bmi and pa were also significant predictors of functional movement (p =.0001, adjusted r2 =.602) with bmi and pa predicting 52.9% and 7.3% of the variance in total fms score, respectively. the results of this study highlight that ambulatory physical activity and weight status are significant predictors of functional movement in british children. scientists and practitioners therefore need to consider interventions which develop functional movement skills alongside physical activity and weight management strategies in children in order to reduce the risks of orthopaedic abnormality arising from suboptimal movement patterns in later life. |
murrah buffalo bulls were divided into two groups : g1 contained four 18-month - old males, and g2 contained three 24-month - old males. the animals were obtained from the department of animal production, faculty of veterinary medicine, unesp - botucatu / sp, which is located at 2253'09 " s, 4826'42 " w and is 804 m above sea level. the buffalo were fed brachiariasp. and mineral salt ad libitum and supplemented with corn silage during the dry periods. endoparasites control was performed with levamisole phosphate, applied 1 ml per 40 kg of liveweight, and the vermifuge (ripercol, fort dodge animal health, campinas, so paulo, brazil) was administered to the animals at the beginning of the wet season (november) and early dry season (june). the vaccines were applied to the animals : the first dose of clostridial was given at four months, with a booster after one month followed by subsequent annual applications ; fmd was administered in may and november. the buffalo used in this study were the same age at the beginning of the experiment. the testes were collected at two time points because the specific age of onset of puberty and spermatogenesis in murrah buffalo bulls raised under the conditions described was unknown. the project was conducted in accordance with the ethical principles for animal experimentation of the animal experimentation ethics committee of the school of animal science of unesp dracena (protocol no. 017/2011). prior to collecting the testes, the animals were physically restrained and anesthetized according to the ethical principles for animal experimentation as recommended by the brazilian college of animal experimentation, and they were weighed using a mechanical balance (acr 1500, 4,000 kg, balanas ares, camb, pr, brazil). after collection, the epididymis was separated from the testis, and the testicular weight (tw), testicular length (tl), testicular width (twd), testicular thickness (tt) and testicular circumference (tc) were determined for each testis. to obtain the tw (g), an analytical balance was used (bk 3000 ; range 0.23100 g). each testis was sectioned into three regions (the capitata, middle and end caudata portions) for tissue fixation. the middle portions of the testicular parenchyma were placed in bouin 's solution for at least 24 h. after dehydration in increasing concentrations of alcohol, followed by infiltration with xylenes, the tissue samples were embedded in paraffin. a microtome (leica rm2145, leica, berlin, germany) was used to section the tissue into 5-mm - thick slices, and these sections were stained with hematoxylin- and eosin (he). the samples were analyzed under a light microscope (leica dm 2500, leica). descriptive analyses of the mean and standard deviation for each testicular biometric parameter were performed with the graphpad prism4 software (graphpad software, la jolla, ca, usa). age, body weight and testicular biometric parameters were tested for correlations using r statistical software (r development core team 2009). murrah buffalo bulls were divided into two groups : g1 contained four 18-month - old males, and g2 contained three 24-month - old males. the animals were obtained from the department of animal production, faculty of veterinary medicine, unesp - botucatu / sp, which is located at 2253'09 " s, 4826'42 " w and is 804 m above sea level. the buffalo were fed brachiariasp. and mineral salt ad libitum and supplemented with corn silage during the dry periods. endoparasites control was performed with levamisole phosphate, applied 1 ml per 40 kg of liveweight, and the vermifuge (ripercol, fort dodge animal health, campinas, so paulo, brazil) was administered to the animals at the beginning of the wet season (november) and early dry season (june). the vaccines were applied to the animals : the first dose of clostridial was given at four months, with a booster after one month followed by subsequent annual applications ; fmd was administered in may and november. the buffalo used in this study were the same age at the beginning of the experiment. the testes were collected at two time points because the specific age of onset of puberty and spermatogenesis in murrah buffalo bulls raised under the conditions described was unknown. the project was conducted in accordance with the ethical principles for animal experimentation of the animal experimentation ethics committee of the school of animal science of unesp dracena (protocol no. 017/2011). prior to collecting the testes, the animals were physically restrained and anesthetized according to the ethical principles for animal experimentation as recommended by the brazilian college of animal experimentation, and they were weighed using a mechanical balance (acr 1500, 4,000 kg, balanas ares, camb, pr, brazil). after collection, the epididymis was separated from the testis, and the testicular weight (tw), testicular length (tl), testicular width (twd), testicular thickness (tt) and testicular circumference (tc) were determined for each testis. to obtain the tw (g), each testis was sectioned into three regions (the capitata, middle and end caudata portions) for tissue fixation. the middle portions of the testicular parenchyma were placed in bouin 's solution for at least 24 h. after dehydration in increasing concentrations of alcohol, followed by infiltration with xylenes, the tissue samples were embedded in paraffin. a microtome (leica rm2145, leica, berlin, germany) was used to section the tissue into 5-mm - thick slices, and these sections were stained with hematoxylin- and eosin (he). the samples were analyzed under a light microscope (leica dm 2500, leica). descriptive analyses of the mean and standard deviation for each testicular biometric parameter were performed with the graphpad prism4 software (graphpad software, la jolla, ca, usa). age, body weight and testicular biometric parameters were tested for correlations using r statistical software (r development core team 2009). at 18 months, the testicular parenchyma contained testicular cords and internal cord compartments associated with the presence of leydig cells, vessels and nerves. within the testicular cords, gonocytes, undifferentiated supporting cells and luminal processes were observed. gonocytes and spermatogonia in the differentiation process were identified near the basal membrane (fig. 1. testis of murrah buffalo bulls at 18 months of age characterized by an immature sexual phase (a and b). we observed solid testicular cords with gonocytes (a) and undifferentiated supporting cells (s). although spermatids were present in high numbers at 18 months in nili - ravi buffalo testes, spermatozoa were not present in the tubule lumens analyzed here. testis of murrah buffalo bulls at 18 months of age characterized by an immature sexual phase (a and b). we observed solid testicular cords with gonocytes (a) and undifferentiated supporting cells (s). the testicular parenchyma at 24 months contained seminiferous tubules with lumens and germinative epithelia present at different stages of the seminiferous epithelial cycle (fig. 2. different cell types in the seminiferous epithelium of the murrah buffalo at 24 months of age. photomicrographs of cross sections of the seminiferous tubules represent the stages of the cycle of the seminiferous epithelium. stage one (a), two (b), three (c), four (d), five (e), six (f), seven (g) and eight (h) represent the following groups of cells : primary spermatocytes at the pre - leptoteno / leptoteno (pl) stage, primary spermatocytes at the pachytene (p) stage, round spermatids (ar), elongated spermatids (al), primary spermatocytes at the zygotene (z) stage, primary spermatocytes at the diplotene (d) stage, residual bodies (cr) and spermatozoa (e). all the stages presented spermatogonia type a (a) and sertoli cells (s). hematoxylin - eosin staining.). the following cell types were identified : sertoli cells, spermatogonia, pre - leptotene / leptotene spermatocytes, pachytene spermatocytes, zygotene spermatocytes, diplotene spermatocytes, round spermatids and luminal spermatozoa (fig. these cells could be classified into eight stages of cellular associations according to the tubular morphology method. bongso noted that the spermatozoa in the tubular lumen of swamp buffalo had the same morphology at 16 months. in crossbred buffalo (mediterranean jaffarabadi), this morphology was observed at 13 months. ohashi showed the onset of the spermatogenic process in buffalo at 9 months. at 24 months, our histological analysis of the spermatogenic process indicated that these animals were not sexually mature. different cell types in the seminiferous epithelium of the murrah buffalo at 24 months of age. photomicrographs of cross sections of the seminiferous tubules represent the stages of the cycle of the seminiferous epithelium. stage one (a), two (b), three (c), four (d), five (e), six (f), seven (g) and eight (h) represent the following groups of cells : primary spermatocytes at the pre - leptoteno / leptoteno (pl) stage, primary spermatocytes at the pachytene (p) stage, round spermatids (ar), elongated spermatids (al), primary spermatocytes at the zygotene (z) stage, primary spermatocytes at the diplotene (d) stage, residual bodies (cr) and spermatozoa (e). all the stages presented spermatogonia type a (a) and sertoli cells (s). hematoxylin - eosin staining. mean body and testicular biometric parameters in murrah buffalo bulls at 18 and 24 months of ageage groupbw (kg)sc (cm)tw (cm)tl (cm)twd (cm)tt (cm)tc (cm)g1 (18 mo)270.25 14.5918.75 1.521.11 2.795.07 0.362.72 0.242.48 0.258.09 0.60g2 (24 mo)446.70 32.5625.30 3.2177.66 38.57.13 1.253.73 0.693.55 0.5112.58 1.71means with different superscript letters are significantly different (p<0.05) according to analysis of variance and the student 's t test (5%). bw = body weight, sc = scrotal circumference, tw = testicular weight, tl = testicular length, twd = testicular width, tt = testicular thickness, tc = testicular circumference.. although the sc and body weight differed significantly between the two age groups analyzed, there was an increase in the sc with increasing body weight. additional testicular biometric parameters are often assessed in domestic animals, particularly those animals used for livestock production because testicular parameters are an important part of the andrological evaluation. among the available biometrics for testes, sc is one of the most commonly employed because it is easy to measure and highly correlated with body weight. means with different superscript letters are significantly different (p<0.05) according to analysis of variance and the student 's t test (5%). bw = body weight, sc = scrotal circumference, tw = testicular weight, tl = testicular length, twd = testicular width, tt = testicular thickness, tc = testicular circumference. as shown in table 2table 2. correlations between age, body weight and testicular parameters in murrah buffalo bulls at 18 and 24 months of age agebwsctwtltwdtttcage0.975 0.854 0.776 0.808 0.777 0.847 0.904bw0.843 0.851 0.827 0.775 0.838 0.904sc0.5170.963 0.932 0.898 0.931tw0.4950.3790.4790.572tl0.982 0.949 0.963twd0.971 0.965tt0.978tcbw = body weight, sc = scrotal circumference, tw = testicular weight, tl = testicular length, twd = testicular width, tt = testicular thickness, tc = testicular circumference. significant (p<0.05)., there was a significant correlation between sc, age and body weight. this result has been described previously in river, swamp and murrah buffalo. bw = body weight, sc = scrotal circumference, tw = testicular weight, tl = testicular length, twd = testicular width, tt = testicular thickness, tc = testicular circumference. significant (p<0.05). the data presented here differ from the findings in cattle, for which the sc is particularly important in identifying bulls with high reproductive capacity. neither scrotal circumference nor body weight should be used alone as a routine and reliable parameter to predict the reproductive capacity in buffalo. the mean values and standard deviation for tw, tl, twd, tt and tc, relative to buffalo age, are shown in table 1. although the average tw did not differ between the two groups, significant differences were observed for tl, twd, tt, and tc (p<0.05). furthermore, these variables exhibited high correlation coefficients, as noted in table 2. body weight, age, sc, tw, tl, twd, tt and tc were positively correlated (p<0.05). this finding differs from observations made in piau pigs, rams and cattle, in which a high correlation was described for all parameters. the strength of the correlations obtained suggests that tl, twd, tc and tt are useful parameters for the selection of breeding animals. under the husbandry conditions described here, both body and testicular development of buffalo occurred more slowly than the typical growth rates for bos taurus. the data for bos indicus, however, suggest a similar rate of body and testicular development to the rates observed for buffalo in the present study. these similarities can be attributed to the fact that zebu cattle are also raised under extensive farming conditions, and therefore suffer from the same nutritional consequences, particularly dietary protein deficiencies, which can lead to developmental delays. in conclusion, our analysis of biometric testicular parameters in murrah buffalo revealed that the tl, twd, tc and tt are useful indicators for the selection of bulls for breeding. at 18 months, the testicular parenchyma contained testicular cords and internal cord compartments associated with the presence of leydig cells, vessels and nerves. within the testicular cords, gonocytes, undifferentiated supporting cells and luminal processes were observed. gonocytes and spermatogonia in the differentiation process were identified near the basal membrane (fig. 1. testis of murrah buffalo bulls at 18 months of age characterized by an immature sexual phase (a and b). we observed solid testicular cords with gonocytes (a) and undifferentiated supporting cells (s). although spermatids were present in high numbers at 18 months in nili - ravi buffalo testes, spermatozoa were not present in the tubule lumens analyzed here. testis of murrah buffalo bulls at 18 months of age characterized by an immature sexual phase (a and b). we observed solid testicular cords with gonocytes (a) and undifferentiated supporting cells (s). the testicular parenchyma at 24 months contained seminiferous tubules with lumens and germinative epithelia present at different stages of the seminiferous epithelial cycle (fig. 2. different cell types in the seminiferous epithelium of the murrah buffalo at 24 months of age. photomicrographs of cross sections of the seminiferous tubules represent the stages of the cycle of the seminiferous epithelium. stage one (a), two (b), three (c), four (d), five (e), six (f), seven (g) and eight (h) represent the following groups of cells : primary spermatocytes at the pre - leptoteno / leptoteno (pl) stage, primary spermatocytes at the pachytene (p) stage, round spermatids (ar), elongated spermatids (al), primary spermatocytes at the zygotene (z) stage, primary spermatocytes at the diplotene (d) stage, residual bodies (cr) and spermatozoa (e). all the stages presented spermatogonia type a (a) and sertoli cells (s). hematoxylin - eosin staining.). the following cell types were identified : sertoli cells, spermatogonia, pre - leptotene / leptotene spermatocytes, pachytene spermatocytes, zygotene spermatocytes, diplotene spermatocytes, round spermatids and luminal spermatozoa (fig. these cells could be classified into eight stages of cellular associations according to the tubular morphology method. bongso noted that the spermatozoa in the tubular lumen of swamp buffalo had the same morphology at 16 months. in crossbred buffalo (mediterranean jaffarabadi), this morphology was observed at 13 months. ohashi showed the onset of the spermatogenic process in buffalo at 9 months. at 24 months, our histological analysis of the spermatogenic process indicated that these animals were not sexually mature. different cell types in the seminiferous epithelium of the murrah buffalo at 24 months of age. photomicrographs of cross sections of the seminiferous tubules represent the stages of the cycle of the seminiferous epithelium. stage one (a), two (b), three (c), four (d), five (e), six (f), seven (g) and eight (h) represent the following groups of cells : primary spermatocytes at the pre - leptoteno / leptoteno (pl) stage, primary spermatocytes at the pachytene (p) stage, round spermatids (ar), elongated spermatids (al), primary spermatocytes at the zygotene (z) stage, primary spermatocytes at the diplotene (d) stage, residual bodies (cr) and spermatozoa (e). all the stages presented spermatogonia type a (a) and sertoli cells (s). hematoxylin - eosin staining. mean body and testicular biometric parameters in murrah buffalo bulls at 18 and 24 months of ageage groupbw (kg)sc (cm)tw (cm)tl (cm)twd (cm)tt (cm)tc (cm)g1 (18 mo)270.25 14.5918.75 1.521.11 2.795.07 0.362.72 0.242.48 0.258.09 0.60g2 (24 mo)446.70 32.5625.30 3.2177.66 38.57.13 1.253.73 0.693.55 0.5112.58 1.71means with different superscript letters are significantly different (p<0.05) according to analysis of variance and the student 's t test (5%). bw = body weight, sc = scrotal circumference, tw = testicular weight, tl = testicular length, twd = testicular width, tt = testicular thickness, tc = testicular circumference.. although the sc and body weight differed significantly between the two age groups analyzed, there was an increase in the sc with increasing body weight. additional testicular biometric parameters are often assessed in domestic animals, particularly those animals used for livestock production because testicular parameters are an important part of the andrological evaluation. among the available biometrics for testes, sc is one of the most commonly employed because it is easy to measure and highly correlated with body weight. means with different superscript letters are significantly different (p<0.05) according to analysis of variance and the student 's t test (5%). bw = body weight, sc = scrotal circumference, tw = testicular weight, tl = testicular length, twd = testicular width, tt = testicular thickness, tc = testicular circumference. as shown in table 2table 2. correlations between age, body weight and testicular parameters in murrah buffalo bulls at 18 and 24 months of age agebwsctwtltwdtttcage0.975 0.854 0.776 0.808 0.777 0.847 0.904bw0.843 0.851 0.827 0.775 0.838 0.904sc0.5170.963 0.932 0.898 0.931tw0.4950.3790.4790.572tl0.982 0.949 0.963twd0.971 0.965tt0.978tcbw = body weight, sc = scrotal circumference, tw = testicular weight, tl = testicular length, twd = testicular width, tt = testicular thickness, tc = testicular circumference. significant (p<0.05)., there was a significant correlation between sc, age and body weight. this result has been described previously in river, swamp and murrah buffalo. bw = body weight, sc = scrotal circumference, tw = testicular weight, tl = testicular length, twd = testicular width, tt = testicular thickness, tc = testicular circumference. significant (p<0.05). the data presented here differ from the findings in cattle, for which the sc is particularly important in identifying bulls with high reproductive capacity. neither scrotal circumference nor body weight should be used alone as a routine and reliable parameter to predict the reproductive capacity in buffalo. the mean values and standard deviation for tw, tl, twd, tt and tc, relative to buffalo age, are shown in table 1. although the average tw did not differ between the two groups, significant differences were observed for tl, twd, tt, and tc (p<0.05). furthermore, these variables exhibited high correlation coefficients, as noted in table 2. body weight, age, sc, tw, tl, twd, tt and tc were positively correlated (p<0.05). this finding differs from observations made in piau pigs, rams and cattle, in which a high correlation was described for all parameters. the strength of the correlations obtained suggests that tl, twd, tc and tt are useful parameters for the selection of breeding animals. under the husbandry conditions described here, both body and testicular development of buffalo occurred more slowly than the typical growth rates for bos taurus. the data for bos indicus, however, suggest a similar rate of body and testicular development to the rates observed for buffalo in the present study. these similarities can be attributed to the fact that zebu cattle are also raised under extensive farming conditions, and therefore suffer from the same nutritional consequences, particularly dietary protein deficiencies, which can lead to developmental delays. in conclusion, our analysis of biometric testicular parameters in murrah buffalo revealed that the tl, twd, tc and tt are useful indicators for the selection of bulls for breeding. | abstract buffalo are an economically important source for meat and milk production, especially in brazil. however, important aspects of their biology remain unknown thus far. herein, we describe the reproductive characteristics of male murrah buffalo (bubalus bubalis) raised under extensive management conditions by applying biometrics associated with testicular weight. we analyzed seven males, divided into two groups : g1, which consisted of four 18-month - old animals, and g2, which consisted of three 24-month - old animals. testicular development occurs slowly in murrah buffalo, suggesting a delay of sexual maturity. the biometric testicular parameters analyzed were scrotal circumference, testicular weight, testicular length, testicular width, testicular thickness and testicular circumference. our data indicate strong correlations between sc, age and body weight, and additional significant relationships were identified between body weight, age and other testicular parameters. thus, these parameters are suitable indicators when selecting bulls for breeding purposes. |
central serous chorioretinopathy (cscr) manifests as neurosensory detachment of the macula and can be attributed to focal or multifocal leakage in the retinal pigment epithelium (rpe). fibrin accumulation in the subretinal space is an unusual and heretofore unreported visually damaging manifestation of severe cscr. the patient was followed up with the use of biomicroscopy, fluorescein angiography, and optical coherence tomography (oct). a 32-year - old woman was referred to our department complaining of metamorphopsia and decreased visual acuity in the right eye. best - corrected visual acuity (bcva) was 20/40 in the right eye and 20/20 in the left eye. oct showed a steep neurosensory retina elevation with a highly reflective material accumulation in the subretinal space, presumably fibrin. since most of these cases resolve spontaneously, the patient was kept under observation ; 1 month later, the fibrin accumulation had expanded subfoveally (bcva 20/200). after the initial injection, bcva improved to 20/50 and, after the 3 injections, to 20/30. two months later (bcva 20/30), fresh leakage was observed at the margin of the original lesion, and an additional intravitreal ranibizumab injection was performed. after another 2 months, bcva stabilized at 20/25 and remained stable throughout the 12 months after the initial injection. prompt recognition of cscr complicated by subretinal fibrin and immediate intervention may result in recovery from this potentially devastating complication. ranibizumab may be an alternative treatment option in the management of refractory cscr complicated by subretinal fibrin accumulation. central serous chorioretinopathy (cscr) manifests as neurosensory detachment of the macula and can be attributed to focal or multifocal leakage in the retinal pigment epithelium (rpe). while described as a disease of healthy, type - a personality men, it has also been reported in women, and is associated with pregnancy. it typically develops in the third trimester and resolves spontaneously within 12 months after delivery. fibrin accumulation in the subretinal space is an unusual visually damaging manifestation of severe cscr [1, 2, 3 ]. schatz., reported 6 cases (average age 40 years) with cscr containing fibrin in the subretinal space that later developed into a subretinal fibrotic scar, with subsequent subretinal fibrosis and severe visual loss (20/400 or worse). in this case report, we present the rapid and impressive response to ranibizumab in a case of cscr complicated by accumulation of subretinal fibrin. a 32-year - old woman was referred to our department complaining of metamorphopsia and decrease of visual acuity in the right eye. best - corrected visual acuity (bcva) was 20/40 and 20/20 in the right and left eye, respectively. in addition, fluorescein angiography (fa) showed partial dye masking in the affected area during the early phase followed by ink - blot leakage and subsequent pooling in the superior margin, not involving the fovea (fig. 1). in the left eye, we observed old window defects and two small rpe detachments. optical coherence tomography (oct) showed a serous elevation of the retina and a shallow rpe detachment, with subretinal accumulation of highly reflective material (presumably fibrin). our initial diagnostic approach was to follow up the patient keeping in mind that this was a case of cscr complicated by fibrin deposition in the subretinal space. after 1 month, bcva decreased to 20/200 and the oct revealed a more irregular subretinal accumulation of highly reflective material that was now involving the fovea (fig. after the first injection, bcva improved to 20/50 while the fa leakage had nearly diminished. two months later, visual acuity was 20/30, while metamorphopsia was still persisting. during the follow - up period, bcva stabilized at 20/25 and remained stable for the following 12 months, without any apparent significant fa leakage (fig. the presence of fibrin with spontaneous resolution in the majority of cscr cases has been previously reported. subretinal fibrin and other extracellular matrix molecules appear to stimulate the rpe to undergo fibrous metaplasia, which results in subretinal fibrotic scar formation and other sequelae, all of which can lead to severe visual loss. in our patient, a steep neurosensory retinal elevation with an extrafoveal subretinal deposition of a fibrin - like material was observed several months after delivery. subsequent fibrinolysis of this meshwork resulted in an amorphous, homogenous subretinal accumulation with involvement of the fovea and significant reduction of the visual acuity. due to the presence of subfoveal fluid, we decided to offer a 3-injection course of intravitreal ranibizumab which resulted in rapid subretinal material (fibrin) absorption and improvement of both bcva and foveal contour confirmed by oct. according to the literature [1, 2 ], in the majority of relevant cases, there is gradual resolution of this fibrin - like material accumulation as a result of a resumption of normal rpe function (in pregnant women, in particular it, resolves spontaneously within 12 months after delivery). schatz., reported 6 cases (average age 40 years) with cscr containing fibrin in the subretinal space which developed into a subretinal fibrotic scar. scar formation was followed by a tenting up of the macula, vascularization of the fibrosis (subretinal neovascularization), or a retinal pigment epithelial rip. four of the 7 eyes with subretinal fibrosis had severe visual loss (20/400 or worse). ranibizumab has been reported to be an effective treatment of choroidal neovascularization related to chronic cscr. however, residual intraretinal or subretinal fluid in addition to increased choroidal permeability persisted in a small case series. ranibizumab has been shown to have no significant direct effect on the choriocapillaris in animal models. conversely, recent evidence from in vitro studies suggests that ranibizumab may decrease rpe barrier function by temporarily enhancing rpe permeability for a small number of days. in this case, it is not clear whether intravitreal ranibizumab contributed to the dissolution of the fibrin - like material, and it is conceivable that resolution may have occurred without any intervention. in contrast, the rapid response to ranibizumab may suggest that this anti - vegf agent may be an alternative treatment option in the management of refractory cscr complicated by subretinal fibrin accumulation. further experimental studies investigating the potential anti - vegf effect on modulating rpe function (e.g. ion pump activation) are required to validate this hypothesis. none of the authors has received financial support for the completion of this study. regarding the results of this study, none of the authors has any proprietary interest. | purposecentral serous chorioretinopathy (cscr) manifests as neurosensory detachment of the macula and can be attributed to focal or multifocal leakage in the retinal pigment epithelium (rpe). fibrin accumulation in the subretinal space is an unusual and heretofore unreported visually damaging manifestation of severe cscr.methodsthe patient was followed up with the use of biomicroscopy, fluorescein angiography, and optical coherence tomography (oct).resultsa 32-year - old woman was referred to our department complaining of metamorphopsia and decreased visual acuity in the right eye. best - corrected visual acuity (bcva) was 20/40 in the right eye and 20/20 in the left eye. biomicroscopy revealed an irregularly shaped foveal elevation and wrinkling in the right eye. oct showed a steep neurosensory retina elevation with a highly reflective material accumulation in the subretinal space, presumably fibrin. our diagnosis was cscr complicated by subretinal fibrin accumulation. since most of these cases resolve spontaneously, the patient was kept under observation ; 1 month later, the fibrin accumulation had expanded subfoveally (bcva 20/200). the patient was offered 3 intravitreal ranibizumab injections. after the initial injection, bcva improved to 20/50 and, after the 3 injections, to 20/30. two months later (bcva 20/30), fresh leakage was observed at the margin of the original lesion, and an additional intravitreal ranibizumab injection was performed. after another 2 months, bcva stabilized at 20/25 and remained stable throughout the 12 months after the initial injection.conclusionsprompt recognition of cscr complicated by subretinal fibrin and immediate intervention may result in recovery from this potentially devastating complication. ranibizumab may be an alternative treatment option in the management of refractory cscr complicated by subretinal fibrin accumulation. |
a evoluo dos aparelhos fotopolimerizadores pode ser notada nos diferentes sistemas introduzidos recentemente no mercado. a tecnologia apresentada pelos aparelhos led promete maior tempo de vida til, no gerar aquecimento e produzir luz especfica para a ativao da canforoquinona. no entanto, ainda so necessrios estudos complementares para se conhecer a real efetividade destes aparelhos na polimerizao dos materiais. neste trabalho foi verificada a microdureza de 4 cores (b-0,5, b-1, b-2 e b-3) da resina composta filtek z-250 (3 m espe) quando polimerizadas com 4 fontes de luz, sendo uma halgena (ultralux - dabi atlante) e trs led (ultraled - dabi atlante, ultrablue - dmc e elipar freelight 3 m espe). os 192 corpos - de - prova foram distribudos em 16 grupos e os materiais foram inseridos em nico incremento em matrizes cilndricas de 4 mm x 4 mm, sendo polimerizados pelo tempo preconizado pelo fabricante. em seguida, foram submetidos ao teste de microdureza na superfcie superior e inferior dos cilindros. os valores de dureza obtidos foram submetidos anlise de varincia e ao teste de tukey ao nvel de 5%. foi observado que a dureza dos corpos - de - prova variou conforme a cor do material e aparelhos utilizados. o aparelho led que emite maior intensidade luminosa proporcionou a obteno dos maiores valores de dureza, com o croma b-0,5 possibilitando a melhor polimerizao. tambm foi observado que a regio do fundo dos corpos - de - prova foi mais sensvel mudana das cores. a intensidade de luz dos fotopolimerizadores led fundamental para seu bom funcionamento, principalmente quando empregadas resinas com croma mais acentuado. the use of composite resins has appeared in dentistry as a response to the expectations to achieve a material that might fulfill the requirements of the oral environment and also allow achievement of restorations with proper function and especially esthetics19. light cured composite resins offer several advantages compared to early restorative systems, and their curing depends on application of light on their surface11,27. curing depth is often considered a primary factor for clinical success of composite resin restorations, since it directly affects the physical properties of materials and longevity of restorations. the factors that may affect the curing of resin materials according to the literature7,17,25,28 include those directly related to the restorative material, including composite resin shade, amount of photoinitiators, organic and filler matrixes. similarly, light curing units also play a fundamental role for proper curing, especially concerning the period of exposure and general status of the equipment. therefore, in an attempt to optimize the utilization of light cured composites, manufacturers of light curing units have developed different curing systems, including those with soft start mechanism, which gradually increase light intensity, and those that emit a constant light intensity higher than 1,000mw / cm. later, light emitting diode (led) light curing units were developed, which apply a lower light intensity than conventional light curing units, yet are able to cure resin materials, because of light emission in specific wavelength for activation of camphorquinone (450 to 480nm)10,14. however, pires,.21 (1993), employing halogen light curing units, observed a strong correlation between light intensity and microhardness, revealing a small depth of curing or reduction in microhardness values when the light intensity applied to the composite was reduced. however, more recent studies (price et.al., 2004)24 revealed that when the energy density (j / cm) is maintained, the increase in intensity is no related with the curing depth. kawaguchi14 (1994) investigated halogen light curing units following guidelines of the international organization standardization (iso) dis 4049, and highlighted that the coefficient of light transmission of different materials also influences their curing, being that some materials may be cured in depth with lower light intensity than others. within this context, the factors that may affect the curing of materials include those related to the restorative material, including the resin shade, amount of photoinitiators, organic and inorganic matrix ; the operator, including the distance and orientation of light beams and restorative technique ; and types of light curing units, concerning the emission spectrum and association between light intensity, period of exposure and general status of the equipment1,7,11. based on these data, doubts have arisen : are led curing units effective ? what is the performance of these units to cure different resin shades ? all these factors and doubts indicate the importance to investigate the real curing ability of different light curing systems available in dental market. moreover, composite resins currently available are highly complex and technically sophisticated materials. therefore, to avoid selection of a certain product by the subjective preference of the operator, it is important to understand its physical and mechanical properties, as well as its performance after utilization of different light curing units17. therefore, the present study evaluated the microhardness of different composite resin shades, after utilization of a halogen light source and three light emitting diode (led) curing units, considering readings performed on the surface close to the light source and also at 4 mm in depth. led units emitting similar wavelengths with different power densities were compared with a conventional halogen light unit that emits a wider light spectrum. the null hypothesis to be tested was that either the shade composite resins or curing equipment would not interfere with the microhardness values. a total of 192 specimens were fabricated, which were distributed into 16 study groups. specimens were fabricated with aid of acrylic cylindrical templates with a central perforation measuring 4 mm in diameter and 4 mm in height. for fabrication of specimens, templates were positioned on a glass slab and completely filled in a single increment, with some excess, with the composite resin filtek z-250 (3 m espe) of different shades (b-0.5, b-1, b-2 and b-3). then, a polyester strip (3 m espe) and another glass slab were positioned on composite resin, with application of force against the composite resin template. after 10 seconds, the top glass slab was removed, the polyester strip was kept in position and each specimen was submitted to the light emitted by the light curing units for 20 seconds, as recommended by the manufacturers. specimens fabricated with the b-0.5 shade were light cured for 30 seconds, as recommended by the manufacturer for this shade. light curing units employed were the ultraled unit (dabi - atlante), with 7 leds and light intensity of 200mw / cm ; ultrablue i unit (dmc), with 7 leds and 140mw / cm ; elipar freelight unit (3 m espe), with 19 leds and 400mw / cm ; and ultralux halogen light unit (dabi atlante) with 380mw / cm. all units were measured with radiometer cure demetron and were connected to a voltage stabilizer revolution ii (sms) to allow maintenance of their power. then, templates filled with composite resin were labeled and marked on the top surface for identification of the region closest to the light source. thereafter, specimens were cleaned with a detergent solution in ultrasound, branson 2210, for 10 minutes, to remove debris occasionally accumulated during handling of specimens. for achievement of a surface with no staining surface microhardness of specimens was measured with hmu-2000 shimadzu, with utilization of a knoop indenter of 20 g for 5s. this way, 3 indents were performed on top surface and further 3 on bottom surface, which was more distant from the light source. considering that each of the 16 study groups had 12 specimens that received 3 indents, original data comprised 576 microhardness values for each surface. statistical analysis was performed by calculation of mean values of 3 measurements performed for each surface, adding up to 192 independent data analyzed for the top surfaces and 192 for the bottom surfaces. results were submitted to analysis of variance ; whenever a significant difference was found, tukey test at the 5% level was applied. table 1 presents original microhardness means and standard deviation of each study group with regard to the top and bottom aspects of specimens. analysis of both surfaces with regard to different light curing units indicated a statistically significant difference for all study groups ; elipar freelight unit (3m - espe) provided highest values of surface hardness, regardless of the composite shade. the lowest values were achieved with the ultrablue unit (dmc), whereas the ultralux (dabi atlante) and ultraled units (dabi atlante), respectively, presented intermediate results (tables 2). dms (top) = 0.0235 dms (bottom)= 0.0242 however, when the composite resin shades were considered at the top surfaces, groups of b-0.5 shade presented highest hardness values, whereas other shades presented similar results. on the other hand, for the bottom surfaces, all had a significant effect on the efficacy of the units. therefore, groups of specimens of b-0.5 shade presented the highest means, followed by shades b-1, b-2 and b-3, respectively (table 3). currently, there has been considerable emphasis on the concentration of photoinitiators and wavelength employed to cure resin materials, since they are in charge of conversion of monomers into resin polymers, which determine the curing of resin material. it is known that concentration of camphorquinone varies between materials and may be altered according to shade and translucency of the material6. thus, curing of resin materials has been subject of several studies with different methodologies 4,5,20. these include microhardness test, which is one of the most often employed methods and is able to provide reliable results and reproducibility to the study. according to shortall and harrington26 (1996), intrinsic characteristics of each material, as composition, hue and shade, may interfere with the curing depth ; an appliance able to transmit adequate light intensity with correct wavelength is required to provide good results11. with regard to the appliances analyzed in the present study, halogen light unit is the most widely employed and, because it works in a wider light spectrum, besides curing the restorative material, it may also heat the tooth and composite resin during the process23. these appliances usually work in wavelengths of 400 nm to 700 nm ; however, only wavelength nearly 470 nm is strongly absorbed by photoinitiator (camphorquinone)23. period of exposure also plays an important role in the curing process, since the surface hardness of composite resin is significantly increased when exposed for longer periods10,15,26. moreover, caughman,.5 (1995) and feilzer,.7 (1995) also agree that light intensity, wavelength and time of exposure are critical variables for achievement of maximum curing of composite resins. considering these observations, analysis of microhardness values achieved i.e. on this may be related to the wavelength emitted by this unit, which according to its manufacturer coincides with the maximum absorption of camphorquinone, explaining its better performance compared to halogen light units. compared to other led units tested, elipar freelight unit probably was more successful because of the higher light intensity emitted. within this context, according to vieira,.34 (1999) and briso4 (2003), light emission in low intensity yields inadequate light curing of composite resins, even if the time recommended by the manufacturers is followed, and may negatively influence physical properties and clinical performance of these materials. similarly, ultralux unit presented better performance than ultraled and ultrablue units because of higher light intensity. the lower mean hardness values found for ultraled and ultrablue units, which are also led units, are assigned to lower light intensity emitted and improper wavelength for curing composite resins employed. frentzen,.9 (2001) compared a halogen light unit and a led unit and reported that led unit achieved higher hardness values ; however, authors did not mention the light intensity emitted by the units, which impairs comparison with the present results. studies as that conducted by pereira,.20 (2001) corroborate the present findings, concluding that units emitting higher light intensities have a stronger ability of curing and thereby allow achievement of higher hardness values. these aspects may also be taken into account for analysis of the ultrablue unit, which was less effective than all other units tested. bosquiroli3 (2003) highlighted that resins may have different photoinitiators, and thus the emission spectrum of the equipment may be na important factor for the final properties of restorations. according to the author, the energy required for each composite should be indicated on the label, and similarly the light curing units should indicate the light intensity emitted and corresponding wavelength. these observations are extremely relevant for led units applied on materials containing camphorquinone as photoinitiator. however, other photoinitiators require energy at other wavelengths and in these cases some units currently available will not effectively cure these materials32. when the influence of shade on microhardness is individually considered, the results demonstrate that shade b-0.5 presented higher values than other shades. this is primarily related to the period of exposure suggested by manufacturer, which is 10 seconds more than recommended for shades b-1, b-2 and b-3. according to baharav,.1 (1997) and pereira,.19 (2000), the time of exposure to light is the most significant aspect influencing higher hardness values and consequently greater curing depth. there was also significant reduction in microhardness on the bottom aspect of specimens in all study groups. despite of that, in general the bottom aspect presented the same tendency observed at the top surface, i.e. units with higher light intensities (elipar freelight and ultralux, respectively), were better than the others (ultraled and ultrablue), with a statistically significant difference in the performance of all units. it should be highlighted that low light intensities may only cure composite superficially, therefore impairing the clinical performance of restorations and giving the false impression that the material is well cured1. this information was demonstrated by the observation that, on the bottom surface of specimens, the composite shade influenced the performance of units. thus, on the bottom surface, shade b-0.5 provided higher hardness values, followed by resins b-1, b-2 and b-3, respectively. this was probably due to the distance from light source, as well as because the curing depth is inversely proportional to hue value. therefore, darker materials possibly impair light penetration into the deepest regions of specimens, as also observed by tanoue.29 (2001). considering the means achieved by the groups, this effect seems to be more significant in specimens submitted to units that emit lower light intensities (g - viii and g - xii). this was also noticed by ferracane,.8 (1997), who related the curing depth of different composite resins to shade and translucency of materials, concluding that lighter shades allow greater curing depth. in fact, the present results are in agreement with the findings of cook and standish6 (1983) and kanca iii13(1986) who stated that pigments employed to provide shades to restorative materials may absorb the light passing through the resin and impair curing. studies conducted by pollack and lewis22 (1981), thirta,.30 (1982), cook and standish6 (1983) and swartz,.27 (1983), also reveal that light cured composite resins of dark shades require longer periods of exposure, and curing in thinner layers is recommended to allow higher hardness values. however, despite the present considerations, it should be highlighted that lohbauer,.16 (2005) tested several photoinitiator equipments and stated that conventional halogen light units allow less loss of mechanical properties of dental composites, even though led units have been presenting encouraging outcomes when employed with compatible resin materials inserted with proper thickness2,12,18,31,33. thus, it seems evident that selection of light curing unit should be considered an important step to conduct clinical work with resin materials. it should also be mentioned that many types of units are introduced in the market every year, whose manufacturers state their proved efficiency, low cost and easy maintenance. however, professionals should base their decisions in well - designed scientific studies, besides searching for constant update, not being limited to reading folders and technical profiles supplied by manufacturers and/or representatives. these aspects demonstrate that correct selection should be based on impartial studies, and materials introduced in the market may not have been adequately tested for utilization in the dental clinic. on the basis of results achieved, the following could be concluded : in both led and halogen light units, the microhardness values ranged according to the shade of resin and appliances employed ; shade was inversely proportional to the surface hardness of resin, with the highest values observed for shade b-0.5 ; led units with low light intensity provided lower hardness values than high intensity units ; aspects distant from the light source were more sensitive to changes in shades. the emission of light at a specific wavelength for activation of camphorquinone by a unit did not lead to successful curing of the materials investigated. | introduction : the evolution of light curing units can be noticed by the different systems recently introduced. the technology of led units promises longer lifetime, without heating and with production of specific light for activation of camphorquinone. however, further studies are still required to check the real curing effectiveness of these units.purpose:this study evaluated the microhardness of 4 shades (b-0.5, b-1, b-2 and b-3) of composite resin filtek z-250 (3 m espe) after light curing with 4 light sources, being one halogen (ultralux dabi atlante) and three led (ultraled dabi atlante, ultrablue dmc and elipar freelight 3 m espe).methods:192 specimens were distributed into 16 groups, and materials were inserted in a single increment in cylindrical templates measuring 4 mm x 4 mm and light cured as recommended by the manufacturer. then, they were submitted to microhardness test on the top and bottom aspects of the cylinders.results:the hardness values achieved were submitted to analysis of variance and to tukey test at 5% confidence level. it was observed that microhardness of specimens varied according to the shade of the material and light sources employed. the led appliance emitting greater light intensity provided the highest hardness values with shade b-0.5, allowing the best curing. on the other hand, appliances with low light intensity were the least effective. it was also observed that the bottom of specimens was more sensitive to changes in shade.conclusion:light intensity of led light curing units is fundamental for their good functioning, especially when applied in resins with darker shades. |
stress echocardiography (stress echo) is widely applied for evaluating cardiac function [1, 2 ]. motion of the left ventricle is assessed by comparing ultrasound images taken in rest and in stress stages. in the past decade, real - time three - dimensional (3d) imaging has become available for quantitative and objective assessment of cardiac function [3, 4 ]. 3d imaging offers the opportunity to overcome the limitations of traditional two - dimensional stress echo. with 3d imaging, non - foreshortened anatomical views however, due to suboptimal image quality, lower spatial and temporal resolution, and the sheer amount of data, quantitative analysis by hand is tedious and subjective. automated analysis methods may allow objective and faster assessment of clinical parameters such as left ventricular volume, ejection fraction and wall motion. in practice, the clinician makes a diagnosis using expert knowledge, gathered by analysing many patient images [6, 7 ]. to develop successful automated methods which can deal with different pathologies and varying image quality, we present automated methods for 3d stress echo which make use of knowledge gained from databases of patient data. the knowledge is encompassed in so - called statistical models. using principal component analysis, patient variability can be represented concisely as mathematical descriptors that form a vector of orthogonal parameters. the parameters to be modelled (for example, spatial coordinates of the endocardial border) essentially, the methods are geared towards fitting the image data at hand to the model. in other words, the variation is estimated according to a set of extra criteria (such as image intensity values, our proposed analysis scheme consists of three steps : identification of correct anatomical views in 3d data, detection of endocardial borders from which motion can be derived, and automated classification of motion. in addition, we present a software package which we have developed dedicated to analysis of 3d stress echo. first we describe a method for detecting four - chamber, two - chamber, and short - axis views in a 3d image. these views are modelled via appearance (image intensity values) and pose parameters (translation, rotation, and scaling). these parameters are identified by fitting the database model to the image at hand within an optimisation framework. the views in a rest image can be used to extract the anatomically corresponding views in a stress image of the same patient. this is achieved via image registration, so that rest and stress images are aligned via minimisation of an objective criterion (in this case the correlation between image intensity values). qualitative and quantitative evaluation in 20 end - diastolic and 20 end - systolic images shows that the views found are in many cases better (18%) or similar (75%) to the manual identification of views. anatomically aligned views are thus obtained automatically, providing more possibilities for accurate visualisation and quantification. detection of endocardial borders provides important clinical parameters such as volume, ejection fraction, and wall motion. here, the active appearance model technique [8, 12 ] is adapted to detect borders in an end - diastolic 3d echocardiogram. the endocardial borders are modelled via spatial coordinates, distributed in a cylindrical and spherical representation which is suited to the shape of the left ventricle (fig. 1). image intensity values are mapped to a normal (gaussian) distribution for appropriate statistical analysis. the model represents variations of ventricular shapes and the typical appearance of the ventricle and myocardium in echocardiograms, including the typical artifacts. the model is trained for image analysis by estimating the relation between model parameters and image change via regression analysis : in the actual matching, the intensity difference between model and image is used to update the model s parameters and drive the model closer to the image (fig. 2). evaluation on 99 patient images shows a successful matching in 91% of cases, with a median surface error of 2.65 mm (average 2.91 mm, standard deviation 1.03 mm). 1modes of variations of an appearance model, created by varying the model descriptors one at a time. the appearance model consists of a shape (spatial coordinates) and a texture (image intensity values) component. the average and the three most principal modes (2 standard deviations) are shownfig. the initial position of the borders and the results after 10, 20, and 39 (final) results are shown modes of variations of an appearance model, created by varying the model descriptors one at a time. shape (spatial coordinates) and a texture (image intensity values) component. the average and the three most principal modes (2 standard deviations) are shown endocardial border detection in a 3d image. the initial position of the borders and the results after 10, 20, and 39 (final) results are shown the borders throughout the cardiac cycle are obtained by tracking the motion of the cardiac wall. a 3d optical flow algorithm is applied, which uses spatial and temporal intensity gradients to track the endocardial border between two time frames. to ensure an overall temporal continuity, the tracking method takes into account physically plausible motion, learned statistically from a patient database. typical image artifacts, such as echo drop - out and near - field artifacts, are detected and dealt with appropriately using an expectation maximisation based classification approach. the method is capable of tracking the endocardial borders accurately (in 35 patients : surface errors : 1.2 0.5 mm, volume errors : 1.4 6.7 ml, ejection fraction errors : 0.9 4.8%). statistical models of the endocardial border motion throughout the cardiac cycle can be used to discriminate between normal and pathological motion patterns. the statistical model is transformed using the orthomax rotation criterion, so that the local wall motion can be represented using fewer descriptors (fig. wall motion classification is demonstrated in 129 two - dimensional echocardiographic sequences. using these models,, the method can be applied directly to 3d borders ; the evaluation is a subject of further investigation. 3modes of variation as calculated by principal component analysis (pca), which depict global variations in endocardial border motion, versus the local modes of variation after orthomax rotation. the local modes are more concise and suitable for automatic classification of wall motion abnormalities modes of variation as calculated by principal component analysis (pca), which depict global variations in endocardial border motion, versus the local modes of variation after orthomax rotation. to enable 3d stress echocardiography in routine clinical practice, we have also developed a software package dedicated to 3d stress echo. the essential functionality for analysing 3d stress echo is investigated : side - by - side, synchronised display of anatomically aligned rest and stress images (fig. 4). interobserver agreement for segmental myocardial ischaemia is better using the dedicated software (96%) than commercially available software (79%) without these functionalities. 4software dedicated to 3d stress analysis, allowing side - by - side viewing of images acquired at rest and in varying stages of stress. anatomical four - chamber, two - chamber, short - axis, and long - axis views are shown software dedicated to 3d stress analysis, allowing side - by - side viewing of images acquired at rest and in varying stages of stress. anatomical four - chamber, two - chamber, short - axis, and long - axis views are shown in this paper, we have presented automated methods for identification of anatomical views, detection of endocardial borders, and classification of wall motion abnormalities. in particular, these methods are better capable of handling typical image artifacts and following expert tracing conventions. we have also demonstrated the advantages of using dedicated software for the analysis of 3d stress echo in the clinical setting. based on this research, opportunities remain for further development, e.g. in the analysis of contrast echocardiograms, technical enhancement of image quality and spatiotemporal resolution, and improvement of clinical workflow. the developed tools can provide useful quantitative and objective parameters to help the clinical expert in the diagnosis of left ventricular function. | real - time three - dimensional (3d) ultrasound imaging has been proposed as an alternative for two - dimensional stress echocardiography for assessing myocardial dysfunction and underlying coronary artery disease. analysis of 3d stress echocardiography is no simple task and requires considerable expertise. in this paper, we propose methods for automated analysis, which may provide a more objective and accurate diagnosis. expert knowledge is incorporated via statistical modelling of patient data. methods for identifying anatomical views, detecting endocardial borders, and classification of wall motion are described and shown to provide favourable results. we also present software developed especially for analysis of 3d stress echocardiography in clinical practice. interobserver agreement in wall motion scoring is better using the dedicated software (96%) than commercially available software not dedicated for this purpose (79%). the developed tools may provide useful quantitative and objective parameters to assist the clinical expert in the diagnosis of left ventricular function. |
(also known as eclipta prostrata roxb.) belongs to the asteraceae family and is commonly known as false daisy in english and bhringoraj or bhringraj in bangladesh and india. it is known in the three major forms of traditional medicinal systems in the indian subcontinent, namely, ayurveda, unani, and siddha, as bhringoraaja, bhangraa, and karissalaankanni, respectively. the full taxonomic hierarchy is shown below in table 1. considering the ethnomedicinal significance of the plant, it was of interest to review the ethnopharmacological reports on the plant and selective phytoconstituents through data base searches (pubmed, scopus, google scholar). the plant and plant parts are used fortreatment of a variety of diseasesby folk medicinal practitioners and tribal medicinal practitioners of the indian subcontinent. ethnomedicinal uses of the plant have been reported from bangladesh, india, nepal, and pakistan (table 3). the available ethnomedicinal reports indicate that although there are a variety of diseases treated with the plant or plant parts, the major uses are limited to treatment of gastrointestinal disorders, respiratory tract disorders (including asthma), fever, hair loss and graying of hair, liver disorders (including jaundice), skin disorders, spleen enlargement, and cuts and wounds. the protective effect of the plant against carbon tetrachloride - induced acute liver damage has been reported. coumestans (wedelolactone and demethylwedelolactone) have been mentioned as the possible components behind the protective effect on liver as well as against liver disorders ; both compounds exhibited antihepatotoxic activity in assays employing ccl4(carbon tetrachloride), galn(galactosamine), and phalloidin - cytotoxicity in rat hepatocytes. alcoholic extract of e. alba was found to have good antihepatotoxic activity as assessed in ccl4-induced liver damage in albino rats through liver to body weight ratio, pentobarbitone sleep time, serum levels of glutamate pyruvate transaminase (gpt) and glutamic oxaloacetic transaminase (got), alkaline phosphatase (alp), and bilirubin. in ccl4-administered rats, there was an increase in liver weight, pentobarbitone sleep time, and elevated got, gpt, salp, and serum bilirubin levels. the alcoholic extract at a dose of 200 mg / kg significantly reversed these effects. the hepatoprotective effect of ethanol / water (1 : 1) extract (ea) of the plant has been studied in ccl4-induced hepatotoxicity in rats. ea significantly counteracted ccl4-induced inhibition of the hepatic microsomal drug metabolising enzyme amidopyrine n - demethylase and membrane bound glucose 6-phosphatase but failed to reverse the very high degree of inhibition of another drug metabolising enzyme aniline hydroxylase. the loss of hepatic lysosomal acid phosphatase and alkaline phosphatase by ccl4 was significantly restored by ea. it was suggested that the hepatoprotective effect of ea may be due to its regulating of the levels of hepatic microsomal drug metabolising enzymes. the ethanolic extract of leaves of the plant has been fractionated into three parts (hot water insoluble (eai), ethyl acetate fraction of hot water soluble (eaii), and remaining hot water soluble fraction (eaiii)) and each fraction studied for hepatoprotective activity against ccl4-induced hepatotoxicity in rats and mice. hepatoprotective activity was determined on the basis of their effects on parameters like hexobarbitone sleep time, zoxazolamine paralysis time, bromsulfalein clearance, serum transaminases (gpt, got), and serum bilirubin. all the experimental parameters were increased by ccl4 ; fraction eaii (1080 mg / kg, p.o.) dose - dependently and significantly reversed these increases. fraction eaii was found to contain coumestan wedelolactone and demethylwedelolactone as major components with apigenin, luteolin, 4-hydroxybenzoic acid, and protocatechuic acid as minor constituents. hepatitis c virus (hcv) inhibitory activity has been reported for e. alba extract. phytochemical analysis of the extract revealed the presence of three compounds, namely, wedelolactone, luteolin, and apigenin. these compounds exhibited dose - dependent inhibition of hcv replicase in vitro, and anti - hcv replication activity in the cell culture system. the results suggest that the plant or individual components have the potential to be used against hcv. ethanol extract of whole plant was tested for hepatoprotective effect against paracetamol - induced hepatotoxicity in mice. treatment with 100 and 250 mg of the extract per 100 kg body weight showed significant reductions in paracetamol - induced serum alanine aminotransferase (alt, also known as got) levels. at the same time, histopathological studies showed marked reductions in paracetamol - induced fatty degeneration and centrizonal necrosis in liver of extract - treated mice. an alcoholic extract of freshly collected eclipta alba exhibited dose - dependent (62.5500 mg / kg p.o.) significant hepatoprotective activity against carbon tetrachloride - induced liver injury in rats and mice as determined through various tests like hexobarbitone - induced sleep, zoxazolamine - induced paralysis, bromsulfalein (bsp) clearance, serum levels of transaminases, bilirubin, and protein. a combination of ethanolic extract of e. alba leaves and p. longum seeds demonstrated better hepatoprotective action against ccl4-induced hepatotoxicity in rats than either extract alone. serum marker enzymes like alanine aminotransferase (alt / got), aspartate aminotransferase (ast, also known as got), acid phosphatase (ap), lactate dehydrogenase (ldh), -glutamyl transferase (ggt), and 5-nucleotidase were elevated with carbon tetrachloride treatment, which were restored towards normalization by the combined extract. at the same time, changes in biochemical parameters like total protein, total bilirubin, total cholesterol, triglycerides, and urea were restored to near normal levels with the combined extract. administration of fresh leaf powder (500 mg / kg) to rats was observed to lead to significant hepatoprotective action in paracetamol - induced liver toxicity in rats. histopathological studies indicated that paracetamol - administered rat liver showed severe congestions, hydropic degeneration, and occasional necrosis, while leaf - administered rat liver showed decreased hepatocyte damage. at the same time, paracetamol - induced elevated levels of serum alt, ast, alkaline phosphatase (alp), ldh, and ggt as well as paracetamol - induced changes in serum proteins, bilirubin, cholesterol, and triglycerides were restored to normal levels with the leaf powder. in ccl4-induced hepatotoxicity in rats, methanol extract of leaves and chloroform extract of roots of e. alba showed significant reductions of lysosomal enzymes in serum from the elevated levels induced by carbon tetrachloride. at the same time ccl4-induced elevated serum got, gpt, alp, and bilirubin levels were also restored towards normalization with administration of both extracts. the hepatoprotective activity of ethanol extract of e. alba whole plants was studied in rats given ethanol for 21 days. ethanol administration led to hepatic damage as manifested by histopathological changes, increase in thiobarbituric acid - reactive substances (tbars), decrease in reduced glutathione (gsh), superoxide dismutase (sod), and catalase (cat), and increase in glutathione peroxidase (gpx) in liver. histopathological changes indicated that, in alcohol - treated animals, liver showed hepatocytic necrosis and inflammation in the centrilobular region with portal triaditis. aqueous extract of leaves of the plant has been found to offer hepatoprotectivity against paracetamol - induced liver damage. paracetamol - induced increases in tbars were reduced by the aqueous extract, and paracetamol - induced decreases in gsh were also reversed by the extract. catalase was also decreased in paracetamol - treated groups, which was also reversed by coadministration of the extract. the alcoholic and aqueous extract of e. alba leaves was tested for hepatoprotective activity against paracetamol - induced liver damage in albino rats. the alcoholic extract - treated rats of group iii revealed marked hepatoprotection as there was significant (p < 0.01) reduction in sgot, sgpt, alp, total bilirubin, and direct bilirubin and a significant (p < 0.01) increase in total protein and albumin as compared to paracetamol treated group. the aqueous leaf extract (85%) of e. alba was examined for hepatoprotective effects in ccl4-induced hepatotoxicity in male albino rats. ccl4 induced oxidative stress in rats resulting in oxidative injury as manifested by increases in tbars and hydroperoxides and augmented levels of serum ast, alt, and alp. at the same time there were depleted levels of sod, cat, gpx, and glutathione - s - transferase (gst). the aqueous extract given at a dose of 250 mg per kg body weight reversed these changes and brought them back to normal levels. the results suggest that increases in oxidative stress play a vital role in development of hepatic injury, which can be ameliorated through administration of aqueous extract of the leaves. a polyherbal formulation (ayush - liv.04) containing e. alba (along with clitoria ternatea, asparagus racemosus, alpinia galanga, and milk tuttam, i.e., copper containing stone) showed hepatoprotective activity against ccl4 and ethanol induced liver damage in rats. elevated levels of serum ast, alt, alp, acid phosphatase, and bilirubin were significantly lowered in the polyherbal formulation - administered rats. the ethanolic extract of a polyherbal formulation containing leaves of melia azadirachta, seeds of piper longum, and whole plants of e. alba has been evaluated for hepatoprotective effects against ccl4-induced hepatic damage in male albino rats. the substantially reduced levels of sod, cat, gpx, gst, and glutathione reductase (gr) due to ccl4 were restored to normal with the extract. hepatoprotective effects of ethanolic extract of e. alba leaves and leaf callus were examined in a model of ccl4-induced acute hepatotoxicity in albino rats. liver damage was assessed by measuring serum parameters like got, gpt, alp, albumin, and total protein, as well as histopathological examination. oral administration of the extract at 250 and 300 mg / kg, respectively, showed hepatoprotective effects as demonstrated by restoring to near normal levels the serum parameters and improving hepatic lesions caused by carbon tetrachloride. petroleum ether and ethanol extract of e. alba has been tested in albino rats for promoting hair growth activity. the extracts were incorporated into oleaginous cream (water in oil cream base) and applied topically on shaved denuded skin of male albino rats. the extracts significantly reduced hair growth time by half, as compared to nontreated control animals. quantitative analysis of hair growth after treatment with petroleum ether extract (5%) exhibited greater number of hair follicles in anagenic phase (69 4) which were higher as compared to control (47 13). the methanol extract of the plant has also been tested for its efficacy for promoting hair growth in pigmented c57/bl6 mice, preselected for their telogen phase of hair growth. in these species, the truncal epidermis lacks melanin - producing melanocytes and melanin production is strictly coupled to anagen phase of hair growth. telogen to anagen transition was assessed following topical administration of the extract. a dose - dependent transition of telogen to anagen phase of hair growth was observed following extract treatment ; with an extract dose of 3.2 mg/15 cm, 87.5% animals showed anagen phase of growth, while with an extract dose of 1.6 mg/15 cm, 50% of the animals showed the transition from telogen to anagen phase. a polyherbal formulation containing e. alba, hibiscus rosa - sinensis, and nardostachys jatamansi exhibited excellent hair growth activity in wistar albino rats. treatment with the formulation resulted in greater number of hair follicles in the anagenic phase. an ayurvedic formulation consisting of withania somnifera, tinospora cordifolia, eclipta alba, ocimum sanctum, picrorhiza kurroa and shilajit, at doses of 100 and 200 mg / kg, p.o. administered once daily for 28 days to streptozotocin- (stz-) induced diabetic male cf strain rats, induced a dose - related decrease in stz hyperglycemia and attenuation of stz induced decrease in pancreatic islet superoxide dismutase (sod) activity. it has been suggested that the stz - induced hyperglycemia was the consequence of decreased islet sod in islets. in alloxan - diabetic rats, oral administration of leaf suspension of e. alba (2 and 4 g / kg body weight) for 60 days resulted in significant reduction in blood glucose (from 372.0 33.2 to 117.0 22.8), glycosylated hemoglobin hba(1)c, a decrease in the activities of glucose-6 phosphatase and fructose 1,6-bisphosphatase, and an increase in the activity of liver hexokinase, all of these activities being beneficial for amelioration of hyperglycemia and other diabetes - related complications. the antidiabetic effect of e. alba ethanolic extract has been investigated for possible beneficial effects against hyperglycemia and diabetic nephropathy in stz - diabetic rats. single dose treatment of the extract was found to significantly lower blood glucose level by 17.6% after 5 h of oral administration at a dose of 250 mg / kg. treatment of stz - diabetic animals for 10 weeks with the above dose level significantly reduced the elevated levels of blood glucose, % hba1c, urea, uric acid, and creatinine and significantly increased the depressed serum insulin level. the extract exerted a significant inhibitory effect on -glucosidase in a noncompetitive manner with an ic50 value of around 54 g per ml and was found inhibitory to eye lens aldose reductase with an ic50 value of about 4.5 g per ml. inhibition of -glucosidase and aldose reductase was postulated to be the reason behind the other observed effects. a bioactivity - guided isolation approach based on -glucosidase inhibition led to the isolation of four echinocystic acid glycosides of which eclalbasaponin vi was found to be the most potent (ic50 54.2 1.3 microm). analgesic activity of alcoholic extract of e. alba has been determined through tail flick, hot plate, and writhing methods in rats and mice. in all three methods, the extract at a dose of 200 mg / kg demonstrated significant analgesic and antinociceptive effects. hydroalcoholic extract of the plant showed significant antinociceptive activity in acetic acid - induced writhing tests in rodent model at a dose of 200 mg / kg p.o. the extract further showed analgesic effects in formalin tests, with the inhibition occurring in the second phase of the response. the analgesic activity of ethanol extract of e. alba whole plants as well as a total alkaloid fraction was seen in experiments with albino mice by using standard experimental models such as the tail clip method, the tail flick method, and the acetic acid induced writhing response. the results from this study showed that both the ethanol extract and the total alkaloids produced good analgesic activity in all the different models of analgesia tested. the anti - inflammatory effect of the plant was evaluated using carrageenan, mediators such as histamine and serotonin induced paw oedema, and cotton pellet induced granuloma tests for their effect on acute and chronic phase inflammation models in rats. the results indicated potent anti - inflammatory activity of the plant in all the models tested. cumulatively, the reports suggest that the plant can prove valuable as both a central and peripheral analgesic agent. leaves of e. alba are used to get rid of ectoparasites in dogs in trinidad and tobago. an ayurvedic formulation containing e. alba powder has been shown to provide complete remission to 22.6% and checked the recurrence of the disease in 89.5% patients of vicharchika (eczema). the antioxidant and protective effect of water extract of e. alba against ultraviolet- (uv-) irradiation - induced damage has been investigated. the extract had a potent effect in scavenging 2,2-diphenyl-1-picrylhydrazyl (dpph), superoxide radicals, and chelating ferrous ion, exhibiting ic50 values, respectively, of 0.23 mg / ml, 0.48 mg / ml, and 1.25 mg / ml. the total phenol content of the extract was 176.45 mg gallic acid equivalents. the extract was also seen to absorb uva and uvb irradiation and demonstrated a dose - dependent protection of hacat human keratinocytes and mouse fibroblasts 3t3 cells against uvb - induced cytotoxicity. the protective effect against skin cell damage was attributed to a synergistic effect between chlorogenic acid and other active components present in the extract. the aqueous and hydroalcoholic extracts of e. alba have been evaluated for sedative, muscle relaxant, anxiolytic, nootropic, and antistress activities at doses of 150 and 300 mg / kg, p.o. the findings indicated nootropic activity of the aqueous extract (300 mg / kg, p.o.) and its hydrolyzed fraction (30 mg / kg, p.o.). the aqueous extract and the hydrolyzed fraction were observed to provide protection against cold restraint induced gastric ulcer formation and also normalized the white blood cell count in the milk induced leukocytosis challenge model. the aqueous extract of leaves of e. alba has been examined for its memory enhancing quality. doses of 100 and 200 mg of extract suspension in water (per kg body weight) were administered to rats to evaluate transfer latency (tl) on an elevated plus maze. spatial habitual learning tests were conducted with mice at the aforementioned two doses. in this method, mice were placed at the center of open - field apparatus to assess spatial habitual learning, observed for 20 minutes for rearing and time spent during rearing for 30 minutes, 24 hours and 96 hours and 144 hours. the extract at both doses produced a significant decrease in tl in rats and the amount of rearing in mice. the results indicate an extract - induced improvement in cognitive functions, which was attributed to the presence of luteolins in the extract. aqueous extract of e. alba has been tested for its ability to reduce aggression through foot shock - induced aggression and water competition tests. minimization of aggression in both tests was observed with the extract at doses of 100 and 200 mg / kg. methanolic extract of e. alba whole plant has been shown to ameliorate oxidative stress - induced mitochondrial dysfunction in an animal (rat) model of alzheimer 's disease (evaluation of short - term memory using elevated plus maze model). synaptosomal fractions of scopolamine hydrobromide - treated rats exhibited a significant decrease in mtt reduction, which was prevented by the extract at a dose of 200 mg / kg. transfer latency of rats using the elevated plus maze model was also dose - dependently decreased (reversal of scopolamine - induced increase) by the extract thus demonstrating increase in memory. the extract showed high phenolic and flavonoid contents, which might have contributed to amelioration of oxidative stress. the methanol and hydrolyzed extract of e. alba has been assessed for its antioxidant potential in both in vitro and ex vivo models. the in vitro antioxidant activity was evaluated through 2,2-diphenyl-1-picrylhydrazyl (dpph) free radical scavenging and nitric oxide radical inhibition activity. the ex vivo antioxidant activity was determined through lipid peroxidation inhibitory activity on mice liver homogenate by thiobarbituric acid - reactive substances (tbars) method. the methanolic extract and hydrolyzed extract both showed potent antioxidant activity in both models in proving to be powerful scavengers of dpph free radicals and nitric oxide radicals, as well as being inhibitors of lipid peroxidation. antioxidant activity as assessed by dpph free radical scavenging methods has also been described for ethanol extract of the plant. methanolic and aqueous extracts of e. alba demonstrated antioxidant activity in hydrogen peroxide scavenging assays, total antioxidant capacity, and through reducing ability assay. the antioxidant potential of the plant methanolic extract has been shown through dpph free radical scavenging and 2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (abts) assays. the ethanolic extract of the plant also demonstrated antioxidant potential in dpph and abts assays. ethanol and ethyl acetate extracts of leaves of the plant showed antioxidant activity in the ferric thiocyanate method ; aqueous and hexane extracts also showed antioxidant effects but less than ethanol and ethyl acetate extracts. the possible cerebroprotective and antioxidant effect of hydroalcoholic extract of e. alba has been evaluated in global cerebral ischemia in rats. the global cerebral ischemia - reperfusion injury was induced by occluding bilateral common carotid arteries (bcca) for 30 min, followed by 4 h reperfusion. bcca caused significant depletion in superoxide dismutase (sod), glutathione peroxidase (gpx), reduced glutathione (gsh), catalase (cat), glutathione - s - transferase (gst), and glutathione reductase (gr) and significant increase in malondialdehyde (mda) in brain. pretreatment with hydroalcoholic extract significantly reversed the levels of biochemical parameters and significantly reduced the edema and cerebral infarct size as compared to the ischemic control group. various solvent (petroleum ether, benzene, chloroform, acetone, methanol, and aqueous) extracts of e. alba were found to be active against clinical isolates from oral cancer cases. these isolates included various bacteria like staphylococcus aureus, escherichia coli, staphylococcus epidermis, pseudomonas aeruginosa, klebsiella pneumoniae, proteus mirabilis, and proteus vulgaris and funguses like candida albicans and aspergillus fumigatus. ethanol and ethyl acetate extracts of leaves of the plant have been found to be active against e. coli, k. pneumoniae, shigella dysenteriae, salmonella typhi, p. aeruginosa, bacillus subtilis, and s. aureus with minimum inhibitory concentrations (mic) ranging from 4.5 to 90 l / ml. hexane extract of aerial parts of the plant reportedly showed antibacterial activity against s. aureus, bacillus cereus, e. coli, s. typhi, k. pneumoniae, streptococcus pyogenes, and p. aeruginosa, whereas acetone, ethanol, methanol, and aqueous extracts showed intermediate activity against s. aureus, b. cereus, e. coli, s. typhi, k. pneumoniae, p. aeruginosa, p. mirabilis, and s. pyogenes. the aqueous extract showed good activity against s. pyogenes, b. cereus, e. coli, and p. aeruginosa. the susceptibility of various extracts of e. alba was tested against nine different test organisms using the well diffusion method. the n - butanol extract showed inhibitory activity against all nine species, namely, b. cereus, b. subtilis, c. albicans, erwinia carotovora, e. coli, k. pneumoniae, p. aeruginosa, s. typhi, and s. aureus. petroleum ether, dichloromethane, methanol and aqueous extracts showed varying levels of inhibition against some of these microorganisms. aqueous extract of leaves, stems, and flowers of e. alba has been screened for inhibitory activity against multiple test organisms. the leaf extract was effective against enterobacter cloacae and k. pneumoniae ; stem extract was effective against e. cloacae, enterococcus faecalis, k. pneumoniae, and staphylococcus saprophyticus, while flower extract was effective against p. vulgaris, s. aureus, and s. saprophyticus. aqueous and ethanolic extracts of leaves also reportedly showed moderate inhibitory activities against s. aureus, e. coli, p. vulgaris, p. aeruginosa, c. albicans, and aspergillus niger, when tested by agar plate disc diffusion method. antimicrobial activity of petroleum ether, ethyl acetate, ethanol and aqueous extract of e. alba were screened for inhibitory activity by agar well diffusion method against b. subtilis, s. aureus, p. mirabilis, b. cereus, e. coli, salmonella enterica serv. maximum numbers of the organisms tested were inhibited by the ethyl acetate fraction with zones of inhibition ranging from 11 1 mm to 22 1 mm, with maximum activity against b. cereus. the ethanol extract demonstrated maximum inhibitory activity against e. coli. petroleum ether extract inhibited only b. cereus, while aqueous extract was found to inhibit b. subtilis, b. cereus, s. aureus, and c. albicans. the antimicrobial activity of methanol, acetone, and aqueous extracts of leaves of three morphotypes of e. alba has been examined by the disc diffusion method against the gram positive bacteria the acetone extract showed more antimicrobial activity against s. aureus, e. coli, and k. pneumoniae than methanol extract. an aqueous extract of leaves of e. alba has been shown to inhibit the fungus fusarium oxysporum as determined by the agar plate disc diffusion method. methanolic extract of aerial parts of the plant showed maximum inhibitory activity against s. epidermis, s. aureus, and salmonella typhimurium. wedelolactone, isolated from the ethyl acetate fraction of aerial parts, showed enhanced antimicrobial activity and as such can be the responsible agent behind the observed antimicrobial effects. eclalbasaponin, another phytochemical constituent of the plant, has been shown to be responsible for the inhibitory activity of the plant against b. subtilis and p. aeruginosa. this inhibitory activity has been attributed to disruption of bacterial cell membrane leading to loss of bacterial cell viability. the antimalarial activity of leaf extract of e. alba has been tested against plasmodium berghei anka strain in mice. the methanolic leaf extract (250750 mg / kg) produced a dose - dependent chemosuppression or schizontocidal effect during early and established infection and high mean survival time (m.s.t.) values particularly in the group administered 750 mg / kg / day of extract. mosquito larvicidal and ovicidal activities of crude hexane, ethyl acetate, benzene, chloroform, and methanol extracts of the leaves of e. alba were tested against the early third - instar larvae of anopheles stephensi (liston) (diptera : culicidae). the highest larval mortality was observed with methanol extract (lc50 = 112.56 ppm, lc90 = 220.68 ppm). thus this plant could prove to be useful in combating malaria through its larvicidal and ovicidal activities. larvicidal and ovicidal activities of benzene, hexane, ethyl acetate, methanol and chloroform leaf extract of e. alba against dengue vector, aedes aegypti, has also been examined. the lc50 values of benzene, hexane, ethyl acetate, methanol and chloroform extract of e. alba against early third- instar larvae of a. aegypti were 151.38, 165.10, 154.88, 127.64, and 146.28 ppm, respectively. the methanol extract was found to be most effective for ovicidal activity against a. aegypti. the adulticidal and repellent activities of crude hexane, ethyl acetate, benzene, chloroform, and methanol extracts of leaf of e. alba were assayed for their toxicity against two important vector mosquitoes, namely, culex quinquefasciatus and aedes aegypti (diptera : culicidae). c. quinquefasciatus is the vector of wuchereria bancrofti, avian malaria, and arboviruses including st. the methanol extract of leaves of e. alba also reportedly demonstrated maximum adulticidal as well as repellent activities against a. stephensi compared to benzene, hexane, ethyl acetate, and chloroform extracts. the effect of administration of dried e. alba leaf powder (3 g per day) has been studied in mild hypertensive subjects. subjects were given six capsules (500 mg powder per capsule) in three doses per day for 60 days. when compared with placebo given control groups, the results showed that eclipta - supplemented group showed a marked reduction in mean arterial pressure by 15%, total cholesterol (17%), low - density lipoprotein fraction (24%), triglycerides (14%), very - low - density lipoprotein fraction (14%), and plasma lipid peroxides (18%). there was a marked increase in urine volume (34%), urine sodium (24%), serum vitamin c (17%), and serum tocopherols (23%) in the eclipta - administered group. the findings indicated that leaf powder possessed diuretic, hypotensive, and hypocholesterolemic properties and helps in the alleviation of oxidative stress - induced complications in hypertensives. ethanolic extract of leaves and leaf calluses of e. alba was examined for cardiac inhibitory activity in isolated frog hearts. the extracts showed negative ionotropic and negative chronotropic effects as well as reduction in cardiac output. callus extract demonstrated a higher cardiac inhibitory effect than leaf extract at 20 mg doses. the immunostimulatory effects of feeding aqueous extract of leaves of e. alba have been studied in tilapia fish (oreochromis mossambicus). fish were fed diet containing extract at 0, 0.01. 0.1, and 1% levels of diet. after each week, nonspecific humoral (lysozyme, antiprotease, and complement) and cellular (myeloperoxidase content, production of reactive oxygen and nitrogen species) responses and disease resistance against aeromonas hydrophila were determined. a. hydrophila are gram - negative straight rods with rounded ends (bacilli to coccobacilli shape) and are regarded as both fish and human pathogens. lysozyme activity significantly increased after feeding fish with aqueous extract for 1, 2, or 3 weeks. reactive oxygen species production and myeloperoxidase content showed significant enhancement after 1 week of feeding with aqueous extract. the percent mortality in fish following feeding the immunomodulatory responses of methanol extract of whole plant of e. alba (containing 1.6% wedelolactone) have been assessed at five dose levels (dose - response relationship) ranging from 100 to 500 mg / kg body wt. using carbon clearance, antibody titer, and cyclophosphamide immunosuppression parameters. the f ratios of the phagocytic index and white blood cell (wbc) count were also significant. methanol extraction of leaf powder of e. alba was evaluated for its antiepileptic activity through maximal electroshock test (mes) in rats. the extract was administered orally to rats for 7 days at doses of 50, 100, and 200 mg per kg body weight. one hour after the last treatment, seizures were induced in rats by delivering electroshock of 150 ma for 0.2 s with an electroconvulsiometer through a pair of ear clip electrodes. a decrease in duration of hind leg extension compared to controls, rats administered extract at different doses exhibited significant decrease in the duration of time spent in extensor phase in a dose - dependent manner. the antiepileptic activity was attributed to wedelolactone, luteolin, and -amyrin present in the extract. the anticonvulsant activity of methanol extract of e. alba leaves was studied using pentylenetetrazole- and picrotoxin - induced seizure models in mice and guinea pigs. the mechanism was further elucidated by studying the extract 's gabaa receptor modulatory activity and its effect on levels of gaba (-amino butyric acid) in mice brain. potent anticonvulsant activity was demonstrated by the extract when administered at doses of 10200 mg / kg with a saturation level at 50 mg / kg. the observed anticonvulsant effect was attributed to positive modulatory effects on gabaa receptors. wedelolactone and luteolin, present in the extract, were hypothesized for giving the observed effect. wedelolactone has been reported to have selectivity and affinity towards bzd (benzodiazepine) binding site on gabaa receptors. also luteolin has neuroprotective activity and has affinity towards bzd binding site on the gabaa receptors. e. alba ethanolic leaf extracts at doses of 50, 100, 200, and 400 mg / kg, p.o., were studied for anticonvulsant and muscle relaxant activity on maximal electroshock - induced seizures (mes), rotarod, and traction test, respectively, in rats. at doses of 200 and 400 mg / kg, the extract reduced seizures induced by mes, decreased the duration of tonic hind limb extension (thle) (by 76.2 and 89.8%, resp.), and decreased motor coordination showing anticonvulsant and muscle relaxant activity. e. alba ethanolic leaf extract has been shown to cause thiopental sodium - induced sleeping time in rats at an early stage and to prolong the duration of sleep at doses of 200 and 400 mg / kg. the extract at 400 mg / kg also decreased locomotor activity in rats, thus showing a sedative effect. ursolic and oleanolic acids, present in the extract, can act as gabaa agonists and this property can be responsible for the central nervous system (cns) depressant effect. notably, cns depressant and antiepileptic activities have been reported for methanolic extract of leaves of ipomoea aquatica. extract of e. alba has been shown to inhibit snake venom phospholipase a2 activity of crotalus durissus terrificus venom. the inhibitory activity has been attributed to the coumestans, wedelolactone, and demethylwedelolactone, present in the extract. the extract inhibited the cell proliferation in dose - dependent manner in hepg2, a498, and c6 glioma cell lines with an ic50 of 22 2.9, 25 3.6, and 50 8.7 g / ml, respectively. the expression of matrix metalloproteinases (mmp) 2 and 9 was downregulated significantly. additionally, downregulation of nuclear factor b (nfb) was also observed. dna damage was observed following 72 h of extract treatment, leading to apoptosis. hydroalcoholic extract of the plant also demonstrated antiproliferative activities in multidrug - resistant dr - hepg2 cells, a hepatocellular carcinoma cell line. juice obtained from e. alba was shown to inhibit the migration of hcc - s102 (hepatocellular carcinoma) cells. in various human cancer cell lines of different tissue origins (liver, lung, and breast), the juice inhibited migration of all the cell lines with ic50 values ranging from 3170 g / ml. antiangiogenic activity was also demonstrated with the juice. the ethyl acetate, methanol, and aqueous extracts of whole dried plants of e. alba were assessed for their inhibitory effects on the human lung epithelial adenocarcinoma cell line (hcc-827) using the mtt assay. dose - dependent reductions in viable cell count were noticed with all three extracts with the ethyl acetate extract showing the most potency. ethinylestradiol is widely used in various contraceptives and for treatment of metabolic and sexual disorders. the treatment of 10 m of ethinylestradiol along with 1.02 10, 2.125 10, 3.15 10, and 4.17 10 g / ml acetone extract of e. alba leaves resulted in a significant dose - dependent decrease in the genotoxic effects induced by the treatment of 10 m of ethinylestradiol in cultured human lymphocytes. crude methanol extract of e. alba has been shown to inhibit growth of colon cancer cells. the methanolic extract of the aerial parts of the plant showed inhibitory activity on the proliferation of hepatic stellate cells or hscs. activity - guided fractionation led to the isolation of five oleanane - type triterpenoids, echinocystic acid, eclalbasaponin ii, eclalbasaponin v, eclalbasaponin i, and eclalbasaponin iii, which are all echinocystic acid derivatives. among the five echinocystic acid derivatives isolated, echinocystic acid and eclalbasaponin ii significantly inhibited the proliferation of hscs in dose- and time - dependent manners. the ethanolic extract of e. alba has been examined for its antiulcer effects in several ulcer models in rats, like cold resistant stress (crs) and pylorus ligation (pl). the extract administered orally twice daily at doses of 50, 100, and 200 mg / kg was found to dose - dependently and significantly reduce ulcerative lesions. at the same time, extract administration led to significant attenuation of lipid peroxidation and elevated levels of catalase activity. antisecretory activity of the extract was evidenced by significant reduction in gastric volume, acid output, and increase in gastric ph when compared to control (without extract) rats. the methanolic extract of e. alba also showed antiulcer activity in ulcers induced in thirty- six - hour fasted sprague dawley rats by aspirin or ethanol or pylorus ligation plus aspirin treatment. in all the three separate experiments the group receiving oral administration of e. alba prior to ulcer induction showed highly significant reduction in the occurrence of gastric ulcers as well as gastric inflammation (after 4 h of treatment) as compared to the control groups. the extract activity was comparable to the activity of the proton pump inhibiting drug rabeprazole. the methanol extract of whole plant of e. alba was evaluated for its anthelmintic potential against the earthworm pheretima posthuma at doses of 25100 mg / ml. the extract exhibited paralysis of worms at doses of 50, 75, and 100 mg / ml and caused death of worms at 75 and 100 mg / ml. 5-lipoxygenase (5-lo) catalyzes the two - step conversion of arachidonic acid to leukotriene a4 (lta4). a study with synthetically prepared wedelolactone and derivatives showed that both the parent compound and most of the wedelolactone derivatives significantly protected primary cultured liver cells from the toxicity of ccl4, galactosamine (galc), and phalloidin and strongly inhibited the activity of 5-lipoxygenase in porcine leukocytes. the synthetic wedelolactone was also found to have stimulatory effect on the rna synthesis in isolated nuclei from hepatocytes. ethanolic extract of the aerial parts of e. alba has been shown to neutralize the lethal activity of the venom of south american rattlesnake (crotalus durissus terrificus) when mixed in vitro before i.p. three phytoconstituents isolated from the plant, namely, wedelolactone (0.54 mg / animal), sitosterol (2.3 mg / animal), and stigmasterol (2.3 mg / animal), were able to neutralize three lethal doses of the venom. aqueous extracts of the plant inhibited the release of creatine kinase from isolated rat muscle exposed to the crude venom. this protection was also observed in vivo, when the venom was preincubated with the extract prior to injection into mice. the antimyotoxic and antihemorrhagic effects of e. alba and three of its constituents, wedelolactone, sitosterol, and stigmasterol, have been investigated for their ability to protect against myotoxicity of crotalid venoms (bothrops jararaca, bothrops jararacussu, and lachesis muta) and purified myotoxins (bothropstoxin, bthtx ; bothropasin ; and crotoxin) through quantification in vitro by the release rate of creatine kinase (ck) from rat or mouse extensor digitorum muscles and in vivo by the plasma ck activity in mice. wedelolactone was more effective than sitosterol or stigmasterol to neutralize in vitro myotoxicity of the crotalid venoms and myotoxins. the in vivo myotoxicity of venoms and myotoxins was also neutralized by preincubation with wedelolactone. intravenous administration of wedelolactone attenuated the increase in plasma ck activity induced by subsequent intramuscular injections of the crotalid venoms or the myotoxins. wedelolactone inhibited the hemorrhagic effect of b. jararaca venom, as well as the phospholipase a2 activity of crotoxin and the proteolytic activity of b. jararaca venom. wedelolactone also antagonized the myotoxic activity in mice of venoms from crotalus viridis viridis and agkistrodon contortrix laticinctus and two phospholipase a2 myotoxins, cvv myotoxin and acl myotoxin, isolated from them. wedelolactone has been found to inhibit lipopolysaccharide- (lps-) induced caspase-11 (an inflammatory caspase) expression in cultured cells by inhibiting nf-b - mediated transcription. it has further been shown that wedelolactone is an inhibitor of ikk (ib, part of the upstream nfb signal transduction cascade), a kinase critical for activation of nf-b by mediating phosphorylation and degradation of ib. wedelolactone also reportedly significantly inhibited the protein expression levels of inos (inducible nitric oxide synthase, produced after activation by endotoxins or cytokines and generating copious amounts of no) and cox-2 (cyclooxygenase-2 converts arachidonic acid to prostaglandins, resulting in pain and inflammation) in lps - stimulated raw 264.7 cells, as well as the downstream products, including no (nitric oxide), pge2 (prostaglandin e2), and tnf- (tumor necrosis factor-). moreover, wedelolactone also inhibited lps - induced nf-b p65 activation via the degradation and phosphorylation of ib- and subsequent translocation of the nf-b p65 subunit to the nucleus. bioassay - guided fractionation for anti - hiv-1 (human immunodeficiency virus 1) integrase activity led to isolation of six compounds from e. alba extract. they were identified as 5-hydroxymethyl-(2,2:5,2)-terthienyl tiglate, 5-hydroxymethyl-(2,2:5,2)-terthienyl agelate, 5-hydroxymethyl-(2,2:5,2)-terthienyl acetate, ecliptal, orobol, and wedelolactone. wedelolactone showed maximum anti - hiv-1 integrase inhibitory activity with an ic50 value of 4.0 0.2 microns. this study supports the use of e. alba in acquired immunodeficiency syndrome (aids) patients. wedelolactone, isolated from wedelia chinensis, has been found to modulate the androgen receptor (ar) activation of transcription from prostate - specific antigen promoter in prostate cancer (pca) cells. the anticancer activity of wedelolactone has also been shown in androgen receptor - negative mda - mb-231 breast cancer cells, where wedelolactone suppressed growth and induced apoptosis. cells were arrested at the s and g2/m phase of the cell cycle with induction of dna damage. wedelolactone was found to interact with dsdna and inhibited the activity of dna topoisomerase ii. in chlamydia trachomatis - infected hep-2 cells (human epithelial type 2 cells, considered to originate from a human laryngeal carcinoma), wedelolactone was found to induce apoptosis in combination with lps and polyi : c (polyinosinic : polycytidylic acid) leading to lesser viability of chlamydia. g protein - coupled receptor-35 (gpr35) has been shown to be a target of the asthma drugs cromolyn disodium and nedocromil sodium. wedelolactone, which is antiallergic, was found to be a potent -arrestin - biased gpr35 agonist and can be considered a potential drug against asthma. adipocyte hyperplasia is associated with obesity and arises due to adipogenic differentiation of resident multipotent stem cells in the vascular stroma of adipose tissue and remote stem cells of other organs. wedelolactone has been observed to inhibit the adipogenic differentiation of human adipose tissue - derived mesenchymal stem cells (hamscs). it has been shown that this process may be mediated through the erk (extracellular signal regulated kinase) pathway. metabolism of arachidonic acid through the 5-lipoxygenase pathway has been shown to play a critical role in the survival of prostate cancer cells. wedelolactone has been found to kill both androgen - sensitive and androgen - independent prostate cancer cells in a dose - dependent manner by inducing apoptosis. wedelolactone - induced apoptosis was dependent on c - jun n - terminal kinase (c - jnk) and caspase-3. apoptosis was triggered via downregulation of protein kinase c but without inhibition of akt (protein kinase b), suggesting that a novel mechanism is at work. notably, jnk plays a critical role in death receptor - initiated extrinsic as well as mitochondrial intrinsic apoptotic pathways through modulating the activities of mitochondrial pro- and antiapoptotic proteins through distinct phosphorylation events. downregulation of protein kinase c has also been shown to occur using tumor necrosis factor - related apoptosis inducing ligand (trail) stimulated glioma cells ; however, in this case reduced expression of akt was also observed. wedelolactone has been reported to synergize with interferon- (ifn-) to induce apoptosis in tumor cells. the compound increased ifn- signaling by inhibiting stat1 (signal transducer and activator of transcription 1 protein) dephosphorylation and prolonging stat1 activation through specific inhibition of t - cell protein tyrosine phosphatase (tcptp), an important tyrosine phosphatase for stat1 dephosphorylation. stat1 has been implicated in modulating pro- and antiapoptotic genes following stress - induced responses. wedelolactone has been reported to exhibit antifibrotic effects on human hepatic stellate cell line lx-2. the compound reduced the cellular viability of lx-2 in a time- and dose - dependent manner. wedelolactone induced apoptosis of lx-2 cells by decreasing the expression of antiapoptotic bcl-2 and increasing the expression of proapoptotic bax. the inhibition of activation of lx-2 cells has been demonstrated and attributed by the authors to a number of reasons including inducing bcl-2 family involved apoptosis, upregulating phosphorylated status of erk and jnk expressions, and inhibiting nuclear factor-b (nf-b) mediated activity. overall, the various reports indicate that the compound has antiinflammatory, hepatoprotective, snake venom neutralizing, anti - hiv, anticancer, and antiasthmatic effects. the antiproliferative effect of eclalbasaponinsisolated from e. alba on hepatic stellate cells has previously been described. eclalbasaponin i isolated from aerial parts of the plant reportedly dose - dependently inhibited the proliferation of hepatoma cell smmc-7721 with ic50 value of 111.1703 g / ml. eclalbasaponin vi, isolated from e. alba, has been shown to demonstrate -glucosidase activity. echinocystic acid isolated from ethyl acetate fraction of 70% ethanol extract of the plant inhibited lps - induced production of nitric oxide and cytokines such as tumor necrosis factor- and interleukin-6 in raw 264.7 macrophages. the compound also inhibited lps - induced inducible nitric oxide synthase expression at the protein level and inducible nitric oxide synthase (inos), tumor necrosis factor- (tnf-), and interleukin-6 (il-6) expression at the mrna level and inhibited lps - induced inos promoter binding activity. additionally, echinocystic acid suppressed the lipopolysaccharide - induced transcriptional activity of nuclear factor-b by blocking the nuclear translocation of p65. reports on -amyrin are few ; however, several reports are present on pharmacological effects of a combination of - and -amyrin (aba) as well as -amyrin derivatives. antilipoxygenase activity has been reported for -amyrin acetate, suggesting its possibly beneficial role in arthritis. in adult male wistar rats made arthritic by subplantar injection of complete freund 's adjuvant, -amyrin palmitate caused increases in serum hyaluronate and blood granulocytes toward nonarthritic levels and corrected the moderate anemia of adjuvant arthritis. the triterpenes, -amyrin and its palmitate and linoleate esters caused growth inhibition of rat osteosarcoma cells and tadpole collagenase digestion of type i (bone) native collagen. aba, isolated from the resin of protium kleinii, caused dose - dependent and significant antinociception against the visceral pain in mice produced by (intraperitoneal) i.p., intracerebroventricular (i.c.v.), or intrathecal (i.t.) administration of aba inhibited both neurogenic and inflammatory phases of the overt nociception caused by intraplantar (i.pl.) treatment with aba was able to reduce the nociception produced by 8-bromo - camp (8-br - camp) and by 12-o - tetradecanoylphorbol-13-acetate (tpa) or the hyperalgesia caused by glutamate. anti - inflammatory effect of -amyrin isolated from p. kleinii has been demonstrated through its ability to inhibit both ear edema and influx of polymorphonuclear cells in response to topical application of 12-o - tetradecanoylphorbol - acetate (tpa) in mice ears. in tpa - induced skin inflammation in mice, topical application of -amyrin inhibited tpa - induced increase of prostaglandin e2 (pge2) levels. in addition, -amyrin also inhibited the activation of upstream protein kinases, namely, erk, p38 mitogen - activated protein kinase (mapk), and protein kinase c (pkc). the anti - inflammatory mechanism has been proposed to be suppression of cox-2 expression through inhibition of erk, p38mapk, and pkc, as well as blocking nf-b activation. aba, isolated from protium heptaphyllum, significantly attenuated cerulein - induced acute pancreatitis in mice. decreases in cerulein - induced increases of tumor necrosis factor tnf-, interleukin-6, lipase, amylase, myeloperoxidase (mpo), and tbars were noted with administration of aba. moreover, aba significantly suppressed the pancreatic edema, inflammatory cell infiltration, acinar cell necrosis, and expressions of tnf and inos. similar protective effect of aba was also seen in acute pancreatitis induced in rats with l - arginine. aba (3100 mg / kg), isolated from protium heptaphyllum resin, demonstrated significant antinociceptive activity against either subplantar (1.6 g) or intracolonic application of capsaicin in mice ; antinociceptive activity of aba has also been reported in mice for cyclophosphamide - induced bladder pain and intracolonic administration of mustard oil. the protective effect of aba has been demonstrated against trinitrobenzene sulphonic acid- (tnbs-) induced colitis in swiss male mice. the results indicated that aba suppresses inflammatory cytokines and cox-2 levels possibly via inhibition of nf-b and creb - signaling pathways. the preventive effect of aba against dextran sulfate - induced colitis in mice has also been demonstrated ; it has been hypothesized that the cannabinoid pathway may be involved. -amyrin acetate, isolated from tylophora hirsuta, tested positive for antispasmodic activity on spontaneous rabbits ' jejunum preparations with ec50 value of (60 2) 10(5) m. the compound also tested positive on kcl induced contractions with ec50 value of (72 3) 10(5) m. the compound thus can prove to be of use against gastrointestinal disorders like diarrhea and dysentery. aba, isolated from the resin of protium heptaphyllum, manifested a hypolipidemic effect in normoglycemic mice and reduced the elevated plasma glucose levels during oral glucose tolerance tests. stz - induced diabetic mice showed significant decreases in blood glucose (bg), total cholesterol (tc), and serum triglycerides (tgs), when treated with aba. in high fat diet- (hfd-) fed mice oral administration of aba (10, 30, and 100 mg / kg), the hfd - associated rise in serum tc and tgs was significantly less, particularly at a dose of 100 mg / kg. at this dose, there were significant decreases in very low - density lipoprotein (vldl) and low- density lipoprotein (ldl) cholesterol and an elevation of high density lipoprotein (hdl) cholesterol. -amyrin acetate, isolated from aerial roots of ficus benghalensis, has been shown to reduce hyperglycemia and improve diabetic conditions in stz - induced diabetic rats and db / db diabetic mice. -amyrin, isolated from rhaponticum carthamoides, has been shown to induce proliferation of human keratinocytes (hacat) by about 18%. -amyrin acetate at concentrations of 1.6% caused 76.9% mortality rate in adult female a. stephensi mosquitoes. in vivo exposure of the mosquitoes to the compound was found to increase mean probing time and decrease blood engorgement time and feeding rate and a declination in fecundity, which reduced the overall survival and reproductive capacity of the malaria vector a. stephensi. aba, from the stem bark resin of protium heptaphyllum, showed anxiolytic and antidepressant effects when tested in mice by the open - field, elevated plus maze, rotarod, forced swimming, and pentobarbital - induced sleeping time. it has been hypothesized that the anxiolytic effect may involve benzodiazepine - type receptors, while the antidepressant effect may involve noradrenergic mechanisms. hepatoprotective action of aba has been reported against acetaminophen - induced liver injury in mice. acetaminophen (500 mg / kg, p.o.) caused fulminant liver damage characterized by centrilobular necrosis with inflammatory cell infiltration, an increase in serum alt and ast activities, a decrease in hepatic glutathione (gsh) and 50% mortality. pretreatment with aba (50 and 100 mg / kg, i.p. at 48, 24, and 2 h before acetaminophen) attenuated the acetaminophen - induced acute increase in serum alt and ast activities, increased the depleted hepatic gsh, and considerably reduced the histopathological alterations. furthermore, aba potentiated the pentobarbital (50 mg / kg, i.p.) the results suggest that the hepatoprotective action of aba involved possible suppression of liver cytochrome p450 and diminution in oxidative stress and toxic metabolite formation in liver. aba, isolated from protium heptaphyllum resin, showed gastroprotective effect against ethanol - induced gastric mucosal damage in mice. maximal gastroprotection was observed with a 100 mg / kg dose of aba, which was almost abolished in mice with their sensory afferents chemically ablated by a neurotoxic dose of capsaicin. it has been suggested that the gastroprotective mechanism of aba involves at least in part the activation of capsaicin - sensitive primary afferent neurons. aba, isolated from protium heptaphyllum resin, reportedly significantly inhibited the scratching behavior induced by dextran t40 and compound 48/80 in mice. the inhibition has been attributed to a stabilizing action on mast cell membrane. the available scientific literature suggests that -amyrin and derivatives by themselves or a combination of and -amyrins have diverse pharmacological activities including antiarthritic, analgesic, anti - inflammatory, antispasmodic, antidiabetic, cholesterolemic, antimalarial, anxiolytic, antidepressant, hepatoprotective, and gastroprotective activities and also may be beneficial against pancreatitis and pruritus. several phytochemical constituents of e. alba have reported multiple and diverse pharmacological activities, which will only be briefly discussed. many of its effects are mediated through activation of the transcription factor nrf2 (nuclear factor erythroid 2-related factor 2). nrf2 can modulate the expression of more than 200 genes that are crucial in the metabolism of drugs and toxins, protection against oxidative stress and inflammation, and maintaining protein stability and degradation. nrf2 can interact with tumor suppressor protein 53 (p53) and so control cell cycle and nf-b. through these interactions, nrf2 plays a major role in protecting against many age - related diseases including cancer and neurodegeneration, as well as increasing longevity. in tumor cells, a recent review has pointed out that the compound can modulate multiple signaling pathways, like nf-b, akt, signal transducer and activator of transcription 3, mammalian target of rapamycin, caspases, intercellular adhesion molecule 1, vascular endothelial growth factor, and poly (adp - ribose) polymerase. as such, oleanolic acid can be a potential preventive as well as a therapeutic agent for cancer. ursolic acid has been shown in various reports to have antioxidative, anticancer, and anti - inflammatory properties, although a recent review has pointed out that proinflammatory properties of the compound have also been reported in normal cells and tissues. a number of reports have shown ursolic acid to have anticancer, cytotoxic, antitumor, antioxidant, anti - inflammatory, antiwrinkle, anti - hiv, acetyl cholinesterase, -glucosidase, antimicrobial, and hepatoprotective activities (reviewed in). oleanolic and ursolic acids have reportedly hepatoprotective, anti - inflammatory, and antihyperlipidemic properties. oleanolic and ursolic acids can also be potentially useful in neurodegenerative disorders like alzheimer 's disease. a review study has described the anticancer effects of ursolic acid to include protection of cellular dna from damage, inhibition of epidermal growth factor receptor / mitogen - activated protein kinase signal or of foxm1 transcription factors, antiangiogenesis (which can inhibit tumor cell growth), inhibition of tumor cell migration and invasion, and inducing apoptosis in cancer cells. the forkhead transcription factor or foxm1 has been shown to bind and regulate a group of genes which are mainly involved in controlling late cell cycle events in the g2 and m phases. inhibition of foxm1 expression has been found to diminish the proliferation and anchorage - independent growth of breast cancer cells. luteolin has also been described to have the ability to block the development of cancer cells both in vitro and in vivo through offering protection from carcinogenic stimuli, inhibiting tumor cell proliferation, and induction of cell cycle arrest and/or apoptosis. furthermore, luteolin can sensitize cancer cells to therapeutic - induced cytotoxicity through a variety of mechanisms including suppression of cell cycle pathways like phosphatidylinositol 3-kinase (pi3k)/akt, nf-b, and x - linked inhibitor of apoptosis protein (xiap) and stimulating apoptosis pathways including those that induce the tumor suppressor p53. modulation of reactive oxygen species (ros) levels, inhibition of topoisomerases i and ii, reduction of nf-b and ap-1 activity, stabilization of p53, and inhibition of pi3k, stat3, insulin - like growth factor 1 receptor (igf1r), and human epidermal growth factor receptor 2 (her2) have also been described as the causes behind the cancer chemopreventive and chemotherapeutic potential of luteolin. it is to be noted that overexpression of her2 is associated with certain aggressive forms of breast cancer. the cardioprotective role of luteolin has been shown in cardiomyocytes following ischemia - reperfusion, suggesting that the compound can form the basis for preventing and treating cardiovascular diseases. a recent review has pointed out that apigenin can inhibit cancer cell growth, sensitize cancer cells to elimination by apoptosis, and hinder the development of blood vessels to serve the growing tumor. apigenin is able to reduce cancer cell glucose uptake, inhibit remodeling of the extracellular matrix, inhibit cell adhesion molecules that participate in cancer progression (like vcam-1), and oppose chemokine signaling pathways that direct the course of metastasis into other locations. for instance, apigenin has been shown to suppress migration and invasion of transformed cells through downregulation of c - x - c chemokine receptor 4 expression. all of these effects have the net result of blocking progression and metastasis of cancer. in head and neck cancers the plant, e. alba, is regarded by traditional medicinal practitioners as a valuable medicinal plant, particularly for the treatment of liver disorders, gastrointestinal disorders, respiratory tract disorders, hair loss, skin disorders, and fever. scientific evidences have validated most of the claims of the ethnomedicinal uses, including that of treatment of snake bite with the plant. these compounds include wedelolactone, eclalbasaponins, -amyrin, ursolic acid, oleanolic acid, luteolin, and apigenin. the available scientific reports indicate that these compounds can form the next generation of drugs to treat cancer, arthritis, liver diseases, hair loss, and snake bites. | eclipta alba can be found growing wild in fallow lands of bangladesh where it is considered as a weed by farmers. traditional medicinal systems of the indian subcontinent countries as well as tribal practitioners consider the plant to have diverse medicinal values and use it commonly for treatment of gastrointestinal disorders, respiratory tract disorders (including asthma), fever, hair loss and graying of hair, liver disorders (including jaundice), skin disorders, spleen enlargement, and cuts and wounds. the plant has several phytoconstituents like wedelolactone, eclalbasaponins, ursolic acid, oleanolic acid, luteolin, and apigenin. pharmacological activities of plant extracts and individual phytoconstituents have revealed anticancer, hepatoprotective, snake venom neutralizing, anti - inflammatory, and antimicrobial properties. phytoconstituents like wedelolactone and ursolic and oleanolic acids as well as luteolin and apigenin can form the basis of new drugs against cancer, arthritis, gastrointestinal disorders, skin diseases, and liver disorders. |
stroke is the third leading cause of death in the us after coronary artery disease and cancer and it is the leading cause of disability. extracranial atherosclerotic carotid artery disease accounts for slightly more than half of the 731,000 strokes per year in the us. a tia is a transient episode of neurological dysfunction caused by focal brain, spinal cord, or retinal ischemia, without acute infarction. symptomatic ischemic strokes are manifested by clinical signs of focal or global cerebral, spinal, or retinal dysfunction caused by central nervous system infarction. hemispheric or focal symptoms relate to a single carotid distribution, causing contralateral hemiparesis or hemiparesthesia, aphasia, and/or ipsilateral monocular blindness (amaurosis fugax). non - hemispheric symptoms that often occur with vertebrobasilar insufficiency include dysarthria, diplopia, vertigo, syncope, and/or transient confusion. carotid endarterectomy (cea) is the currently established surgical procedure for stroke prevention in patients with extracranial carotid artery disease. some of cea 's technical issues such as the benefits of an intraoperative shunt or of a patch closure versus primary repair continue to be debated. the comparability of data from highly selected patient populations enrolled in clinical trials of cea with results obtained in everyday practice has been questioned. there were markedly higher mortality rates in medicare patients who underwent cea at clinical trial hospitals than in the selected patients treated in clinical trials. caution is advised in translating the efficacy of carefully controlled studies of cea to effectiveness in everyday practice. there has been significant variability or heterogeneity in the reporting of cea outcomes in the literature, making comparison of studies difficult and confusing. in a meta - analysis of cea in symptomatic patients (n = 51 studies), the strongest predictor of stroke or death was who (neurologist or surgeon) performed the post - operative assessment. when a neurologist evaluated post - operative patients, the risk of 30-day stroke and death was 7.7%, but when a single author who was a surgeon performed the evaluation, the reported risk was only 2.3%. there is a strong bias in favor of cea in the current literature comparing medicare or specialty outcomes data [4 - 6 ]. the bias is one of ascertainment ; that is, independent neurological examination is mandated for reimbursement for most carotid artery stent (cas) procedures but is rarely done for cea. to obtain medicare reimbursement, all but a very few cas procedures must conform to us food and drug administration (fda) protocols, which require independent neurological examination. an independent neurological examination markedly increases the number of events that are detected following a procedure. when comparing cea and cas, it is critical that the methodology for detecting events (the ascertainment of events) is similar, or the outcomes will be unfairly slanted. patients with high - surgical - risk features (table 1) treated with cas have been proven to have outcomes non - inferior ' to cea in sapphire (stenting and angioplasty with protection of patients with high risk for endarterectomy), a randomized controlled trial (figure 1). additional supporting peer - reviewed and published evidence include a meta - analysis and multiple pre - market [10 - 18 ] and post - market [19 - 23 ] surveillance trials. additionally, a multi - specialty endorsed professional society document from the american college of cardiology (acc) is consistent with the conclusions of the randomized controlled trial (sapphire) and supports the benefit of cas in patients with high - risk features for both symptomatic (> 50% stenosis) and asymptomatic (> 80% stenosis) patients. recently, three very large, post - market surveillance trials evaluating cas in a real - world ' environment were published. the primary objective of the sapphire world - wide (sapphire ww) post - market approval registry was to evaluate 30-day outcomes after cas was performed in high - surgical - risk patients by cas operators of varying experience. the investigators reported 30-day safety and efficacy outcomes in 2,001 symptomatic and asymptomatic high - surgical - risk patients treated by carotid stent operators with varying clinical experience. the overall, independently adjudicated, 30-day stroke and death rate for cas in 2,001 high - surgical - risk patients was 4.0%. the results of more than 6,000 high - surgical - risk patients treated by cas operators with varying levels of experience in two large prospective, multi - center, fda - mandated post - market surveillance trials (emboshield and xact post - approval carotid stent trial [exact ] [n = 2,145 ] and carotid acculink / accunet post - approval trial to uncover unanticipated or rare events-2 [capture-2 ] [n = 4,175 ]) were recently published and demonstrated excellent outcomes. both trials included independent neurological assessment of outcomes to reinforce the rigor for ascertaining adverse events. the overall rates of incidence of 30-day stroke and death were 4.1% for the 2,145 exact patients and importantly, for patients who would have been comparable to patients included in the 2006 american heart association (aha) published guidelines (50% stenosis) and asymptomatic (> 80% stenosis) patients. recently, three very large, post - market surveillance trials evaluating cas in a real - world ' environment were published. the primary objective of the sapphire world - wide (sapphire ww) post - market approval registry was to evaluate 30-day outcomes after cas was performed in high - surgical - risk patients by cas operators of varying experience. the investigators reported 30-day safety and efficacy outcomes in 2,001 symptomatic and asymptomatic high - surgical - risk patients treated by carotid stent operators with varying clinical experience. the overall, independently adjudicated, 30-day stroke and death rate for cas in 2,001 high - surgical - risk patients was 4.0%. the results of more than 6,000 high - surgical - risk patients treated by cas operators with varying levels of experience in two large prospective, multi - center, fda - mandated post - market surveillance trials (emboshield and xact post - approval carotid stent trial [exact ] [n = 2,145 ] and carotid acculink / accunet post - approval trial to uncover unanticipated or rare events-2 [capture-2 ] [n = 4,175 ]) were recently published and demonstrated excellent outcomes. both trials included independent neurological assessment of outcomes to reinforce the rigor for ascertaining adverse events. the overall rates of incidence of 30-day stroke and death were 4.1% for the 2,145 exact patients and importantly, for patients who would have been comparable to patients included in the 2006 american heart association (aha) published guidelines (< 80 years of age), the cas results met the threshold recommendations for 30-day stroke and death rate at 5.3% (benchmark for cea 6%) for symptomatic patients (i.e., those with 50% stenosis) and 2.9% (benchmark for cea 3%) for asymptomatic patients (i.e., those with 80% stenosis) (figure 2). these studies demonstrate equipoise for cas and cea in community settings on the basis of data collected during the decade of the 1990s with the large cea versus best medical therapy trials, and the aha expert consensus panel suggested that the perioperative risk of stroke and death should not exceed 3% for asymptomatic patients, 6% for symptomatic patients, or 10% for repeat cea. capture, carotid acculink / accunet post - approval trial to uncover unanticipated or rare events ; exact, emboshield and xact post - approval carotid stent trial. published data have suggested that very old patients (75 - 80 years of age) are at increased risk for not only a higher complication rate of cea but also worse outcomes for cas. however, three peer - reviewed manuscripts published in the past year have reported excellent outcomes in high - surgical - risk patients 80 years of age undergoing cas [31 - 33 ]. the very favorable overall 30-day stroke and death rates with independent neurological assessment in these octogenarians were 3.3%, 2.7%, and 0.8% [31 - 33 ]. the authors emphasized the importance of operator experience and careful case selection to avoid difficult aortic arch access, excessive lesion tortuosity, and heavy calcification. the improved outcomes for octogenarians are consistent with the data reported in the capture-2 and exact trials demonstrating reduced cas complications with expanding operator experience. the published peer - reviewed evidence does not support denying cas to very old patients but does show that the best results are obtained with careful patient selection and experienced operators. the eva-3s (endarterectomy versus angioplasty in patients with symptomatic severe carotid stenosis) trial randomly assigned 527 symptomatic (60%) low- or average - surgical risk patients to cas or cea. the 30-day incidence rates of stroke or death were 3.9% for cea and 9.6% for cas. early in the trial, the use of embolic protection devices (epds) was not required and this generated a stroke rate of 25% (5 of 20). the inexperience of the interventionalists, particularly the surgical operators, is a significant limitation of this study. the patients in eva-3s had risk profiles similar to those of crest (carotid revascularization endarterectomy versus stenting trial) roll - in patients, but crest required at least 20 cases of carotid stent experience with audited results and mandated the use of an epd. in contrast to the high rate of stroke and death in eva-3s, the most recent report of 1,246 lead - in patients demonstrated a 30-day stroke and death rate of 5.6% for symptomatic crest lead - in registry patients. physician specialty - specific data from crest were presented by donald v heck at the 2009 society of neurointerventional surgery meeting. he reported 30-day stroke and death by subspecialty during the lead - in phase of the crest trial. subspecialty training in catheter - based techniques - cardiology, radiology, and neuroradiology - had a statistically lower event rate than did the non - catheter - based specialty of vascular surgery. vascular surgeons had a statistically significant, twofold increase in their complication rate (stroke and death) compared with the physicians trained in catheter - based techniques. the space (stent - supported percutaneous angioplasty of the carotid artery versus endarterectomy) trial showed no difference between cea and cas in average surgical risk in symptomatic patients with optional use of epds. the 30-day stroke and death rates were 6.8% for cas and 6.3% for cea and were not clinically or statistically different. one drawback of the study was the lack of epd use in 73% of the study subjects. after 2 years of follow - up, there continued to be no difference in outcomes between cea and cas ; however, for patients who were younger than 69 years of age at randomization, cas was significantly better (30-day stroke and death and ipsilateral stroke for 2 years ; 4.8%) compared with cea (8.0% ; p < 0.005). a major impact on the field has been made with proximal embolic occlusion (peo) devices by lowering post - procedural complication rates. these peo devices have an advantage in that when the carotid lesion is crossed with a guidewire for the entire procedure, no antegrade flow occurs, thus the patient is protected against procedure - related emboli. a recent trial reported a 1.4% 30-day stroke and death rate with a peo in 1,288 consecutive patients. the risks of 30-day stroke and death were less than 1% in asymptomatic patients and near 3% in symptomatic patients. there now exists the highest level of evidence (aha / acc class i, level of evidence a) that there is clinical equipoise between cas and cea for patients at increased surgical risk for cea. this applies to both symptomatic and asymptomatic patients with anatomic or comorbid features that place them at increased risk for cea. this conclusion is supported and reinforced by the three recently published post - market surveillance trials (capture-2, exact, and sapphire ww). in these patients at high risk for cea (both symptomatic and asymptomatic patients) the current recommendation for patients at increased risk for cea is that cas should be considered a reasonable alternative for stroke prevention. there is no consensus regarding the relative outcomes of cas versus cea in average - risk patients. clinical trials in this patient population over the past 2 years have ranged from eva-3s, which strongly favored cas over cea, to the most recently reported peo system with extremely low stroke and death rates in both symptomatic and asymptomatic patients. the space trial split the difference, showing a benefit for cas in patients younger than 69 years old and an advantage for cea in older patients. the results of crest, a large randomized controlled trial in average - surgical - risk patients, will be reported within the next few months and will go a very long way toward informing our recommendations in the low- or usual - surgical - risk population. there now exists the highest level of evidence (aha / acc class i, level of evidence a) that there is clinical equipoise between cas and cea for patients at increased surgical risk for cea. this applies to both symptomatic and asymptomatic patients with anatomic or comorbid features that place them at increased risk for cea. this conclusion is supported and reinforced by the three recently published post - market surveillance trials (capture-2, exact, and sapphire ww). in these patients at high risk for cea (both symptomatic and asymptomatic patients) the current recommendation for patients at increased risk for cea is that cas should be considered a reasonable alternative for stroke prevention. there is no consensus regarding the relative outcomes of cas versus cea in average - risk patients. clinical trials in this patient population over the past 2 years have ranged from eva-3s, which strongly favored cas over cea, to the most recently reported peo system with extremely low stroke and death rates in both symptomatic and asymptomatic patients. the space trial split the difference, showing a benefit for cas in patients younger than 69 years old and an advantage for cea in older patients. the results of crest, a large randomized controlled trial in average - surgical - risk patients, will be reported within the next few months and will go a very long way toward informing our recommendations in the low- or usual - surgical - risk population. the author is the national principal investigator for the cabana (carotid stenting boston scientific surveillance program) trial, a carotid stent trial sponsored by boston scientific (natick, ma, usa). | revascularization of the extracranial carotid arteries is a commonly performed surgical procedure to prevent stroke. open surgery (i.e., carotid endarterectomy [cea ]) is a well - established stroke prevention procedure but is being challenged ' by a less invasive percutaneous procedure (i.e., carotid artery stent [cas ] placement). clinical trials comparing cas and cea for average - surgical - risk patients have demonstrated mixed results, whereas the data for cas compared with cea in high - surgical - risk patients have demonstrated non - inferiority. the impending carotid revascularization endarterectomy versus stenting trial (crest) results will have a major impact on the utility of cas relative to cea in average - surgical - risk patients. |
water molecules are a key component of biological systems and act as ordered structural elements at binding interfaces. the mediation of ligand binding by water molecules can have important consequences for binding affinity and specificity. numerous examples of water - mediated protein ligand interactions are known, including peptide binding in tyrosine kinase (src), binding of inhibitors to proteases, and carbohydrate - binding proteins. the consideration of individual water molecules in ligand design hinges on an accurate assessment of opposing thermodynamic contributions. this includes the entropic gain of displacing a highly ordered water molecule and the enthalpic loss of breaking water however, assessing the role of individual water molecules at the binding interface is a complex problem, as highlighted by the prediction that there is no direct correlation between the free energy of water molecules in the binding site and the affinity of bound ligands. despite the difficulty in predicting and interpreting the roles of such water molecules, several attempts have been made at classifying binding site water molecules with respect to the likelihood of their displaceability. high displaceability in this context corresponds to displacement of the water molecule by a suitable chemical group on the ligand with an accompanying favorable change in the binding affinity. there are a variety of methods for identifying and ranking water molecules in binding sites, including physics - based methods and empirical methods. a physical method based on the double decoupling method employing thermodynamic integration (ti) to replica exchange monte carlo simulations was found to be successful in classifying water molecules as displaceable or conserved. however, such an approach requires extremely time - intensive calculations that must be performed on each water molecule individually. this drawback also affects free - energy perturbation (fep) techniques, which have also been used to successfully predict the ease of displacement of ordered water molecules in protein binding sites. fep predictions were found to correlate with the change in affinity for structural modifications that displaced the water molecules. however, the study also noted that a complete thermodynamic analysis is needed in order to accurately compute the effects of ligand modifications. another approach employed machine learning to create a probabilistic water classifier. this was found to be very good at predicting the location of water molecules and shown to have reasonable predictive power in classifying water molecules as displaceable or conserved. the structural features of water molecules in x - ray crystallographic structures, such as b - factors, accessibility to bulk solvent, number, and strength of protein - water hydrogen bonds, were used to develop multivariate logistic regression models for predicting the displacement of water molecules, yielding a prediction efficiency of 67%. an empirical method has also been developed that employs pseudo - bayesian statistical analysis on predictions from the hint scoring function and the rank algorithm, which is based on the number and geometric quality of hydrogen bonds for each water molecule. the method was found to be particularly useful for identifying strongly conserved water molecules. an alternative to the approaches described above is the use of inhomogeneous fluid solvation theory (ifst). ifst can be used to create a thermodynamic profile of the solvent surrounding a protein, which can be used to identify binding hotspots. the free energies calculated by ifst are the contributions of regions of three - dimensional space to the solvation free energy of the solute. when this region is a highly occupied hydration site, g can be thought of as the contribution of a specific water molecule to the solvation free energy of the solute. watermap has been used to explore the hydrophobic effect, understand sar, explain kinase selectivity, and predict binding affinities. the advantage of ifst over other physics - based methods such as ti and fep is that one simulation yields predictions for all the water molecules in a system. recent work on ifst has focused on binding site prediction, discretization on a cartesian grid, and investigating the effect of using different water models. in this study, we employ ifst to predict the experimentally observed displacement of water molecules in a protein binding site. when displacing a water molecule from a binding site in molecular design, consideration must be given to the functional group that replaces it. multiple copy simultaneous search (mcss) is one of the oldest methods to predict energetically favorable positions of functional groups in protein binding sites. the mcss scoring function is based on the charmm force field with interaction energies calculated from the difference between the bound and unbound states. the solvent effects are commonly taken into account by an approximation of dielectric screening from a distance - dependent dielectric model. an alternative approach to account for solvent effects is to couple the molecular mechanical energy component with polar and nonpolar solvation free energies derived from implicit solvent formalism in mm - pb / sa or gb / sa methods. implicit solvent approaches have gained widespread application in virtual screening, rescoring of docking poses, and estimation of ligand binding energies in ligand series. the sorting of mcss minima based on mm - pb / sa derived free energy estimates has yielded encouraging results. mcss analysis using probe molecules with different chemical characters provides what have been termed functionality maps. these functionality maps have been used for construction of ligands for numerous protein targets. other approaches have also been developed for computational solvent mapping and probe analysis. ftmap searches for favorable probe positions on protein surfaces using an empirical energy function including a desolvation term. a clustering procedure is used to identify consensus probe binding sites which are identified as fragment - binding hotspots. the integral equation theory of liquids, known as the three - dimensional reference interaction site model (3d - rism) has also been used to find most probable positions of functional groups and small ligands on protein surfaces. this method has been applied to thermolysin with solvent probes for which experimental data were available, and reasonable agreement with experimental results was obtained. more recently, methods employing molecular dynamics (md) simulations using a mixture of explicit water and functional group probes have been introduced. guvench and mackerell used a solvent mixture consisting of propane benzene water to construct probability density maps of probe binding preferences which corresponded to known ligand functional group preferences. in other studies, more diverse sets of probe molecules have been used to assess the druggability of allosteric protein targets and binding hotspots. an important feature of these developments was the combined use of probe clustering and water displacement as indicators of hotspots. as summarized above, md simulation based protocols using solvent - probe mixtures have been used previously to identify binding hotspots and predict target druggability. however, a direct assessment of relative displaceability of ordered water molecules has not been addressed in these studies. an effective method for providing such assessments would prove valuable in the hit - to - lead and lead optimization stages of drug development. in addition, an accurate ranking of water molecules making bridging interactions between the protein and the ligand can be also be used to make judicious choices about keeping or removing water molecules prior to molecular docking methods for virtual screening. the estimation of thermodynamic properties of water molecules at binding interfaces can provide very useful information for ligand design. the main aim of this study was to investigate the combination of hydration site free energies calculated by ifst with the mcss scores of probe molecules in order to predict the observed displacement of water molecules. to calculate an experimental measure of displaceability, we used a large collection of ligand - bound structures of heat shock protein 90 (hsp90). the observed displacement was calculated from the frequency with which water molecules from the apo structure were displaced by a ligand. hsp90 is well - known for its function as a molecular chaperone. due to its important role in assisting protein folding, preventing self - aggregation, and cell cycle progression, the structure of hsp90 contains a highly conserved n - terminal domain that is linked, via a highly flexible linker, to a middle domain and a c - terminal domain. the n - terminal domain contains an adenosine binding pocket, responsible for its atpase activity, and has an unusual motif known as bergerat fold, which is characterized by a rigid adenosine binding site and a flexible loop in phosphate binding region. the atpase activity of the n - terminal domain drives structural transitions required for chaperone functioning, and therefore it has been targeted in several drug discovery campaigns. water molecules have been a key aspect of structural studies on hsp90, and a key consideration in ligand design programs has been the role of four key water molecules in the binding site that are highly conserved in several complex structures. their systematic displacement and the resulting consequences on ligand optimization have also been the subject of recent investigations. these factors make hsp90 an ideal test case to assess the predictions of ifst. a data set consisting of 103 ligand - bound x - ray crystallographic structures of hsp90 (n - terminal domain) was compiled, based on roughley and hubbard s work. the structures can be organized into four groups : those based on a resorcinol substructure (41), those based on a (benz)-amide substructure (14), those based on an aminopyr(im)idine substructure (39), and those based on second - site fragments (9). due to modifications around central scaffolds, this data set the set of crystal structures used in this work is reported in the supporting information. in addition, a crystal structure of the unbound closed - state conformation of hsp90 (1yer) was used as the apo form of the receptor. all the structures were moved to the same reference frame by a rigid - body superimposition onto the ligand - bound crystal structure from 1uy6. from the superimposition, any water molecule in the apo structure lying within 2.0 of any of the ligand atoms in any structure was selected for subsequent analysis. for each of these reference water molecules, the observed displacement fraction (d) was defined from the fractional conservation (f).12 f is obtained by counting the number of ligand - bound structures where a corresponding water molecule was present within 1.0 (nhydrated) and then representing it as a fraction of total structures in the data set (ntotal). the orientations of asparagine and glutamine residues were then checked using schrodinger s preparation wizard. residues asn40, asn105, gln123, gln133, and gln194 were altered by switching the nitrogen and oxygen atoms to improve the hydrogen - bonding patterns. histidine residues were also checked for orientation and protonation state using schrodinger s preparation wizard. residues his154, his189, and his210 were flipped and assigned as epsilon protonated. all remaining histidines were assigned as delta protonated, but residue his77 was flipped. the residues lysine, arginine, aspartate, glutamate, cysteine, and tyrosine were also analyzed to check their protonation state. there was no evidence of any unusual protonation states, and thus all lysine and arginine residues were assigned as positively charged, all aspartate and glutamate residues were assigned as negatively charged, and all cysteine and tyrosine residues were assigned as neutral. the hydrogen - atom positions were then built using the hbuild facility of charmm with the charmm27 energy function, and the atomic charges were assigned from the charmm27 force field. the prepared protein structure was then subjected to mcss calculations using eight different probe molecules. the probes were chosen to ensure that energetically favorable positions of polar, charged, hydrophobic, and aromatic functional groups were sampled. two representative probes from each group were selected : methanol and ammonia (polar), ammonium ions and acetate ions (charged), methane and propane (hydrophobic), plus phenol and benzene (aromatic). the binding site region for mcss calculations was defined as a 12.0 sphere around the centroid of all overlaid ligands. for each probe molecule, 500 copies were randomly placed in this sphere where any copy within 1.2 of any protein atom was removed. energy minimizations were carried out in stages, and duplicate copies (within 0.2 of another copy) were removed at the end of each stage. the first stage was 500 steps of steepest descent ; the second stage was 300 steps of steepest descent, and the final stages were 20 repetitions of 500 steps of conjugate gradient minimization. all minimizations were performed using a distance - dependent dielectric model with the default dielectric constant of 1.0. at the end of the mcss run, probe copies with charmm interaction energies greater than + 10.0 kcal / mol were removed. finally, probe copies within 1.0 of each other were clustered together, and the copy with the lowest interaction energy was selected as the cluster representative. all of the above calculations were performed using the mcss implementation in accelrys discovery studio 3.1. the raw probe scores from mcss consist of the charmm interaction energy between the probe molecule and protein. these raw scores were size - normalized by multiplying by the ratio of water to probe molecular volume. this allowed us to compare the scores of probes with different sizes together. in two variations of the protocol, the normalized mcss scores were combined with two different desolvation penalties : the experimental hydration enthalpy (mcss - he) and the experimental hydration free energy (mcss - hfe). the experimental values of hydration enthalpy and hydration free energy for each probe were obtained from the literature. the mcss derived probe positions were compared with the reference water molecules using the following procedure. a probe position was identified as corresponding to a reference water molecule if any of the probe heavy atoms was less than 1.5 from the oxygen atom of the water molecule. when multiple probe positions were obtained for a given water molecule, the best scoring pose was selected. in a variation of the scoring protocol for mcss - derived probe positions, the binding energy of each probe was calculated using an mm - gbsa approach:3 the free energy of each of the above terms is calculated from4 emm is the energy calculated from the standard charmm energy function, which includes bonded and nonbonded terms. gelec represents the electrostatic component of solvation energy and was calculated using a variation of the standard gbsa model, referred to as the generalized born method with molecular volume integration (gbmv). the nonpolar contribution (gnp) to solvation free energy was calculated on the basis of the surface area (sa) model, which assumes a linear relationship between gnp and the solvent accessible surface area. a value of 5.0 cal / mol / was used for the surface area coefficient in the sa model. the change in the solute entropy (ts) terms was assumed to be zero in this case. the mm - gbsa scores were also size - normalized using the method detailed above. for the md simulation, solvation was performed with the solvate program, version 1.0 from the max planck institute to generate a sphere of radius 50 around the protein center. the system was then cut to form a rhombic dodecahedron (rhdo) with an edge length of 60 using the charmm program (version 34b1). water molecules were modeled with the tip3p - ewald, tip4p-2005, and tip5p - ewald water models in three separate systems. the water model has been shown to affect free - energy calculations in recent studies. six sodium ions were included in all three systems to yield a net charge of zero. the ions were placed far from the hsp90 binding site. during all md simulations, the heavy atoms were harmonically restrained using a 1.0 kcal / mol / harmonic force, the rhdo was treated using periodic boundary conditions, and the electrostatics were modeled using the particle mesh ewald method. the system was first subjected to md equilibration for 100 ps in an npt ensemble. this stage of preparation was undertaken to equilibrate the density of the water molecules. the density of the water molecules plays an important role in ifst and is thus important to equilibrate. the system was then subjected to md equilibration for 1 ns in an nvt ensemble. we ensured that the system was brought to equilibrium before continuing by verifying that the system reached a point where the energy fluctuations were stable. production simulations were performed for 5.0 ns at 300 k. all md simulations were performed using the namd program version 2.8 with the charmm27 force field allowing an md time step of 2.0 fs. electrostatic interactions were modeled with a uniform dielectric and a dielectric constant of 1.0 throughout the setup and production runs. van der waals interactions were truncated at 11.0 with switching from 9.0. all md simulations were performed using namd compiled for use with the cuda - accelerated gpus. the 5.0 ns md simulations required approximately 15, 20, and 25 h for the tip3p - ewald, tip4p-2005, and tip5p - ewald water models on two four - processor gpu cores. the first stage of the ifst analysis was to identify regions within the binding site with a high number density of water, termed hydration sites. we defined the center of the binding site as the centroid of the two water molecules w331 and w440 and considered the region within 12.5 of this centroid. we employed a radius of 1.2 for these hydration sites, in line with prior work. the first method was to place hydration sites at crystallographically determined water positions, and the second was to cluster water molecules from the md simulation. for clustering, hydration sites were selected by sampling 5000 equally spaced snapshots from the md trajectory and calculating the number density at cartesian gridpoints within the binding site. a grid with a resolution 0.5 was generated around the centroid of the binding site. the number of water molecules within 1.2 from all 5000 snapshots was used to calculate the number density, and hydration sites were selected iteratively, starting at the gridpoint with the highest number density. no gridpoints were selected within 2.4 of an existing gridpoint, and the iteration terminated when the remaining gridpoints had a number density lower than 50% of the bulk value. the hydration site positions were then calculated as the mean position of all water molecules within 1.2 of each selected cartesian gridpoint. the clustering required approximately 21, 27, and 34 h for the tip3p - ewald, tip4p-2005, and tip5p - ewald water models on a single processor. the interaction energy of each hydration site was calculated by sampling 1000 snapshots with one every 5.0 ps from the 5.0 ns simulation. the average interaction energy of every water molecule within the site was computed from the interaction with the solute (esw) and the other water molecules (eww). this was then combined with the average interaction energy of a water molecule determined from the bulk water simulation (ebulk) to calculate the energy difference (e) and binding energy difference (ebind) as shown in eqs 5 and 6, respectively:56 the average number of water molecules in a hydration site is termed n. from a corresponding simulation of bulk water, ebulk takes values of 9.81 kcal / mol, 11.33 kcal / mol, and 9.67 kcal / mol for the tip3p - ewald, tip4p-2005, and tip5p - ewald water models. the binding energy can also be termed the normalized energy or the per water energy. the entropy of each hydration site was calculated by sampling 250 000 snapshots with one every 20 fs from the 5.0 ns simulation. the entropy difference was calculated from the contributions of the solute water term (ssw) and the water the variable r represents the position of the water molecule with respect to the center of the hydration site, and the set of angles represents the orientation of the water molecule in the fixed protein reference frame. as in previous work, only contributions from two particle correlations were considered, and vibrational entropy changes are not considered. furthermore, the solute - water term was separated into translational (ssw, trans) and orientational (ssw, orient) contributions, and the orientational distributions were assumed to be independent of the position within the sites. these terms can be calculated for each hydration site as shown in eqs 7 and 8, respectively:78 in these equations, k is boltzmann s constant, is the number density of bulk solvent, and is the integral over the angles. the solute water translational correlation functions were calculated using a bin size of 0.03 for the radial component, 10 for the angle and 1/18 for cos. the solute water orientational correlation functions were calculated using a bin size of 10 for the and angles and 1/18 for cos. the water water reorganization term was also separated into translational (sww, trans) and orientational (sww, orient) contributions. the water water translational pair probability densities were calculated as previously between water molecules in the hydration site and a set of subvolumes surrounding the hydration site. a sphere of radius 3.6 around the hydration site was split into subvolumes using a bin size of 0.1 for the radial component, 12 for the angle and 1/15 for cos. the 3.6 cutoff is used because contributions to the translational entropy outside the first solvation shell are expected to be small. due to the vast amount of data required for calculation of the solute water water triplet correlation function, a bulk water water radial distribution function gww(r) was assumed as previously.9 gsw(r) is the translational probability density with respect to bulk water within a subvolume. for each hydration site, sww, orient was calculated from the mutual information (iww) between the orientations of water molecules in the hydration site and the orientations of water molecules in subvolumes surrounding the hydration site. a sphere of radius 3.6 around the hydration site was split into subvolumes using a bin size of 1/4 for cos and a bin size of 45 for the other euler angles. the mutual information was then calculated between the hydration site and each of the subvolumes:101112 sww(rel) is the water water relative orientational entropy, where rel is the relative orientation of two water molecules using a bin size of 10. the full five - dimensional relative orientational entropies were estimated by using the second order entropy approximation generated by a truncation of the mutual information expansion. water terms were then compared with the entropy of a water molecule determined from the bulk water simulation (sbulk) due to other water molecules within 3.6 to calculate the entropy difference (s) and entropy of binding (sbind) using eqs 13 and 14, respectively:1314 tsbulk takes values of + 3.54 kcal / mol, + 3.94 kcal / mol, and + 3.85 kcal / mol for the tip3p - ewald, tip4p-2005, and tip5p - ewald water models based on the calculations described here. it is important to note that the values of a half in eq 13 have been removed from the definitions for sww, trans and sww, orient in eqs 9 and 10 (which has been the convention in previous work) such that eqs 13 and 14 correspond to eqs 5 and 6. for each hydration site, the difference in free energy (g) and the free energy of binding (gbind) is then calculated using eqs 15 and 16, respectively:1516 e is assumed to be a good approximation for h, as the pv term is expected to be negligible. we hypothesized that the combination of best probe score and the corresponding water score should be reliable predictors of the observed displacement measure defined in this study. in order to assess this relationship, multiple linear regression analysis was performed in the r statistical computing package:17where o, 1, and 2 are coefficients and e represents the error term. several models of this form were constructed using different formulations of the probe and hydration site scores. the probe scores corresponded to mcss, mcss - he, mcss - hfe, and mm - gbsa scores. for the hydration site score, seven ifst calculated properties were used : g, gbind, e, ebind, s, sbind, and esw. the predictive power for each model was expressed as the adjusted coefficient of determination (r), which is given by18where n is the number of samples and p is the number of variables in the model. the use of r corrects for improvement in correlation arising just by the increase in number of variables. in all other instances, where correlation between any two variables is reported, we used the standard r values. in the initial ifst analysis, hydration sites were placed on each crystallographic water molecule. the results of this analysis using water model tip4p-2005 are summarized in table 1. all of the crystallographic water positions bound favorably to the protein surface, as indicated by the corresponding hydration sites with negative g values. favorable g values have also been noted in previous applications of ifst to proteins and model systems. the free energy of hydration sites exhibited a moderately strong correlation with the observed displacement as indicated by an r value of 0.57. interestingly, some of the hydration sites with a highly favorable free energy corresponded to water molecules (e.g., w301 and w323) that are involved in bridging interactions and are retained by most of the ligands (figure 1). among the other thermodynamic properties of hydration sites calculated from ifst, esw and e also demonstrated strong correlation with the observed displacement, with r values of 0.62 and 0.58, respectively. this is not unexpected, as the binding quantities do not consider the occupancy of the hydration site, which can be low or high. however, the predictions are that enthalpic contributions tend to outweigh entropic contributions in determining the free energy. this is an interesting result that has also been noted in previous applications of ifst to proteins and model systems. however, it may not be general for all systems or for all force fields. it is interesting to note that one of the hydration sites (w336) consistently appeared as an outlier in all correlation plots (figure 2). the removal of this hydration site from the data set resulted in substantial improvement of correlation between observed displacement and thermodynamic properties, as indicated by r values of 0.78, 0.82, and 0.81 for g, esw, and e, respectively. a clear explanation for why this hydration site is an outlier can be found by comparing w336 with w301. w301 is very close to w336 and hydrogen bonds to the same aspartate residue (asp93). interactions with asp93 yield strongly favorable free energies for both hydration sites in comparison with hydration sites near uncharged residues. however, w301 makes a significantly more favorable contribution to the solvation free energy than w336 (11.81 kcal / mol versus 8.28 kcal / mol). this corresponds to a significantly lower observed displacement for w301 compared to w336 (0.05 versus 0.99). importantly, displacement of w336 is only achieved by a positively charged ligand moiety, presumably because the strong electrostatic interactions are necessary to overcome the strong interactions made by the water molecule. thus, displacement of w301 must overcome even stronger water interactions and is rarely observed. this explanation provides the rationale for employing mcss calculations, to allow the strength of interactions with water to be compared with the strength of interactions with probe molecules. water molecules in the binding site of hsp90 n - terminal domain in the apo form (1yer). each water molecule is labeled with the observed displacement fraction based on the analysis of 103 ligands considered in this study. a subset of four highly conserved water molecules (labeled w1w4) is involved in several hydrogen bonds with the binding site. one of these water molecules (w2) was not seen in 1yer, and its position was derived by superposition of a liganded structure (1uy6). correlation plots between the observed displacement from eq 2 and g calculated from eq 15. ifst predictions were made using the water models tip3p - ewald, tip4p-2005, and tip5p - ewald. seven different ifst predictions are reported for hydration sites determined from crystallographic water molecule locations and the ifst free energy prediction is reported for hydration sites determined by clustering of water molecules from the md trajectory. we also compared the effect of other water models in obtaining these quantities for hydration sites. the detailed results for each water model are given in the supporting information (table s2). a comparison of the linear correlation between observed displacement and g values from different models is shown in figure 2. all three water models produced very similar results, with tip3p - ewald producing a very slightly lower correlation and tip4p-2005 producing a very slightly higher correlation. from this point onward, the discussion of ifst results is based entirely on the tip4p-2005 model for this reason. in addition to placing hydration sites at crystallographic water positions the density cutoff was set to half the bulk density in order to identify all hydration sites. it was noted that some crystallographically observed water molecules have approximately the same number density as bulk water and were not predicted using a higher cutoff. such hydration sites exhibit clustering of water molecules, leading to translational ordering and an unfavorable entropy contribution, but have a weakly favorable enthalpic component and thus a free energy that is approximately the same as bulk water. in terms of reproducing the crystallographic water positions from the simulation, the success rate (expressed as a percentage of crystallographic water molecules reproduced within 1.0) was similar across different water models with tip4p-2005 having the highest success rate of 80% (table 1 and table s2). clustering identified 18 of the 20 displaceable water molecules within 1.5 and 16 within 1.0. the failures all corresponded to crystallographic water positions with low occupancy and small free energy contributions as computed by ifst from the experimentally determined positions. this resulted in a slightly lower correlation with observed displacement (r of 0.50) and suggests that placing hydration sites at crystallographic water positions may be more predictive. the results of the mcss calculations are summarized in table 2. for each water position, the raw mcss scores reflect the charmm interaction energy between the probe and the protein, and desolvation penalties were applied to these raw scores. we tested the use of the hydration enthalpy (mcss - he) and the hydration free energy (mcss - hfe) as desolvation penalties. the resulting scores were then normalized according to the relevant probe molecular volume. the original mcss output indicated a strong preference for charged groups for the majority of the hydration sites. this is most likely due to the crude approximation of solvent - screened electrostatic interactions in the distance - dependent dielectric model. the addition of desolvation penalties somewhat corrected for this trend by replacing nonpolar or polar probes in at least eight water positions. the mm - gbsa scores demonstrated the most significant differences, identifying polar molecules such as methanol and phenol as the most suitable probes for the majority of the hydration sites. the important point to note from the mcss analysis was that none of the different formulations had a significant correlation with observed displacement (table 3). this is not unexpected, since capturing displaceability of water molecules solely from probe binding is unlikely, especially when probe scores contain crude approximations for solvation and entropic effects. the correlation between the different formulations of probe score and the predicted thermodynamic properties of water molecules was also mostly weak or insignificant. volume - corrected mcss scores showed r values of 0.29 and 0.39 with g and gbind of the corresponding hydration site. when best scoring mcss probes are divided into polar and nonpolar categories, the correlation of scores with most of the ifst based properties is in opposite directions. for instance, polar probes have a correlation of 0.21 and 0.24 with g and h, respectively. on the other hand nonpolar probes have a correlation of 0.60 and 0.59 with g and h, respectively. this partially explains the nearly insignificant correlation when probes are considered together. in general, it was noticed that hydration sites with a large enthalpic component were associated with charged or polar probes whereas hydration sites with a small enthalpic component were associated with nonpolar probes. in order to qualitatively assess the mcss results, we compared the distribution of probes in the binding site with that of the ligands. for each hydration site, ligand atoms within 1.0 of the crystallographic water molecule were obtained, which provided the distribution of atoms that most likely displaced it. these atoms were then divided into polar (n, o, s, p) and nonpolar (c) groups. the same procedure was then repeated for probes which provided the predicted distribution of functional groups around each hydration site. in figure 3, these distributions are represented as an occupancy map on a 1 grid, where each grid point contains the amount of time it was occupied by an atom, represented as a fraction of the total number of atoms within 1 of the hydration site. first, we selected a set of hydration sites with highly favorable free energy (figure 3a and b) and relatively high observed displacement. for at least three of these hydration sites (w325, w346, and w412), polar atoms comprised more than a 1/3 fraction of the displacing groups. w324 and w529 were displaced less frequently by polar groups. when compared with probe occupancies, an important difference was that probe polar atoms more frequently occupied w324 and w529. on the other hand, probe polar atoms were less frequently observed for w346, which is frequently displaced by a polar ligand atom. the overlap between the ligand and probe occupancy maps was more pronounced for a set of less favorable hydration sites (figure 3c and d), which were most frequently displaced by nonpolar functional groups (both in crystallographic ligands and probes). this can be rationalized on the basis that the optimal van der waals interactions between (ligand / probe atoms and protein atoms) are more accurately reproduced by mcss than the electrostatic interactions. taken together, these results showed that hydration sites with highly favorable free energy (owing mostly to a strong enthalpic component) were displaced frequently by polar ligand atoms, and mcss - based predictions for probes showed a similar trend. conversely, hydration sites with less favorable free energy (with a decreased enthalpic but comparable or even large entropic component) were mostly displaced by nonpolar ligand and probe atoms. the number of distinct probes occupying the same energetically favorable site has been used before as an indicator of binding hot spots. prior to selection of the best scoring probe, we obtained the number of distinct probes identified at each hydration site and compared it with the observed displacement. hence, a clear relationship between diversity of probe binding and the likelihood of water displacement was not established from these data. a comparison of polar and nonpolar atom densities derived from crystallographic ligands (a and b) and mcss - derived probe positions (c and d) in the hsp90 binding site. atoms around a given hydration site are represented as an occupancy map on a 1 grid, with grid points contoured at points where 30% of the total number of atoms within a 1 region around the hydration site are of the given type). (a and c) a set of highly favorable hydration sites with corresponding polar - atom occupancy maps compared across crystallographic ligands and probes. (b and d) a set of relatively less favorable hydration sites with corresponding nonpolar atom occupancy maps compared across crystallographic ligands and probes. the results obtained from ifst calculations showed that the predicted free energy of hydration sites was a fairly reliable predictor of observed displacement. in order to assess whether the combination of ifst and mcss probe analysis could improve the predictions, we used multiple linear regression models based on different combinations of hydration site thermodynamic properties and probe scores. the resulting data are summarized in table 4, where the adjusted coefficient of determination is reported for each model. in most cases, the use of desolvation penalties derived from experimental data also did not lead to any improvement in predictions. however, as noted above, when the outlier w336 is removed from the data set, the adjusted r values increased in some cases. for example, the highest value of adjusted r (0.80) was obtained from a model combining esw values from ifst and probe gbsa scores. this also indicated that the use of gbsa to estimate probe desolvation penalties provided better results than constant desolvation penalties based on experimental hydration enthalpy or free energy values. each model is derived from two predictors, indicated by row and column titles, and the value reflects strength of correlation with observed displacement. for comparison, r s for ifst values (without mcss) are also given. an important consideration in ligand design is the prioritization of ordered water molecules in the binding site for displacement and concomitant affinity gains. we selected a set of four water positions to assess the applicability of solvation thermodynamics and probe analysis in order to address such questions (figure 1). three of these water positions are already included in the set of reference water molecules derived from the apo structure (1yer). however, one additional water molecule is visible only in ligand bound hsp90 (e.g., 1uy6). for consistency with previous studies, we refer to these water positions as w1, w2, w3, and w4. the free energy of hydration sites corresponding to these water molecules, the best scoring probes (based on gbsa scores), and associated data are summarized in table 5. these water molecules form a buried and tightly coordinated network of hydrogen bonds with the protein and/or ligand (figure 1). the combined regression model based on ifst and mcss predicts w2 to be the most displaceable in this group, whereas w2 and w3 are predicted to be moderately displaceable, and w1 is predicted to be very difficult to displace. the probe analysis predicts that w2 and w4 could be replaced by methane, whereas the w1 and w3 sites were most favorably filled by ammonia and methanol, respectively (figure 4b and d). a collective picture emerges from the probe analysis that the binding site region containing w2 and w4 accommodates functional groups of nonpolar character, but w3 needs to retain hydrogen bonding interactions made by the displaced water molecules. this is also suggested by the good interaction energy (esw) for the w3 site. (b) w2 and w3 were predicted to be replaced by methane and methanol as most favorable probes. (c) in x - ray crystallographic ligands, w2 and w3 have been displaced by nitrile and methyl groups, respectively. (d) the best scoring probes corresponding to w1 and w4 were ammonia and methane, respectively. (e) in x - ray crystallographic ligands, w4 has been replaced by an ethyl group, but no reported examples were found for the displacement of w1 (it is not present in some structures but never overlaps with the ligand). w1, w3, and w4 correspond to 301, 328, and 303 from 1yer, whereas w2 is missing from 1yer. thus, all ifst results are from corresponding hydration sites using the clustering protocol. the ifst predictions agree well with experimental data on binding affinities for a pyrrolopyrimidine scaffold. commensurate with this is a high predicted displacement, and indeed displacement of w2 by a nitrile group was shown to increase binding affinity approximately 250-fold (figure 4c). mcss suggests a methyl probe is the most favorable replacement for w2 (with an mmgbsa score of 4.55 kcal / mol), but the methanol probe also scores highly (with an mmgbsa score of 4.13 kcal / mol), and this is consistent with displacement by a nitrile group that makes a hydrogen bond with asn51. thus, the experimental observations can be explained by ifst and mcss due to the low g for w2 in concert with a good probe score (table 5). for the nitrile derivative, further displacement of w3 by a methyl group did not increase binding affinity. this was attributed to the attenuation of affinity gain by the loss of hydrogen bonding interactions for w3 and the clash of the new side chain. the rationalization based on ifst predictions is that the lack of significant gain in affinity is because w3 is not as easy to displace as w2 and that a nonpolar group is not the optimal replacement.. further displacement of w4 by extending the methyl group to an ethyl group was shown to increase binding affinity 3-fold (figure 4c). ifst and mcss again rationalize this, with w4 predicted to be moderately displaceable by a nonpolar group such as methane. in addition, there is a small nonpolar cavity behind w3 that does not contain a water molecule. such evacuated regions can provide unexpected boons in binding affinity, and extension of the ethyl group to an isopropyl group may prove beneficial. it is interesting to note that the model predicts that w2, w3, and w4 are significantly more displaceable than has been observed experimentally. the recent experimental data suggest that ifst is able to identify untapped potential at these sites. this would be very useful information in other established drug development projects. in order to further investigate the relationship between x - ray crystallographic ligands and thermodynamic properties of hydration sites along with associated functional groups, we selected a subset of unfavorable hydration sites including w338, w357, w379, w381, w385, w405, w435, w476, w536, and w547. the choice of these hydration sites was based on the fact that most of these are almost always displaced by the ligands (table 1), and their calculated free energies lie in the range 0.5 kcal / mol to 2.5 kcal / mol. the positions of the corresponding x - ray crystallographic water molecules and the most favored mcss probes, based on mm - gbsa scores, are shown in figure 5. the majority of sites are occupied by nonpolar groups, though some lower energy hydration sites are occupied by polar groups. this suggests an overall trend of prioritizing lower energy hydration sites for displacement with polar groups and high energy hydration sites with nonpolar groups. we performed a retrospective analysis of the different ligand series used in this study to see if a similar trend could be observed. first, we observe that w338 and w547 are commonly displaced by nonpolar groups in known inhibitors, which is consistent with the nonpolar mcss probe positions. w338 is displaced by a nonpolar group in figures 6a c and 7a, b, whereas w547 is displaced by a nonpolar group in figures 6c, 7a, and 8a c. similarly, for w357 the methanol probe position is consistent with the polar functionalities of known inhibitors in figures 6a c, 7a, b, and 8a, b. a subset of crystallographic water molecules in the hsp90 binding site, labeled with the ifst - based solvation free energies in kcal / mol and colored from yellow to red representing low to high solvation free energy. representative ligands from aminopyri(mi)dine series overlaid on the crystallographic water molecules labeled with ifst - based solvation free energies in kcal / mol and colored from yellow to red representing low to high solvation free energy. these compounds represent hits from fragment and virtual screening (a and b) and an optimized clinical candidate (c). representative ligands from resorcinol (a, b) and benzamide (c, d) series overlaid on the crystallographic water molecules labeled with ifst - based solvation free energies in kcal / mol and colored from yellow to red representing low to high solvation free energy. ligand series overlaid on crystallographic water molecules labeled with ifst - based solvation free energies in kcal / mol and colored from yellow to red representing low to high solvation free energy. in addition, we noticed that w405 and w435, which are relatively favorable hydration sites in this subgroup, are located in a hydrophobic cavity lined by ala55, ile96, gly97, and ile107. in the x - ray structure, the only hydrogen bond observed for this pair of water molecules is between w435 and the backbone carbonyl of leu107. the mcss predicted probes reproduce this pattern by favoring a methane in place of w405 and a phenol group in place of w435, retaining the hydrogen bond of w435 with the backbone carbonyl of leu107. such binding motifs where hydrogen bonding functionalities are enclosed in a hydrophobic environment can provide binding hotspots. the combination of ifst and mcss can provide valuable information in this regard. many of the potent hsp90 inhibitors displace this pair of water molecules with a combination of polar and nonpolar groups, as predicted by probe analysis (figure 6b, 7c, d). figures 68 provide different scenarios in ligand design where predicted displacement of water alongside probe analysis could prove useful. we emphasize that binding site hydration was not explicitly addressed during ligand design in these examples from the literature. however, the results are useful in understanding the utility of ifst and mcss. in figure 6, representative ligands from various stages of design (fragment / virtual screening hits 6a and b plus the clinical candidate 6c) are shown, which highlight the observation that stepwise disruption of binding site hydration (although not attempted in this particular ligand series) is a suitable route toward optimization. it is notable that all of the ligands in this study displace w357, which ifst predicts to make the least favorable contribution the solvation free energy among the 20 waters studied. w536 is predicted to make the second least favorable contribution to the solvation free energy and also appears to be a clear binding hotspot. the weaker inhibitors in figure 6a and b do not displace it, whereas the high affinity clinical candidate in figure 6c does displace it. this suggests that further affinity can be gained by molecules that do not displace w536, such as the inhibitor in figure 8b. a systematic approach to ligand optimization should involve an assessment of binding site water molecules and the chemical functionalities suitable for displacing them. figure 7 provides an example where core scaffolds from two distinct chemotypes (resorcinol in figure 7a and b plus benzamide in figure 7c and d) bind at similar locations, displacing the same hydration sites, w357 and w405, representing potent hydration sites for displacement. apart from these two sites, different functional groups in each of these optimized candidates displace different water molecules. consequently, the similarities between these functional groups and mcss - predicted probes are interesting, e.g., methanol probes at w385 and w536 compared against the ligand in figure 7a and phenol at w381 compared against the ligand in figure 7c. finally, in fragment linking approaches (shown in figure 8a and b), the choice of linker can benefit from consideration of probes favorably displacing a water molecule. for example, the methylsulfonamide linker between the two fragments shown in figure 8a displaces w381 in figure 8b. mcss favors a polar phenol probe at this site (figure 6b), and the overlap of polar atoms between ligand and probe is noteworthy to summarize, the results suggest that ifst and mcss can be favorably combined to provide a useful binding site profile. the clustering of probes at a given location on the protein surface has been used as an indicator of binding hotspots, which can give rise to substantial gains in affinity for protein ligand interactions. however, the use of probe calculations alone can result in false positives as they do not accurately consider the effects of solvation. ifst provides explicit accounting for water molecules, and hence a combination of both approaches can lead to better identification of hotspots in concert with advantageous functional groups for binding. conversely, high scoring probe positions from mcss that correspond to energetically favorable hydration sites can indicate strategically important sites where designing bridging interactions will be more appropriate. in this study, we have combined ifst calculations of solvent thermodynamics with mcss probe analysis to predict the observed displacement of water molecules from protein hydration sites on hsp90 and the best type of functional moiety to replace them, in the context of small molecule ligand design. ifst calculations of the hydration site free energies yielded a moderate correlation with the observed displacement, an encouraging result given the number and variety of ligands considered. interestingly, the correlation with the total interaction energy (e) is slightly better than the correlation with the free energy (g), and the correlation with the protein interaction energy (esw) is even better. this can be rationalized on the basis that the enthalpic contributions to the solvation free energies are calculated to be significantly higher than the entropic contributions and that the loss of water water interactions is less significant in the context of a ligand in the binding site that has already displaced the neighboring water molecules. this result suggests that approaches such as mcss, which can calculate the interaction energy of water molecules with the protein, should work well in predicting observed displacement. however, there are two issues with this. the first issue is that the protein interaction energy from ifst is calculated from an explicit water simulation, and calculations based on a single water molecule that is not part of an ensemble will be different. the second issue is that the protein interaction energy from ifst is scaled by the occupancy of the site, and a calculation of the highest scoring single water molecule is not. such a calculation is more analogous to the ifst binding quantities, which we have shown to have a weaker correlation with observed displacement. to explore the application of ifst, we also calculated predictions based on three different water models, and in this case the results are very similar. all three water models predict hydration sites at very similar locations, and the r between free energy predictions for the different water models is greater than 0.9 in all cases. in addition to predicting the observed displacement of water molecules, we considered whether md simulations can reproduce crystallographic water positions. for tip4p-2005, 18 out of the 20 crystallographic water positions were reproduced within 1.5 (table 1). it is important to note that a complete reproduction of crystallographic water positions is not always to be expected, as crystallographic water positions rely on an assignment of density from sometimes ambiguous data. the first was to place hydration sites at the positions of crystallographic water molecules, and the second was to place hydration sites by clustering the positions of water molecules from the md simulations. the two approaches yield hydration sites at very similar positions with almost identical thermodynamic properties. this is shown by the high correlation between the free energies of the set of 18 equivalent hydration sites that are common between the two approaches (r = 0.99). however, placing hydration sites at crystallographic water positions is slightly more predictive of the displacement, because the clustering procedure failures to identify all the crystallographic hydration sites. the ifst calculations show another interesting result, which is that the enthalpic contributions to the total free energy are commonly of greater magnitude than the entropic contributions. this has been noted previously and in contrast to the results of ifst, mcss proved unsuccessful at predicting displacement and was unable to improve the predictions of ifst when the two were used in concert. however, mcss does demonstrate a synergy with ifst when applied to ligand design. when ifst identifies a hydration site as highly displaceable, mcss can predict the ligand functionality that should be used to displace it. such information is harder to glean from ifst calculations alone, although two points should be noted. the first is that the positions of hydration sites predicted to have a highly favorable free energy are commonly found to overlap with polar ligand atoms. this is easily rationalized, as strong hydrogen bonding is the cause of both effects. in addition, the predicted orientation of the hydrogen bonding interactions for a water molecule is often recapitulated by the geometry of ligands, and this observation could be used in design. conversely, hydration sites predicted to have a small free energy are commonly found to overlap with nonpolar ligand atoms. water displaceability is a difficult metric to capture from experimental data because displacement is boolean for any given complex. in fact, almost every water molecule should be displaceable by some ligand, but this may be deleterious to the binding affinity. for example, displaceability data may be confounded if medicinal chemists have deliberately attempted to displace particular water molecules in cases where this is deleterious to the binding affinity. this will increase the frequency of displacement for a given water molecule in a case where displacement is not advantageous. for this reason, thermodynamic data may prove more useful in quantifying displaceability, but one still can not quantify the specific contribution of a single water molecule to the binding affinity. thus, we consider that the frequency of displacement from a large and varied data set of complexes is the best experimental data available. however, such data are still imperfect due to the bias of design by medicinal chemists. in addition, there are many cases where water molecules are subtly shifted rather than displaced, and this blurs such data. despite these issues, ifst has proven to be a useful method for studying solvation thermodynamics, providing a perspective that is not available using other computational methods by decomposing solvation free energies into contributions from specific spatial regions. a similar perspective can be gained from fep, by decomposing solvation free energies into contributions from specific species, including the water molecules. fep can also be used to selectively excite the individual degrees of freedom of water molecules and assess their contribution to the physicochemical characteristics of water. in this work, we have shown that ifst can be used to identify binding hotspots and predict which water molecules should be advantageously displaced from a protein binding site. while a larger study would be needed to show that this is a general result, early results are promising and suggest that such an analysis performed on a new drug target would provide very useful information for ligand design. it may also show utility in identifying untapped potential for well - known drug targets. as noted previously, accurately predicting the effect of a ligand modification upon binding affinity can not be achieved by considering any one factor and requires a comprehensive thermodynamic analysis of the ligand binding. however, given the vast number of potential ligands to be considered, there is a significant advantage in deriving useful information from ifst, which can be calculated from a single md simulation. | intermolecular interactions in the aqueous phase must compete with the interactions between the two binding partners and their solvating water molecules. in biological systems, water molecules in protein binding sites cluster at well - defined hydration sites and can form strong hydrogen - bonding interactions with backbone and side - chain atoms. displacement of such water molecules is only favorable when the ligand can form strong compensating hydrogen bonds. conversely, water molecules in hydrophobic regions of protein binding sites make only weak interactions, and the requirements for favorable displacement are less stringent. the propensity of water molecules for displacement can be identified using inhomogeneous fluid solvation theory (ifst), a statistical mechanical method that decomposes the solvation free energy of a solute into the contributions from different spatial regions and identifies potential binding hotspots. in this study, we employed ifst to study the displacement of water molecules from the atp binding site of hsp90, using a test set of 103 ligands. the predicted contribution of a hydration site to the hydration free energy was found to correlate well with the observed displacement. additionally, we investigated if this correlation could be improved by using the energetic scores of favorable probe groups binding at the location of hydration sites, derived from a multiple copy simultaneous search (mcss) method. the probe binding scores were not highly predictive of the observed displacement and did not improve the predictivity when used in combination with ifst - based hydration free energies. the results show that ifst alone can be used to reliably predict the observed displacement of water molecules in hsp90. however, mcss can augment ifst calculations by suggesting which functional groups should be used to replace highly displaceable water molecules. such an approach could be very useful in improving the hit - to - lead process for new drug targets. |
a total of 28 volunteers, consisting of 16 males (age range : 25 - 29 years ; mean age : 26.9 1.2 years) and 12 females (age range : 20 - 38 years ; mean age : 27.8 5.5 years), were included in this study. all the subjects were right - handed with no history of neurological or psychiatric illness. the subjects were informed of the procedure and matters that required attention prior to the experiment, and written informed consent was obtained. after completion of the fmri study, each subject was asked to rate their emotional status while viewing the natural and urban sceneries on a 3-point scale : 1, suffocating ; 2, accustomed ; 3, comfortable. the study was approved by the chonnam national university hospital institutional review board (irb). the visual stimulation paradigm consisted of three times rest condition and two times activation condition, each lasting for 30 and 120 seconds, respectively. the natural and urban scenic views were presented for 3 seconds each and repeated two times during the activation condition. the rest conditions were a thin white cross mark in the block background screen. prior to the fmri examination, a total of 10 subjects were each requested to pick up 20 natural and urban scenic views from a pool of 300 pictures. pictures for visual stimulation were collected for every 20 natural and urban environment scenic views related to human habitation from a variety of web sites on the world wide web. natural scenic views included themes such as natural landscapes, mountains, natural parks, forest, and so on. urban scenic views included themes such as city landscapes, tall buildings, and so on. the illuminance levels were measured with a digital illuminance meter (illuminance meter, tektronix, beaverton, or). the visual stimuli were generated on a pc and projected via a liquid crystal display (lcd) projector onto a screen located on the head coil in front of the subject 's forehead. a functional mri was performed on a 3.0 t magnetom trio mr scanner (siemens medical solutions, forchheim, germany) with a bird cage head coil. the functional images were acquired from 25 transverse slices parallel to an ac - pc (anterior commissure to posterior commissure) line using a gradient - echo echo planar pulse sequence with the following parameters : repetition time (tr)/echo time (te) = 2,000 ms/30 ms, flip angle = 90, field of view (fov) = 2222 cm, matrix size = 6464, number of excitations (nex) = 1, and slice thickness = 5 mm, giving a total of 4,125 images. in addition, two phases of dummy scans were supplemented to circumvent unstable fmri signals, for a total acquisition time of 330 seconds. also, high - resolution anatomical images, t1-weighted images (tr / te = 500 ms/8 ms), and t2-weighted images (tr / te = 3,500 ms/88 ms), were acquired with the following parameters : fov = 2222 cm, matrix size = 192192, nex = 2, slice thickness = 5 mm, and slice gap = 2 mm. functional images were analyzed using the spm99 software (statistical parametric mapping 99, the wellcome department of cognitive neurology, university college london, uk). at first, images were realigned within and across the scans to correct for head movement. next, whole - brain normalization was applied to transform all images according to the montreal neurological institute (mni) template and each volume was resliced with 2 mm. normalized images were then smoothed with a spatial gaussian filter with an 8 mm full - width - at - half - maximum (fwhm). next, the activated areas were identified by a multiple regression analysis of the time series of the mri signal intensities in each voxel. the preprocessed date was analyzed using the standard general linear model (glm) approach of spm99 with the boxcar model. to analyze the individual bold signal in a voxel with a dimension of 2 2 2 mm statistical activation maps were obtained for the contrast of ' natural versus rest ' and ' urban versus rest '. this analysis was performed in order to identify brain areas with an increased bold signal while viewing the natural and urban scenery in relation to the rest periods. for the group analysis of natural and urban groups, the differential activation maps, which correspond to the contract of natural versus urban and the urban versus natural, were obtained from the two sample t - test. significant activation maps for these contrasts were identified by a whole - brain analysis with a statistical threshold of p < 0.05. a total of 28 volunteers, consisting of 16 males (age range : 25 - 29 years ; mean age : 26.9 1.2 years) and 12 females (age range : 20 - 38 years ; mean age : 27.8 5.5 years), were included in this study. all the subjects were right - handed with no history of neurological or psychiatric illness. the subjects were informed of the procedure and matters that required attention prior to the experiment, and written informed consent was obtained. after completion of the fmri study, each subject was asked to rate their emotional status while viewing the natural and urban sceneries on a 3-point scale : 1, suffocating ; 2, accustomed ; 3, comfortable. the study was approved by the chonnam national university hospital institutional review board (irb). the visual stimuli were presented in a block design fashion. the visual stimulation paradigm consisted of three times rest condition and two times activation condition, each lasting for 30 and 120 seconds, respectively. the natural and urban scenic views were presented for 3 seconds each and repeated two times during the activation condition. prior to the fmri examination, a total of 10 subjects were each requested to pick up 20 natural and urban scenic views from a pool of 300 pictures. pictures for visual stimulation were collected for every 20 natural and urban environment scenic views related to human habitation from a variety of web sites on the world wide web. natural scenic views included themes such as natural landscapes, mountains, natural parks, forest, and so on. urban scenic views included themes such as city landscapes, tall buildings, and so on. the illuminance levels were measured with a digital illuminance meter (illuminance meter, tektronix, beaverton, or). the visual stimuli were generated on a pc and projected via a liquid crystal display (lcd) projector onto a screen located on the head coil in front of the subject 's forehead. a functional mri was performed on a 3.0 t magnetom trio mr scanner (siemens medical solutions, forchheim, germany) with a bird cage head coil. the functional images were acquired from 25 transverse slices parallel to an ac - pc (anterior commissure to posterior commissure) line using a gradient - echo echo planar pulse sequence with the following parameters : repetition time (tr)/echo time (te) = 2,000 ms/30 ms, flip angle = 90, field of view (fov) = 2222 cm, matrix size = 6464, number of excitations (nex) = 1, and slice thickness = 5 mm, giving a total of 4,125 images. in addition, two phases of dummy scans were supplemented to circumvent unstable fmri signals, for a total acquisition time of 330 seconds. also, high - resolution anatomical images, t1-weighted images (tr / te = 500 ms/8 ms), and t2-weighted images (tr / te = 3,500 ms/88 ms), were acquired with the following parameters : fov = 2222 cm, matrix size = 192192, nex = 2, slice thickness = 5 mm, and slice gap = 2 mm. functional images were analyzed using the spm99 software (statistical parametric mapping 99, the wellcome department of cognitive neurology, university college london, uk). at first, images were realigned within and across the scans to correct for head movement. next, whole - brain normalization was applied to transform all images according to the montreal neurological institute (mni) template and each volume was resliced with 2 mm. normalized images were then smoothed with a spatial gaussian filter with an 8 mm full - width - at - half - maximum (fwhm). next, the activated areas were identified by a multiple regression analysis of the time series of the mri signal intensities in each voxel. the preprocessed date was analyzed using the standard general linear model (glm) approach of spm99 with the boxcar model. to analyze the individual bold signal in a voxel with a dimension of 2 2 2 mm statistical activation maps were obtained for the contrast of ' natural versus rest ' and ' urban versus rest '. this analysis was performed in order to identify brain areas with an increased bold signal while viewing the natural and urban scenery in relation to the rest periods. for the group analysis of natural and urban groups, the differential activation maps, which correspond to the contract of natural versus urban and the urban versus natural, were obtained from the two sample t - test. significant activation maps for these contrasts were identified by a whole - brain analysis with a statistical threshold of p < 0.05. the questionnaire results evaluating the subjects ' emotional status by self - report, while viewing the natural scenery were as follows : comfortable (93%), accustomed (4%), and suffocating (4%). on the other hand, the results of subjects exposed to the urban scenic views were as follows : comfortable (0%), accustomed (50%), and suffocating (50%). figure 1 shows the differential brain activation patterns between natural and urban scenic views, which were analyzed by two sample t - test (p < 0.05). the predominant activation areas in natural scenic views in contrast with urban views consist of the superior and middle frontal gyri, superior parietal gyrus, precuneus, basal ganglia, superior occipital gyrus, anterior cingulate gyrus, superior temporal gyrus, and insula. conversely, the predominant activation areas in urban scenic views in contrast with natural scenic views consist of the middle and inferior occipital gyrus, parahippocampal gyrus, hippocampus, amygdala, anterior temporal pole, and inferior frontal gyrus (table 1) (fig. the aim of this study was to compare the differential brain centers associated with psychological preference between natural and urban scenic views by using the bold - based fmri. our questionnaire results are consistent with previous studies (6, 16), which suggested that people generally have a preference for natural scenic views over urban views. we assume that such emotional status and behaviors are connected with the activation of the specific brain areas. in our current study, the predominant brain activation areas following exposure to urban views in contrast to natural scenic views were observed in the middle and inferior occipital gyri, parahippocampal gyrus, hippocampus, amygdala, anterior temporal pole, and inferior frontal gyrus. (12), who had shown the brain activation associated with unpleasant emotion in healthy women using pet (positron emission tomography). they suggested that unpleasant emotion was associated with significant increases in the occipito - temporal cortex, parahippocampal gyrus, hippocampus, and amygdala. 17) suggested that unpleasant stimuli induced the activation of the amygdala and visual cortex in contrast with the pleasant stimuli. in our study, the primary visual cortex and its adjacent areas, which consist of the middle and inferior occipital gyri, showed higher activities in urban views compared to natural views. however, the superior occipital gyrus showed a higher degree of activity in viewing the natural scenic view. the fmri studies (13, 18, 19) suggested that the primary visual cortex played an important role in visual information perception, and the visual association cortex showed greater enhancement of signal intensities during the processing of emotional pictures. moreover, increased activity of the primary and association visual cortex was found in the unpleasant / pleasant pictures comparisons (12, 17). it should be noted that the activation of the common areas of the visual cortices evoked by both natural and urban scenic views were eliminated by the statistical analysis with the two sample t - test. moreover, assuming that viewing the urban scenery leads to an increased emotional arousal compared to natural views, the activation of the middle and inferior occipital gyri noted in our study could be related to a greater arousal level than for mood valence, including pleasant or unpleasant. we also observed the activation of the parahippocampal gyrus, hippocampus, and amygdala with urban views only. the limbic system, including these areas is related with emotion excitation and affective behavior. the hippocampus and parahippocampal gyrus play the important roles of perception, memory, and recall by visual stimulus (12, 20). in addition, activation in the parahippocampal gyrus and amygdala are associated with recall and re - experiencing of emotional distress as well as distraction for visual stimuli (21). in particular, the parahippocampal gyrus is implicated in the encoding of complex visual pictures (22, 23). the activation of the parahippocampal gyrus noted in our study may reflect encoding of the perceptual aspects related to the visual complexity of urban environments. the amygdala is one of the most important structures in the evaluation of brain activation with negative acquirement and the expressions of anxiety, fear, aversion, and unpleasantness. in our study, various studies with humans and animals have suggested that the amygdala responds to aversive stimuli (12, 14, 18, 24). the subjects ' self - reported ratings supported that the subjects felt suffocated when viewing the urban scenery compared to than the natural scenery. (25) demonstrated that normal participants experienced negative emotions from negative visual stimuli, whereas urbach - wiethe patients with no amygdala did not experience negative emotions from the same negative stimuli. such evidence supports that the brain activation in our study may be due to an underlying original nature related to unpleasant emotion while viewing urban scenic views. the activation of the anterior temporal pole was induced by the urban scenic view only. the anterior temporal pole has reciprocal connections to the amygdala, hippocampus, and prefrontal cortex (26). this area is activated during subjective emotional responses (27), which are associated with negative emotions, including anger (28, 29) and unpleasantness (30). this finding is correlated with self - reported ratings of their unpleasant emotions in viewing the urban scenery over the natural scenic views. activation of the anterior temporal pole may be related to the appraisal process of negative emotional reactions induced by urban views. the hippocampus, parahippocampal gyrus, amygdale, and anterior temporal pole showed greater activity when exposed to urban scenery, which may be due to the specific role of these areas in processing the unpleasant emotional arousal than the comfortable arousal induced by natural scenic views. contrary to the parahippocampal gyrus, hippocampus, and amygdala, the anterior cingulate gyrus, which is also a part of the brain 's limbic system, showed greater activity for the natural views. activation of the anterior cingulate gyrus in our study is typical for mood induction elicited by recall or imagery, as well as during emotional tasks with cognitive demand (31, 32), and is presumably caused by the regulation of tasks with cognitive and affective components when viewing the natural environment. the parietal lobe is often associated with visual and spatial attention. the precuneus is activated when paying attention to a visual, and is also related to episodic memory which generates a visual image during the stimulus (33 - 35). in our study, the superior parietal gyrus and precuneus were activated while viewing the natural scenery. these results suggest that the subjects showed a tendency to be more concentrated in viewing the urban scenic pictures than the natural scenery, and they did not pay attention to higher visual processing of unpleasant emotion when viewing the stuffy urban scenic pictures. another interesting finding in our study is the activation of the insula, which is one of the internal components of the limbic system. the insula showed significant activation in the natural scenic views in contrast with the urban scenery. some previous neuro - imaging studies (36 - 38) have demonstrated that the insula is related to a variety of emotional functions. in particular, the insula is preferentially involved in the evaluative, experiential, or expressive aspects of ' internally generated ' emotions (37, 39). also, bartels and zeki (40) found significant activation of the insula when viewing the preferred pictures. conversely, other studies (41, 42) suggested that the insula responded to negative emotional stimuli such as pain and trauma. the basal ganglia include the globus pallidus and caudate nucleus, and its activation is observed in response to happiness - induced recall (38, 43) and pleasant pictures (13, 18, 44). in our study, this area was predominantly activated when viewing the natural scenic pictures only. these findings suggest that the activation of these areas reminds of past experienced memories or recollection by paying greater visual attention to the natural views than the urban scenery. in our study, the predominant activation areas of the frontal lobe for natural scenic views in contrast with urban scenery included the superior and middle frontal gyri. these areas play an important role in emotional cognitive processes and are thought to be sensitive to the approach - withdrawal emotion specifically (12, 15, 17). the inferior frontal gyrus, another significant area in our study, showed greater brain activity in urban scenic views than natural views. this area may be related with the decision - making and emotional processes associated with urban views. kross. (45) suggested that the rejection sensitivity is associated with a significantly greater level of activity in the inferior frontal gyrus related to self - monitoring. (13) also suggested that the distraction was associated with activation of the inferior frontal gyrus. in particular, the combination of the prefrontal cortex and the limbic system is very important in its brain structure and function. this cortex has a high number of interconnections with the limbic system and thus plays a role in the regulation and expression of emotions and feelings when viewing their favorite or least favorite scenic pictures. however, our study dealt with only the visual stimulation with no regard to other olfactory and auditory stimuli. in addition, we evaluated a subject 's emotional status with a self - report while viewing the natural and urban scenery. we did not measure the arousal level of the pictures and the physiological indices reflecting heart rate and blood pressure in this study. therefore, it is unclear whether the activation areas were related to high or low arousal elicited by natural and urban scenic views. this study dealt with the evaluation of differential activation patterns of the human brain in response to natural and urban scenic views. the brain activation patterns are presumably associated with personal preference of the scenic views, reflecting a given subject 's emotional status and feelings based on psychology. these findings can be utilized as a neural index for the assessment of objective preference of the living environments on human habitation. | objectiveby using a functional magnetic resonance imaging (fmri) technique we assessed brain activation patterns while subjects were viewing the living environments representing natural and urban scenery.materials and methodsa total of 28 healthy right - handed subjects underwent an fmri on a 3.0 tesla mri scanner. the stimulation paradigm consisted of three times the rest condition and two times the activation condition, each of which lasted for 30 and 120 seconds, respectively. during the activation period, each subject viewed natural and urban scenery, respectively.resultsthe predominant brain activation areas observed following exposure to natural scenic views in contrast with urban views included the superior and middle frontal gyri, superior parietal gyrus, precuneus, basal ganglia, superior occipital gyrus, anterior cingulate gyrus, superior temporal gyrus, and insula. on the other hand, the predominant brain activation areas following exposure to urban scenic views in contrast with natural scenes included the middle and inferior occipital gyri, parahippocampal gyrus, hippocampus, amygdala, anterior temporal pole, and inferior frontal gyrus.conclusionour findings support the idea that the differential functional neuroanatomies for each scenic view are presumably related with subjects ' emotional responses to the natural and urban environment, and thus the differential functional neuroanatomy can be utilized as a neural index for the evaluation of friendliness in ecological housing. |
in both diabetic and non - diabetic patients, albuminuria has been recognised as an important renal and cardiovascular risk factor. although, using chemical methods, albuminuria had been detected in patients without primary renal disease more than 100 years ago, it was only after the introduction of immune detection methods and the recognition that albuminuria is a predictor of diabetic nephropathy in type 1 and type 2 diabetes [4, 5 ] that there was an explosive growth of information in this field. the issue whether there is a safe threshold value of albuminuria has recently provoked intense discussion, mainly because in both non - diabetic and diabetic patients, urinary albumin concentrations in the upper normal range have been found to predict both cardiovascular and renal plus cardiovascular events in high risk and low risk populations. for these and other reasons it has been proposed by some authors to abandon microalbuminuria as a diagnostic category and to treat urinary albumin excretion (uae) as a continuous variable, much like bp or serum cholesterol concentration. a great number of past and recent studies have evaluated the evolution of uae from normoalbuminuria to microalbuminuria and its correlates in adolescent [1012 ] and adult [13, 14 ] type 1 diabetic patients. information on type 2 diabetic patients is scarcer and is available only for some small cohorts. the roadmap (randomised olmesartan and diabetes microalbuminuria prevention) study started in 2004 and provided a unique opportunity to investigate the factors which correlate with albumin excretion rates across the range of normoalbuminuric values in a large cohort of type 2 diabetic patients. knowledge of these factors is of interest because albuminuria is correlated with cardiovascular and renal risk. it is the goal of the roadmap study to provide further information on the selection of strategies for primary prevention of diabetic nephropathy. in normoalbuminuric type 2 diabetic patients, it has already been shown that an ace inhibitor reduces the risk of de novo onset of microalbuminuria, but, as recently emphasised in a cochrane review, there is a deficit of information on angiotensin receptor blockers (arbs). the aim of the roadmap study is to provide evidence of whether or not it is possible to prevent the development of microalbuminuria by administration of the arb olmesartan medoxomil. in the present study, we analysed the baseline albuminuria levels in the roadmap cohort and determined which factors correlate with the degree of albuminuria within the so - called normoalbuminuric range. the design of the roadmap study has been described in detail previously. in the following details relating to the baseline data study design and organisation roadmap is a randomised, double - blind, placebo - controlled, parallel - group, multicentre phase 3 study that is being conducted in 262 collaborating centres in 19 european countries. the study protocol, which complies with the principles of good clinical practice and the declaration of helsinki, has been approved by the relevant ethics committee at each participating centre. study population the study has recruited 4,449 white patients (2,054 male and 2,395 female ; age range, 1875 years) with : type 2 diabetes (fasting plasma glucose 7.0 mmol / l and hba1c 6.5%, or treatment for diabetes) ; normoalbuminuria (35 mg [women ] or 25 mg [men ] albumin / g urinary creatinine) ; and at least one additional cardiovascular risk factor, including a lipid disorder defined as > 5.2 mmol / l cholesterol or statin treatment, hdl - cholesterol 1.70 mmol / l, high systolic bp (sbp) (130 mmhg) and diastolic bp (dbp) (80 mmhg) or antihypertensive medication, obesity (bmi 28 m / kg), high waist circumference (> 102 cm [men ], > 88 cm [women ]) or smoking more than five cigarettes per day.exclusion criteria included documented renal and/or renal vascular disease, estimated gfr (egfr) 200 mmhg and/or dbp > 110 mmhg) and recent (130/80 mmhg or antihypertensive treatment ; 67.5% of patients were receiving antihypertensive treatment ; 71.6% had lipid disorders. table 1patient data at baselinecharacteristictotaltotal4,449women, n (%) 2,395 (53.8)age (years)57.7 8.7 (58)known duration of diabetes (years)6.1 6.0 (4.3)insulin treatment, n (%) 821 (18.5%)oral hypoglycaemic agents3,752 (84.3)hba1c (%) 7.6 1.6 (7.3)fasting blood glucose (mmol / l)9.1 3.1 (8.4)sbp (mmhg)140.8 16.3 (140.0)dbp (mmhg)84.0 9.8 (84.0)bmi (kg / m)31.0 4.9 (30.5)central obesity, n (%) 3,181 (71.5)metabolic syndrome, n (%) 3,528 (79.3)current smokers, n (%) 831 (18.7)egfr (ml min 1.73 m)84.5 16.8 (83.8)haemoglobin (g / l)142 13 (141)triacylglycerol (mmol / l)2.1 1.3 (1.8)total cholesterol (mmol / l)5.3 1.1 (5.2)ldl - cholesterol (mmol / l)3.2 0.9 (3.1)hdl - cholesterol (mmol / l)1.2 0.3 (1.2)uric acid (mol / l)321 83 (321)known family history of diabetes, n (%) 2,194 (49.3)known family history of cv events, n (%) 1,678 (37.7)values are meanssd (median) for continuous variables, or n (%) for categorical variableshigh waist circumference : > 102 cm in men, > 88 cm in womenncep atp iii criteriamore than five cigarettes per daycv, cardiovascular patient data at baseline values are meanssd (median) for continuous variables, or n (%) for categorical variables high waist circumference : > 102 cm in men, > 88 cm in women ncep atp iii criteria more than five cigarettes per day albuminuria at screening in these patients the albumin / creatinine ratio in morning spot urine tests was loge normally distributed with a median of 0.44 mg / mmol creatinine (interquartile range 0.280.81 ; fig. 1). in men, the median was 0.41 mg / mmol creatinine (interquartile range 0.240.75, mean 0.58 0.78, geometric mean 0.43) and in women the median was 0.46 mg / mmol creatinine (interquartile range 0.310.88, mean 0.67 0.54, geometric mean 0.52). 1relationship of uacr with quintiles of night - time sbp (a), hba1c (b) and egfr (c)the median uncorrected albumin concentration in morning spot urine tests in men was 4.2 mg / ml (interquartile range 2.07.7, mean 6.11 10.26, geometric mean 4.2) and in women 3.2 mg / ml (interquartile range 1.56.2, mean 5.04 5.27, geometric mean 3.6). the albumin / creatinine ratio is higher in women, as expected, because of the higher uacr exclusion values for female patients (3.96 mg / mmol) than for men (2.83 mg / mmol) ; however the absolute uncorrected albumin concentration is lower in women as a result of markedly lower urinary creatinine concentrations in women. relationship of uacr with quintiles of night - time sbp (a), hba1c (b) and egfr (c) ambulatory bp (holter monitoring) in a sub - cohort of patients the values of 24 h abpm prior to taking the study medication were available in a sub - cohort of 1,234 of the 4,449 patients (567 men, 667 women). the average bp was 139.6/81.5 mmhg, confirming the results obtained in the office bp cohort. table 2twenty - four hour data from abpmreadingvalue (n = 1,234)24 h sbp139.6 15.4 (138.9)24 h dbp81.5 8.1 (81.1)daytime sbp143.6 15.6 (143.4)daytime dbp84.3 8.5 (83.8)night - time sbp133.8 17.9 (131.7)night - time dbp77.4 9.4 (6.9)24 h pulse pressure58.1 10.3 (57.6)daytime pulse pressure59.3 10.7 (59.0)night - time pulse pressure56.5 11.7 (55.1)mean daytime sbp minus mean night - time sbp9.7 12.1 (10.0)mean daytime dbp minus mean night - time dbp6.9 7.3 (6.9)values are means sd (median) twenty - four hour data from abpm values are means sd (median) correlation of albuminuria with continuous variables the correlations for different continuous variables were calculated. the strongest correlation was observed for night - time sbp and hba1c levels (table 3 and fig. multivariate analysis in the total cohort and in the sub - cohort of patients with abpm confirmed that hba1c and sbp had the best correlation with uacr (tables 4 and 5). table 3spearman rank correlation coefficients (rs) by univariate analysis with uacr (morning spot urine) as the dependent variablevariablersp valuenight - time sbp0.19 102 cmhigher0.0171 women > 88 cmhighernsmetabolic syndrome (yes) (ncep - atp - iii)higher 140 mmhg or dbp > 90 mmhghigher 130 mmhg or dbp > 85 mmhghigher 60 ml min 1.73 m). as the inclusion and exclusion criteria were fulfilled by the majority of the screened patients, this study population is representative of the majority of type 2 diabetic patients. moreover, our data on uacr are based on the measurement of albuminuria in three morning spot urines. it should be noted that only 10% of the total variance of the uacr could be explained by the degree of blood glucose control, bp variables, lipids and age. analysed the correlation of albumin excretion (uae) with cardiovascular risk factors in 7,841 patients of the prevend cohort. in their cohort, taken from the general population, only 3.5% had diabetes and 14% microalbuminuria at baseline. they found a correlation between uae and male sex, age, sbp, dbp, fasting glucose levels, bmi, smoking and creatinine clearance. in a multivariate analysis only 22% of the variance of uae since all patients in the roadmap study had diabetes, this observation might explain why we were only able to explain 10% of the variance of the loge uacr. the relatively modest correlation may also be because of the fact that most of the patients were in the low normoalbuminuric range at baseline. in this low range the intra - individual variability of the three repeated uacr measurements was 55% in the roadmap cohort. therefore, the correlation observed points more to the direction (thus providing a potential target of treatment) rather than reflecting the magnitude of the relationship. the identified variables correlate not only with the degree of albuminuria within the normoalbuminuric range but might also be predictors for the development of microalbuminuria (table 8). table 8determinants of baseline values for the development of microalbuminuriavaluestudydirect hope benedict agen.d.+n.d.male sex++n.d.sbpn.d.+=dbpn.d.==pulse pressuren.d.n.d.=bmi kg / mn.d.+n.d.cholesteroln.d.=n.d.low hdl - cholesteroln.d.=n.d.smokingn.d.+n.d.baseline uae+n.d.n.d.retinopathy+n.d.n.d.hba1c+n.d.n.d.n.d., not determined ; +, positive correlation ; =, no significant correlationthe diabetes incidence after renal transplantation (direct) study : 3,326 and with type 1 and 1,905 type 2 diabetes were followed for 4.7 yearsthe heart outcomes prevention evaluation (hope) study : 9,043 patients with and without type 2 diabetes were followed for 4.5 years and determinants of albuminuria assessedthe bergamo nephrologic diabetes complications trial (benedict) : 1,204 patients with type 2 diabetes were followed for 3.6 years and risk factors for the development of microalbuminuria analysed determinants of baseline values for the development of microalbuminuria n.d., not determined ; +, positive correlation ; =, no significant correlation the diabetes incidence after renal transplantation (direct) study : 3,326 and with type 1 and 1,905 type 2 diabetes were followed for 4.7 years the heart outcomes prevention evaluation (hope) study : 9,043 patients with and without type 2 diabetes were followed for 4.5 years and determinants of albuminuria assessed the bergamo nephrologic diabetes complications trial (benedict) : 1,204 patients with type 2 diabetes were followed for 3.6 years and risk factors for the development of microalbuminuria analysed studies in normoalbuminuric diabetic patients have additional limitations. first, on the one hand in diabetic patients albuminuria is certainly correlated with the severity of glomerular lesions, but the correlation is not strict, particularly in type 2 diabetes. on the other hand, diabetic glomerular lesions may even precede the onset of albuminuria. second, microalbuminuria is present in 16% of patients at the time of diagnosis of type 2 diabetes. microalbuminuria frequently precedes the onset of overt type 2 diabetes and one potential explanation may be the relatively strong correlation between albuminuria and the metabolic syndrome, a prediabetic state with insulin resistance. in conclusion, the present baseline data of the roadmap study suggest that albuminuria is a continuous variable, and that even in normoalbuminuric type 2 diabetic patients albumin excretion rates are correlated with a number of factors which are potentially susceptible to therapeutic intervention, although certainly correlation does not necessarily imply causality. | aims / hypothesisin contrast to microalbuminuric type 2 diabetic patients, the factors correlated with urinary albumin excretion are less well known in normoalbuminuric patients. this may be important because even within the normoalbuminuric range, higher rates of albuminuria are known to be associated with higher renal and cardiovascular risk.methodsat the time of screening for the randomised olmesartan and diabetes microalbuminuria prevention (roadmap) study, the urinary albumin / creatinine ratio (uacr) was 0.44 mg / mmol in 4,449 type 2 diabetic patients. the independent correlates of uacr were analysed.resultsindependent correlates of uacr during baseline were (in descending order) : night - time systolic bp (rs = 0.19) ; hba1c (rs = 0.18) ; mean 24 h systolic bp (rs = 0.16) ; fasting blood glucose (rs = 0.16) ; night - time diastolic bp (rs = 0.12) ; office systolic bp, sitting (rs = 0.11), standing (rs = 0.10) ; estimated gfr (rs = 0.10) ; heart rate, sitting (rs = 0.10) ; haemoglobin (rs = 0.10) ; triacylglycerol (rs = 0.09) ; and uric acid (rs = 0.08 ; all p 0.001). significantly higher albumin excretion rates were found for the following categorical variables : higher waist circumference (more marked in men) ; presence of the metabolic syndrome ; smoking (difference more marked in males) ; female sex ; antihypertensive treatment ; use of amlodipine ; insulin treatment ; family history of diabetes ; and family history of cardiovascular disease (more marked in women).conclusions / interpretationalthough observational correlations do not prove causality, in normoalbuminuric type 2 diabetic patients the albumin excretion rate is correlated with many factors that are potentially susceptible to intervention.trial registration : clinicaltrials.gov i d no. : nct00185159funding : this study was sponsored by daichii - sankyo. |
trillions of microorganisms among bacteria, archaea, viruses, and fungi inhabit our gastrointestinal tract (git). bacterial cells largely outnumber our own cells, and the gut microbiota is a prolific example of a symbiotic relationship as it plays a crucial role in host 's physiology and health. studies based on gnotobiotic models and fecal microbial transplants (fmt) have provided unequivocal evidence that perturbations in bacterial communities play a key role in the pathophysiology of obesity and insulin resistance. the gut microbiota is the product of a complex interaction between host 's genetics and environment, and diet is one of the main driving forces shaping intestinal bacterial communities. the so - called western obesogenic diet (i.e., rich in saturated / trans fat and simple sugars and poor in fibers) is associated with specific modulation of taxonomic profiles that are functionally linked with a more proinflammatory milieu and disrupted intestinal barrier. disturbance of intestinal homeostasis then leads to excessive bacterial fragments / products internal diffusion, which promotes inflammation in key insulin - responsive tissues, resulting in insulin resistance. the current knowledge suggests that gut bacterial profiles may represent new disease predictors and that manipulation of the gut microbiota could be a promising approach for the prevention and management of metabolic diseases. were the first group to demonstrate a positive correlation between alteration of gut microbiota population, the increase of intestinal permeability and the development of metabolic endotoxemia that is characterized by the translocation of bacterial lipopolysaccharides (lpss) into the systemic circulation and induction of inflammatory pathways in mice fed obesogenic diet. since metchnikoff 's era, the field of probiotics - live microorganisms that, when administered in adequate amounts, confers a health benefit on the host (food and agriculture organization of the united nations, 2002 ; updated by hill.) -continues to grow thanks to the recent access to investigate the role of an increasing number of potential probiotic strains in host 's physiology. according to this definition, the safety and efficacy of a given strain must be scientifically demonstrated in order to be considered as a probiotic. here, we propose a critical review of the most recent studies concerning the effects of probiotic bacterial strains in the prevention or treatment of metabolic disorders such as obesity, insulin resistance, diabetes mellitus and its comorbidities. most currently used probiotics belong to bifidobacteria, lactic acid bacteria (lab), dairy propionibacteria, yeasts (saccharomyces boulardii), bacillus, and the gram - negative escherichia coli strain nissle 1917. lab represent a heterogeneous group of microorganisms broadly present in the diet, particularly by the use of non - human strains in the fermentation of dairy products being also normal inhabitants of the gastrointestinal and urogenital tract. most of them are members of the phylum firmicutes, while bifidobacterium, also considered as lactic - producing bacteria, belong to actinobacteria phylum. probiotic administration has been shown to stimulate the immune response, improve lactose tolerance, help prevent diarrhea, have an anti - inflammatory effect and even restore obesity - linked gut dysbiosis. given the relationship between obesity - related disorders and gut homeostasis, probiotics may be of interest to supplement the limited arsenal of therapies against the metabolic syndrome. the diversity of reported studies in tables 1 and 2, shows that the positive effects of probiotics are strain - specific and the idea of a " universal strain, " that would provide at once all the benefits associated with probiotics, is unrealistic, even for strains of the same species. in the context of obesity and metabolic disorders, probiotic supplementation may help to reduce hyperphagia, improving control of weight gain, fat mass loss and glucose tolerance. on the contrary, such positive effects could also be obtained without modulation of caloric intake, as demonstrated by most of the reported studies. to demonstrate the beneficial effect of probiotics in improvement of metabolic disorders, researchers have access to a variety of assays such as plasma and liver cholesterol, free - fatty acids, alanine and aspartate transaminases (hepatotoxicity markers), gene and protein expressions (involved in inflammatory and metabolic pathways), etc. (tables 1 and 2 for details). for example, dairy products supplemented with propionibacterium, a well - known promising non - lab genus, may exert a probiotic effect in the colon by producing metabolites such as short - chain fatty acids (scfa), vitamins (b8, b9, and b12), and 1,4-dihydroxy-2-naphtoic acid, bifidogenic and anti - inflammatory product (dhna). shermani js has anti - inflammatory effects on high fat diet - induced inflammation in apoe3leiden mice, with a decrease of intestinal mast cell numbers and a demonstrated intestinal but also systemic anti - inflammatory potential. finally, although it needs further investigation, multiple strain probiotics could confer a more effective strategy than single - strain probiotics against diet - induced obesity (dio). interestingly, vsl#3, a mixture of eight different strains of bacteria, has shown efficacy in prevention but also in the treatment of obesity and type 2 diabetes. probiotic administration is frequently associated with important shifts in gut bacterial composition, along with beneficial effects on metabolism and inflammatory tone. indeed, within the gut, probiotic are in competition for nutrients, metabolites and also for antimicrobial proteins, altering gut microbiota population diversity in several ways. however, it remains unclear if the modulation of gut microbiota is the cause or the consequence of probiotic treatment, or whether the mechanisms are partially or totally interdependent. the probiotic components associated with positive effects are a variety of cell constituents as polysaccharides, peptidoglycan, dna, teichoic acids and certain cell - surface bound and secreted proteins as well as organic acids, bacteriocins, polyphosphate, and fatty acids (fa), which can modulate host responses, inhibit pathogens or interact with the intestinal microbiota. furthermore, whereas a disrupted intestinal barrier contributes to the pathogenesis of metabolic diseases, the underlying causes remain unclear. indeed, it may include changes in nutritional factors, infections (i.e., helicobacter pylori infection leading to an increased rate of incident diabetes), exposure to toxins, lack of exposure to microbes in early childhood, as well as impaired function and diversity of the gut microbiota. moreover, probiotic strains can not only affect the intestinal microbiota directly but also affect other organs by modulating intestinal inflammation and permeability. several potential mechanisms underlying the beneficial effects of probiotics are illustrated in figs 1 and 2. the " obese microbiome " is thought to display an increased capacity to harvest energy from the with a decreased ability to stimulate the production of gut factors that inhibit fat deposition. furthermore, the beneficial effect of probiotics to decrease dio is both highly strain- and model - specific. for example, the gut microbiota could promote storage of triglyceride in adipocytes through suppression of intestinal expression of a circulating lipoprotein lipase (lpl) inhibitor, the angiopoietin - like 4. nevertheless, storage of excess fa is the result of unbalanced lipid absorption involving lpl and lipid catabolism. in fact, yoo., treated dio - mice with a combination of probiotics that resulted in a decreased expression of genes involved in fa transport and -oxidation (table 1). another potential mechanism by which probiotics can counteract the negative effect of obesogenic diet is by interaction with commensal bacteria and altering expressions of microbial enzymes, especially those involved in carbohydrate metabolism or butyrate synthesis pathways. butyrate, with acetate and propionate, are the most abundant scfa produced by some colonic bacteria as end - products from the breakdown of non - digestible carbohydrates that pass unaffected through the small intestine. among major bacterial phyla, bacteroidetes are recognized as acetate and propionate producers, whereas firmicutes are more butyrate - producing bacteria. the butyrate - producer strain miyairi 588 has shown promising effects on liver homeostasis and insulin resistance in a rat model of choline - deficient diet - induced non - alcoholic fatty liver disease (nafld). as reported in table 1, ritze. have also shown that lactobacillus rhamnosus gg protects against nafld through specifically reducing liver fat mass loss in association with modulation of the carbohydrate - responsive element - binding protein pathway. moreover, in many studies, the beneficial effects allocated to probiotics on body fat mass, could be explained by complex and still unclear mechanisms that may or may not involve change in caloric intake (tables 1 and 2). have demonstrated that the vsl#3 probiotic promoted the release of the hormone glucagon - like protein-1 (glp-1), resulting in reduced food intake and improved glucose tolerance, which was correlated with scfa production leading to l - cell stimulation and glp-1 production and the modulation of several genes involved in food intake regulation. the modulation of the intestinal immune system is also thought to ameliorate insulin sensitivity even without decreased fat mass accumulation. the intestinal barrier is a functional entity separating the gut lumen from the inner host. it comprises elements that are mechanical (mucus, epithelial layer), humoral (defensins, immunoglobulin a), cellular or cell - mediated (lymphocytes, innate immune cells), muscular and neurological. this barrier is maintained by the expression of adherens junctions and tight junctions (tj) molecules, including cadherins, claudins, occludin, and junctional adhesion proteins, which seal adjacent cells together. moreover, the intestinal mucosa is the primary site where the mucosa - associated lymphoid tissue is exposed to and interacts with the external environment. gut barrier integrity is influenced by both exogenous (i.e., toxins, stress, diet, vitamins, pro- and prebiotics, antibiotics, exercise) and endogenous factors (i.e., inflammatory mediators, defensins, serotonin, proteases, mucus quality, and the endocannabinoid system). in obese individuals, decrease in tj protein abundance, myosin light chain kinase activation and cytoskeletal modulation (zo1 interacts directly with actin, occludin, claudins, or other proteins) have all been proposed to mediate cytokine - induced loss of tj barrier function. it is well documented that lab are able to sense the environment, to produce bacteriocins which can directly modulate gut microbiota populations (figs 1 and 2), but also organic acids (i.e., lactic and acetic acids) that indirectly inhibit pathogen colonization by decreasing intestinal ph or increasing peristalsis. by preventing the invasion of undesirable microorganisms, beneficial probiotic effects indeed, mucosal permeability is adaptable and may be directly regulated in response to extracellular stimuli, such as nutrients and bacteria. these interactions can result in the variation of gene expression of receptors involved in numerous and diverse pathways leading to the production of cytokines and other active molecules, secreted from epithelial cells into the lumen inducing gut microbiota modulations. it was recently demonstrated that dio - mice display low - grade systemic inflammation and metabolic perturbations, in association with reduced intestinal bifidobacteria and increased plasma levels of endotoxin (lps), a trait strongly correlated with disrupted intestinal barrier integrity. moreover, plasma citrulline and intestinal fa - binding protein levels (markers of gut barrier integrity) are significantly elevated in severely obese diabetic individuals, which was associated with increased small - intestinal enterocyte mass and increased enterocyte turnover. furthermore, there are several families of innate receptors which are involved in the recognition of microbe - associated molecular patterns (including toll - like receptors, nod - like receptors, or inflammasomes). moreover, changes in gut microbiota modulate endotoxemia by a mechanism that affects gut barrier function and increases intestinal permeability, which may involve the disruption of tj. cani 's group recently focused on the probiotic effect of akkermansia muciniphila, an interesting mucin - degrading member of the verrucomicrobia phylum, and found that its administration to dio mice decreases metabolic endotoxemia and adipose tissue inflammation by improving intestinal mucosal barrier function, a trait linked to an increased mucus layer thickness. among other potential mechanisms involved in the maintenance of intestinal homeostasis, butyrate production has also been suggested to alleviate intestinal bowel diseases (ibds) through its ability to inhibit histone deacetylases and to activate g - coupled protein receptors, leading to enhanced protective immunity and improved gut barrier. inoculation of mice with the butyrate - producers clostridium cluster iv and xiva or butyrate administration per se were both capable of expanding the colonic population of regulatory t cells (treg), which increases the production of the anti - inflammatory cytokine interleukin 10 and reduces the colonic population of the pro - inflammatory cd4 + t cells. similarly, oral administration of butyricicoccus pullicaecorum, whose presence was found to be lower in ibd patients compared with healthy subjects, attenuated intestinal inflammation in a rat model of colitis. while the resolution of obesity - induced intestinal inflammation is a valuable strategy to improve whole - body metabolism, butyrate can also act at the systemic level to exert anti - obesity and anti - inflammatory effects. interestingly, given the large body of literature supporting the beneficial effects of butyrate, the administration of butyrate producer strains such as faecalibacterium prausnitzii, roseburia intestinalis, or anaerostipes caccae may confer predictability and safeness to potential probiotic - based treatments of several pro - inflammatory disorders. the concomitant use of probiotics with specific prebiotics, known as sources of " non - digestible compound that, through its metabolization by microorganisms in the gut, modulates composition and/or activity of the gut microbiota, thus conferring a beneficial physiological effect on the host " should also be considered as mean of improving health status. prebiotics can improve probiotic effects on body weight loss and maintenance, when they are co - administrated to the host organism. moreover, inulin - type fructans (itfs) have been shown to affect gut ecology and stimulate immune cell activity, as well as decreasing body weight gain and fat mass in obese individuals. it also appears that polyphenols can, in conjunction with a probiotic strain of bacillus, stimulate the growth of anti - inflammatory bacterial species belonging to the genus barnesiella and improve the bioavailability of certain health beneficial polyphenols. in the context of obesity, the use of relatively new prebiotics such as arabinoxylan (ax) and arabinoxylan oligo - saccharides (axos) may be promising candidates to counteract related metabolic disorders, since ax and axos have been linked to adiposity reduction and lower metabolic endotoxemia in obese mice, respectively. furthermore, there is growing evidence that the bifidogenic and butyrogenic effects of ax and axos are reflected in potential cross - feeding mechanisms, such as for itf, in which primary degraders such as bifidobacterium selectively and competitively degrade these fructose polymers to produce acetate and lactate that are consumed by secondary degraders such as roseburia to produce butyrate. interestingly, ax administration in rodents has also been involved in gut microbiota modulation ; firstly by increasing bifidobacterium and roseburia in dio mice, and secondly by shifting mucin degradation from the caecum to the colon where a higher abundance of mucolytic a. muciniphila may locally produce beneficial metabolites such as propionate. another growing concept is to genetically engineer bacterial strains in order to reinforce a pre - existing probiotic capacity or to increase their effectiveness. in fact, lab have been genetically manipulated in order to target the delivery of antioxidant and anti - inflammatory molecules produced by probiotics (i.e., enzymes, cytokines) for the treatment of ibd. since the use of gut anti - inflammatory agents is promising against the metabolic syndrome, it would be interesting to test whether these engineered lab originally conceived to counter ibd could also exert positive effects on obesity and associated metabolic disorders. indeed, duan. recently reported the successful application of an engineered probiotic that secretes the inactive full - length form of glp-1 to reprogram intestinal cells into glucose - responsive insulin - secreting cells for the treatment of type 1 diabetes. another interesting potential strategy is the genetic modification of the probiotic e. coli nissle 1917 to produce n - acylphosphatidylethanolamines, which is converted quickly after meals into potent appetite - suppressing lipids, know as n - acylethanolamines. the aforementioned examples show the great potential of engineered strains as a strategy to treat obesity and its metabolic consequences. altogether, various studies (tables 1 and 2) demonstrate that probiotic administration may confer beneficial effects in the prevention and treatment of obesity, inflammation and other associated metabolic disorders through various mechanisms including direct effects on mucosal barrier and surrounding cells in particular, that can impede on chronic inflammation (figs. 1 and 2). currently, researchers are on the path to uncover beneficial and detrimental gut microbiota phylotypes that could lead to the use of living probiotics in order to reshape gut bacterial communities in beneficial ways to the host. the major issue that hampers a meta - analysis comparison of all the potential probiotic strains is the considerable heterogeneity between protocols used in many studies (model, dose, treatment, and times). for the same reason, research on probiotics are still confronted with an apparent lack of conclusive results, further limited by the small number of trials where the application of probiotics was evaluated in double - blinded large - scale cohorts studies, particularly in the context of obesity prevention. indeed, even if fmt showed very good results in recent human trials, the fact that potential adverse effects have also been reported, calls for caution because probiotics are already used for obesity management. this is particularly true for specific groups (i.e., neonates infants or individuals with immune deficiency) that may be a greater risk for adverse effects of probiotics. moreover, a better understanding of how environmental factors (i.e., culture conditions, product formulations, storage time, host metagenome and genotype and variability of consumer - associated factors) influence probiotic function would ultimately be useful for unraveling the significant inter - individual variation in response to probiotic bacteria among human subjects and for comparing outcomes of different clinical studies. despite the methodological and regulatory issues raised above, the field of probiotics is evolving based on a growing body of research, which is paving the way for a successful strategy against obesity and its related comorbidities, using strains capable of producing well characterized molecules, or using engineered bacteria that ensures safety of use. moreover, the increased interest in the role of the gut microbiota in host 's physiology is revealing novel potential probiotic strains while triggering a regain of interest in probiotics as a tool to manipulate intestinal bacterial communities and therefore treat / prevent intestinal and systemic diseases. | over the past decade, growing evidence has established the gut microbiota as one of the most important determinants of metabolic disorders such as obesity and type 2 diabetes. indeed, obesogenic diet can drastically alter bacterial populations (i.e., dysbiosis) leading to activation of pro - inflammatory mechanisms and metabolic endotoxemia, therefore promoting insulin resistance and cardiometabolic disorders. to counteract these deleterious effects, probiotic strains have been developed with the aim of reshaping the microbiome to improve gut health. in this review, we focus on benefits of widely used probiotics describing their potential mechanisms of action, especially their ability to decrease metabolic endotoxemia by restoring the disrupted intestinal mucosal barrier. we also discuss the perspective of using new bacterial strains such as butyrate - producing bacteria and the mucolytic akkermansia muciniphila, as well as the use of prebiotics to enhance the functionality of probiotics. finally, this review introduces the notion of genetically engineered bacterial strains specifically developed to deliver anti - inflammatory molecules to the gut. |
isolates - ninety - one gas isolates recovered from oropharynx secretions were included in this study. clinical specimens were processed during routine diagnoses by one clinical laboratory in rio de janeiro from january 2008-july 2012. the subjects ages varied from two-56 years, but 46% of the isolates were recovered from children two-11 years of age and 51.6% were from women. in our laboratory, these isolates were cultured on blood agar plates (difco laboratories, usa) and submitted to conventional tests (pyr test, bacitracin susceptibility and streptococcal serogrouping) to confirm species identification. antimicrobial susceptibility testing - all isolates were submitted to susceptibility tests to ceftriaxone (30 g), clindamycin (2 g), erythromycin (15 g), levofloxacin (5 g), penicillin (10 u), tetracycline (30 g) and vancomycin (30 g) (cecon, brazil) using the disk diffusion method on mueller - hinton blood agar (difco) according to clsi guidelines (2013). macrolide resistance phenotypes were determined by the double disk test using erythromycin (15 g) and clindamycin (2 g) disks placed 12 mm apart (clsi 2013). the erythromycin minimum inhibitory concentration (mic) was determined in all resistant and intermediate isolates by the agar dilution method (clsi 2009). investigation of erythromycin resistance - associated genes - dna preparation was performed as previously described (dmitriev. briefly, suspensions with turbidity adjusted to mcfarland standard 3.0 were prepared in 300 l of 10 mm tris - edta buffer and boiled for 5 min. the presence of erma, ermb and mefa / e genes was investigated in erythromycin - resistant isolates using specific polymerase chain reaction (pcr) protocols (sutcliffe. cycling was carried in a geneamp 9700 thermocycler (applied biosystems, usa). determination of emm types - emm types were determined by a sequence - based protocol (cdc.gov/ncidod/biotech/strep/protocol_emm-type.htm) using bigdye terminator cycle sequencing kit (applied biosystems). sequences were edited using bioedit software v.7.0 and compared with reference sequences using the blast algorithm (blast.ncbi.nlm.nih.gov/blast.cgi). sequences that did not match with 100% similarity to any sequence deposited in genbank were submitted to the centers for disease control and disease (cdc) emm sequence database (cdc.gov/ncidod/biotech/strep/strepblast.htm) for assignment to new emm subtypes. analysis of dna restriction patterns by pfge - all 44 isolates belonging to the five prevalent emm types (emm1, emm4, emm12, emm22 and emm81) were ana- lysed by pfge after the dna was digested with smai according to a previous protocol (teixeira. a 300-l aliquot of bacterial suspension in piv buffer was mixed with an equal volume of low melting point agarose (promega, usa) and distributed into plug moulds. plugs were incubated in 2 ml of lysis solution containing 5 mg / ml lysozyme. smai digestion was performed according to the manufacturer s recommendations (invitrogen, usa). dna fragments were separated by the chef - driii system (bio - rad laboratories, usa). the dice coefficient was calculated by visual analysis and dendrograms based on unweighted pair group method with arithmetic mean were constructed using genomes.urv.cat/upgma. statistical analyses - the discriminatory power of emm typing, regarding the overall population as well as erythromycin and tetracycline resistance, was measured using simpson s index of diversity (sid) by calculating the 95% confidence intervals (ci) (hunter & gaston 1988, grundmann. conventional tests identified all isolates as s. pyogenes. by the disk diffusion method, tetracycline - resistant and intermediate isolates comprised 18.7% and 2.2% of isolates, respectively. clindamycin resistance was observed in 15.4% of isolates, while erythromycin - resistant and intermediate isolates comprised 14.3% and 1.1% of isolates, respectively. the erythromycin mic varied from 8 - 256 g / ml and therefore, the resistance rate was 15.4%. the genetic determinant ermb was predominant and was detected in 78.6% of the erythromycin - resistant isolates, alone or in association with erma (64.3%). neither the m phenotype nor the mefa / e gene was observed in this study. erythromycin mic values, the distribution of macrolide resistance phenotypes and genotypes and emm types of erythromycin - resistant isolates are shown in table. tablephenotypic and genotypic characteristics of macrolide resistant isolatesisolate numberyear of recovery emm typemic (g / ml)phenotypemacrolide resistance genes erma ermb mefa / e 186200868.1 > 256cmlsb -+-274200858 > 256imlsb + --302200858 > 256imlsb + --40220091 > 256cmlsb -+-418200958.148imlsb + --425 20093.316cmlsb -+-536201022 > 256cmlsb -+-637a20111 > 256cmlsb + + -638201176.7 > 256cmlsb + + -710201128 > 256cmlsb -+-749201173 > 256cmlsb + + -75020116 > 256cmlsb + + -780201211 > 256cmlsb + + -798201211 > 256imlsb + + -a : this isolate was intermediate by disk diffusion ; cmlsb : constitutive mlsb phenotype ; imlsb : inducible mlsb phenotype ; mic : minimal inhibitory concentration (susceptible 1 g / ml) ; + : presence of the gene ; - : absence of the gene. : this isolate was intermediate by disk diffusion ; cmlsb : constitutive mlsb phenotype ; imlsb : inducible mlsb phenotype ; mic : minimal inhibitory concentration (susceptible 1 g / ml) ; + : presence of the gene ; - : absence of the gene. thirty - two emm types or subtypes were identified among 86 of the 91 gas isolates. the most frequent types were emm1, emm4, emm12, emm22 and emm81, accounting for 48% of all isolates. three new sequences were designated by the cdc streptococcus laboratory as new emm subtypes (emm1.74, emm58.14 and emm76.7) and deposited in the emm sequence database (cdc.gov/pub/infectious_diseases/biotech/tsemm) and in genbank (accession km364527-km364529). these new subtypes differed from their parental types in 1 - 2% of the nucleotide sequence. the emm typing revealed a high level of diversity among the overall population (sid = 0.941 ; 95% ci, 0.917 - 0.965). the frequency of each emm type is shown in fig. 1. fig. 1 : distribution of emm types and subtypes among isolates of streptococcus pyogenes recovered from oropharynx secretion. fourteen different emm types or subtypes were found among 19 tetracycline non - susceptible isolates, while 11 distinct emm types or subtypes were found among 14 erythromycin and clindamycin - resistant isolates. the sid values calculated for erythromycin - resistant (0.967 ; 95% ci, 0.929 - 1.000) and tetracycline non - susceptible (0.959 ; 95% ci, 0.915 - 1.000) isolates were higher than those calculated for erythromycin (0.929 ; 95% ci, 0.898 - 0.960) and tetracycline (0.898 ; 95% ci, 0.855 - 0.940) susceptible isolates. pfge analysis of 44 isolates belonging to the five prevalent emm types generated 20 restriction profiles, which are shown in fig. three profiles, whose similarities varied from 50 - 85%, were observed among emm1 isolates. the most frequent profile was shared by eight isolates, including one resistant to erythromycin. regarding emm4, four profiles were observed among seven isolates, ranging from 85 - 96% similarity. among emm22 and emm81 isolates (6 isolates each), unique pfge profiles were observed for each isolate, varying from 70 - 95% similarity. 2 : pulsed - field gel electrophoresis profiles, dendrograms, year of isolation and macrolide resistance features of prevalent emm types isolates. in this study, 91 gas isolates recovered from oropharynx secretions of residents of rio de janeiro were submitted to susceptibility testing and typing methodologies. despite the lack of data regarding the clinical conditions of the individuals, these results are relevant because both infected and colonised individuals can transmit this species to susceptible subjects. isolates were fully susceptible to beta - lactams and glycopeptides, as observed over decades of antibiotic usage. they were also susceptible to levofloxacin, despite previously detected resistance to this antimicrobial (smeesters. the tetracycline resistance rate was lower than that observed in a study conducted in brazil one decade ago, in which the authors reported 43.1% resistance (doliveira. 2003). instead, it was very similar to recent data from southern brazil and portugal (torres. however, 73% resistance to this antimicrobial was reported in a recent study from india (mathur. these findings may reflect that, while a consistent trend of decreasing tetracycline resistance has been recently observed in brazil, circulating gas isolates from other regions are highly resistant to this antimicrobial. regarding erythromycin and clindamycin resistance, the rates found here far exceeded those previously reported in the same geographical area (doliveira. 2011), but they are very similar to those described in some recent studies conducted in europe (fries. 2013). while a trend of decreasing erythromycin resistance has been recently observed in spain and taiwan (huang. inducible and constitutive mlsb phenotypes, which are associated with erma and/or ermb genes, were observed among isolates belonging to a variety of emm types, reflecting the polyclonal origin of such isolates. it is worth noting that the m phenotype and the mefa / e genotype, associated with emm12, were common in brazil before the year 2000 (torres. 2011), but they have not been detected since that time, either by those authors or in this study. in contrast, mlsb emerged after the 2000s and is linked to a variety of emm types, such as emm11, emm22, emm28 and emm73 (torres. in contrast to this local replacement of the m phenotype with the cmlsb phenotype, the m phenotype has been prevalent over 12 years of survey (1998 - 2010) in taiwan (huang. 2014). however, the authors observed the replacement of emm12 with emm22 as the prevalent type associated with macrolide resistance, as well as a decreasing rate in macrolide resistance from 53.1% before 2000 to 10.7% from 2006 - 2010. these variations demonstrate how dynamic the bacterial population is worldwide and also highlight the need for changing therapeutic recommendations in geographical areas where mlsb phenotypes predominate. a wide variety of emm types, including new alleles, the diversity of emm types and the detection of new alleles from gas isolates recovered from oropharynx secretions have been described in previous studies from brazil (teixeira. these findings reflect the genetic heterogeneity of such isolates and contribute to the expansion of the emm type database. the most frequent emm types found here are among the prevalent types in high - income countries, where the majority of studies have been performed (steer.. however, they differ significantly from those types observed in recent studies from india and the pacific region (baroux. 2014, mathur. 2014). due to the limited amount of data from latin america, our results contribute to a better understanding of emm type distribution in a poorly studied area. this issue is particularly important considering that m protein - based vaccines are under development (dale. 2013). when comparing the two most promising 26 and 30-valent m vaccines, 49% and 64% of our isolates, respectively, the potentially low impact of these vaccines can be explained by the fact that the m protein fragments for the 26-valent m vaccine were selected based on emm type distribution in north america (steer. there was strong agreement between emm type and pfge clustering, as previously observed (torres. no identical pfge profile was observed among isolates belonging to distinct emm types, which could be related to the horizontal transfer of emm genes (whatmore. regarding erythromycin - resistant isolates, a great diversity of pfge profiles and emm types were observed, illustrating that this characteristic is not due to the expansion of a specific clone. moreover, both erythromycin - susceptible and erythromycin - resistant emm1 isolates shared a single pfge profile. this finding suggests that the emm type, not erythromycin resistance, is more likely to be a determinant of clonality. in conclusion, susceptibility testing and epidemiological typing techniques revealed an incidence of macrolide resistance not yet observed in this area and a great diversity of emm types, including new alleles, among gas isolates circulating in brazil. these data contribute to the improvement of prevention and treatment strategies of gas infections. | streptococcus pyogenes is responsible for a variety of infectious diseases and immunological complications. in this study, 91 isolates of s. pyogenes recovered from oropharynx secretions were submitted to antimicrobial susceptibility testing, emm typing and pulsed - field gel electrophoresis (pfge) analysis. all isolates were susceptible to ceftriaxone, levofloxacin, penicillin g and vancomycin. resistance to erythromycin and clindamycin was 15.4%, which is higher than previous reports from this area, while 20.9% of the isolates were not susceptible to tetracycline. the macrolide resistance phenotypes were cmlsb (10) and imlsb (4). the ermb gene was predominant, followed by the erma gene. thirty - two emm types and subtypes were found, but five (emm1, emm4, emm12, emm22, emm81) were detected in 48% of the isolates. three new emm subtypes were identified (emm1.74, emm58.14, emm76.7). there was a strong association between emm type and pfge clustering. a variety of pfge profiles as well as emm types were found among tetracycline and erythromycin - resistant isolates, demonstrating that antimicrobial resistant strains do not result from the expansion of one or a few clones. this study provides epidemiological data that contribute to the development of suitable strategies for the prevention and treatment of such infections in a poorly studied area. |
acute retinal necrosis (arn) syndrome is clinically characterized by peripheral necrotizing retinitis, retinal arteritis, and a prominent inflammatory reaction in the vitreous and anterior chamber.1 the pathogenesis of arn is presumed to be associated with herpes virus infection, varicella - zoster virus (vzv), herpes simplex virus (hsv), cyto - megalovirus (cmv), or epstein barr virus (ebv).2 polymerase chain reaction (pcr) is a sensitive and specific method for detecting viral dna and is usually performed to assist in the diagnosis of arn from the vitreous or the aqueous humor.3 herpes zoster ophthalmicus has been linked to vzv reactivation within the trigeminal ganglion, and asymptomatic viral shedding of vzv from saliva and tear fluid has been reported.46 however, asymptomatic viral shedding in patients with arn has not been described. in the present case report, we simultaneously identified the vzv and cmv genomes in the aqueous humor, tears, saliva, and swabs of the auricular and forehead skin areas in sequential samples assessed by pcr in an otherwise healthy patient with arn. a 65-year - old otherwise healthy japanese woman presented at another hospital with blurred vision in the left eye. the left eye showed inflammation in the anterior chamber and white patches in the posterior pole. she was given a full medical examination, and the results of blood tests, including angiotensin converting enzyme, syphilis test, chest x - ray, tuberculin skin test, and cerebrospinal fluid examination, were all within normal limits. her decimal visual acuity on the left eye was 0.15 and her intraocular pressure was 13 mmhg. on examination, there were 2 + aqueous cells in the anterior chamber and mutton - fat keratic precipitates, and the following were also observed : 3 + vitreous cells, vasculitis in the temporal arcade, and white patches of retinal necrosis in the temporal part of the posterior pole and peripheral retina (figure 1). the right eye was normal. according to these findings and the rapid progression of necrosis, a clinical diagnosis of arn serum vzv, hsv, and cmv eia - igm antibody levels were not detected. serum eia - igg antibody levels were hsv 4.0, vzv 35.2, and cmv 3.7 arbitrary units ml, respectively. antibody test was negative and she had no previous history of diabetes mellitus, leukopenia, cytotoxic chemotherapy, corticosteroid use, or non - cytotoxic immunosuppressive medication use. her full blood count, glucose, renal function, and liver function tests were within normal limits. the patient was admitted to our hospital, and intravenous acyclovir 500 mg three times a day, oral prednisone 40 mg / day, and aspirin 100 mg / day were given. recent reports have described that prophylactic vitrectomy may help to eliminate inflammatory factors and reduce the incidence of retinal detachment;7,8 we therefore performed prophylactic vitrectomy, entire circumferential scleral buckling, silicone oil tamponade, and cataract surgery on day 2 of admission. the undiluted vitreous was sampled from the patient at the beginning of the vitrectomy and tested by pcr. the results of the pcr of vitreous were positive for vzv and cmv and negative for hsv, ebv, and human herpes virus 6 and 7 (figure 2). although cmv was positive, there were fewer copies than for vzv, and since the patient was otherwise healthy, we continued treatment with intravenous acyclovir. although the patient was given only acyclovir, the white patches gradually disappeared or cicatrized (figure 3). an intravenous acyclovir dose of 500 mg three times a day was given for 12 days. on day 12 of admission, the visual acuity of the left eye improved to 0.3 and the intraocular pressure was 10 mmhg. the treatment was later changed to oral valacyclovir 1,000 mg, three times a day for 3 months, and oral prednisone was tapered slowly over 3 months. at 1 month after hospitalization, rhegmatogenous retinal detachment of the peripheral retina occurred and a reoperation was performed. there was neither a new focus of infection nor a relapse of inflammation following the reoperation. at the end of the follow up, the visual acuity of the left eye was 0.1 and intraocular pressure was 12 mmhg. swabs of the auricular and forehead skin areas and tears were collected bilaterally and sequentially, and pcr was performed at various time points during the treatment period. nucleic acid from clinical samples was extracted and purified using a qiaamp dna blood mini kit (qiagen nv, venlo, the netherlands) for amplification of the hsv-1, hsv-2, vzv, ebv, cmv, hhv (human herpes virus)-6, and hhv-7 gene by pcr, as described previously.6,9,10 pcr analysis was performed using the primer pairs shown in table 1. the vzv genomes were detected in the vitreous and saliva, and the swabs of the auricular and forehead skin areas. the left eye was affected and the viral copy numbers were more abundant in the tears and skin taken from the ipsilateral side than in those from the contralateral side on days 25, although the difference decreased with time. cmv was detected in the tears, saliva, and skin from the initial samples with an increasing copy number on day 8 and later, but no corneal or skin lesions were observed over the observation period. arn is caused by hsv and vzv in otherwise healthy patients and by cmv in immunosuppressed patients, but simultaneous detection of cmv has not been reported in patients with either hsv - induced arn or vzv - induced arn. lau reported that the ebv - positive eyes of their patients were also positive for vzv.11 to our knowledge, this is the first case report of simultaneous detection of cmv in a patient with vzv - induced arn. ocular cmv infections usually present as anterior uveitis.12 arn is usually caused by vzv and hsv, and more rarely by cmv in some immunocompetent patients.2 tajunisah reported the case of an immunocompetent patient with cmv arn in whom the vitreous tap was positive for cmv without detecting vzv and hsv.13 in their case, the patient had anterior chamber inflammation, marked vitritis, and anterior retinal necrosis. initially, they treated the patient with 500 mg of intravenous acyclovir three times daily, but the necrotic retinitis lesions enlarged. schneider presented five cases of cmv necrotizing retinitis in non - hiv patients.14 they described that the cases had both characteristic arn and classic cmv retinitis, in addition to panretinal occlusive vasculitis, moderate intraocular inflammation, and slowly progressive granular retinitis. these cases were treated with ganciclovir or valganciclovir, but they required prolonged antiviral therapy to achieve a complete resolution of retinitis, which contrasts with the rapid treatment response normally observed in the cases of arn caused by hsv or vzv. in our case, the patient had inflammation in the anterior chamber, vitritis, occlusive vasculitis, and retinal necrosis in the temporal region of the posterior pole and the peripheral retina. the retinal necrosis expanded within 1 week, unlike the slowly progressive retinitis described by schneider.14 although previous studies of cmv necrotizing retinitis have reported successful treatment with ganciclovir or valganciclovir, in our patient the retinal lesion was improved only by acyclovir, which elicited a rapid treatment response. in consideration of these clinical findings and the responses to acyclovir, we conclude that cmv did not contribute to the pathogenesis of arn in this case. as shown in table 2, the viral dna was detected by pcr even during antiviral therapy. the inhibition of dna replication might not have been complete in the infected cells because the inhibition of dna synthesis depends on the ratio between synthesized guanosine triphosphate and incorporated acyclovir triphosphate. vzv dna was detected in the vitreous, tears, saliva, and skin, indicating that vzv reactivation occurred in the tears, saliva, and skin of the auricular and forehead regions, a broad area of the skin and mucous membranes of the face, as shown in table 2. since the patient did not present any lesions related to vzv or cmv except in the left eye, asymptomatic shedding of vzv and cmv might have occurred in the facial area. if so, such shedding might suggest that some stimuli caused viral reactivation in the various ganglia focusing on the left trigeminal ganglion. we could not find any specific trigger in the patient history, but we simultaneously detected vzv and cmv in the vitreous and in the facial area in the patient with vzv - induced arn. however, while the vzv reactivation caused arn in the left eye, it caused no skin lesions. the cause of the vzv reactivation was unclear, although it might have been due to a breakdown of the immune system. this suggests that asymptomatic reactivation of vzv and cmv might occur in patients with arn. | backgroundto report a case of simultaneous detection of cytomegalovirus (cmv) in acute retinal necrosis (arn) due to varicella - zoster virus (vzv) in an immunocompetent adult.methodsa 65-year - old healthy woman presented with necrotizing retinitis. vitreous, tears, saliva, and swabs of the auricular and forehead skin areas were collected and tested by polymerase chain reaction analysis at various time points during the treatment period.resultsvzv and cmv dna were detected in the vitreous, tears, saliva, and skin. cmv was present in fewer copies than vzv in the vitreous. the retinal lesion clinically improved rapidly only by acyclovir. according to the clinical findings and the clinical responses to acyclovir, cmv seems not to cause arn in this case.conclusionvzv and cmv dna were detected in the vitreous of a patient with vzv arn. cmv reactivation appeared to be asymptomatic. |
video assisted thoracic surgery (vats) was introduced in the early 1990s [1, 2 ] and is now widely considered to be the gold standard for suspected malignant lung nodule resection. the technique might reduce patient discomfort, complications, the need for higher level of care, hospital stay, and cost compared to open thoracotomy. in 1999, a review of vats lung nodule resection demonstrated that 54% of the cases required conversion to open thoracotomy to complete resection. the study identified that nodule localization failure was the cause for the majority of conversions (46%). the challenge increases further with small deeply placed lung nodules as the visceral pleura may not demonstrate any altered shape or colour, making these nodules even more difficult to locate thoracoscopically. a number of differing methods of preoperative marking have been reported in the literature with varying degrees of success, complications, cost, specialised equipment, and required expertise. we revisit a simple but safe technique using computed tomography (ct) guided marking using methylene blue injection prior to vats pulmonary metastasectomy described in both the adult and the paediatric populations dating back to the 1990s [5, 6 ] but subsequently underutilized. using our technique, nodules are identified on a low dose ct (120 kv, modulated ma and 6.0 mm acquisitions) prior to marking. a metallic skin marker is used to aid in localisation and to plan an appropriate cutaneous needle insertion point. to dilute the methylene blue safely, 5 ml of the patients ' venous blood is withdrawn and mixed with methylene blue aseptically in a ratio of 1 part methylene blue to 5 parts autologous blood. then, under local anaesthesia and aseptic conditions, the nodule is then marked using ct fluoroscopy. in our institute, we use manually controlled pulsed carevision (siemens somatom) ct fluoroscopic guidance (120 kv and a mas of 30). the nodule is approached with a 16/18 g spinal needle in a way akin to lung biopsy (figure 1). on reaching the nodule, the methylene blue blood mixture is injected immediately adjacent to the nodule and along the needle tract right up to the pleural surface as the needle is retracted, leaving a methylene blue / blood tract (figure 2). once in the operating department, the surgeon initially reviews the coronal reformatted images and can then locate the dye on the pleural surface under direct vision using the thoracoscope, guiding the approach to the parenchymal nodule. when the needle is placed slightly superior or inferior to the nodule due to technical difficulties, such as the approach being hindered by overlying ribs, the dye may not be immediately adjacent to the nodule. in this case, ct reconstructions and dialogue with the operating surgeon are essential. the mean patient age was 68 (5181), with a bmi of 25 (2228) and a mean fev1 98% of predicted (82115%). the mean nodule size was 8 mm (418 mm) located at a mean depth of 17 mm (642 mm), distributed through both lungs (right = 17 (upper = 5, lower = 12) and left 13 (upper = 8, lower = 5)). the mean volume of dye injected was 3 ml (24 ml). dye was detected at the pleural surface in 97% (29/30) of the patients and at the nodule in 93% (28/30). there were no major complications, but two small pneumothoraces with no clinical significance, and one patient suffered discomfort ; this was resolved with secondary local anaesthetic injection. there was no adverse effect on general anesthesia, and thoracoscopic resection was possible in 90% (27/30) of the cases. one of the cases converted to open resection which was due to technical difficulties with the surgery rather than being related to the marking. in the other two cases in which thoracoscopic resection was not possible the location of the nodule made it difficult to ensure complete resection with vats resection, and this only became apparent during the surgery computed tomography guided radiological marking techniques described include wire marking [7, 8 ], lipiodol marking, barium marking, and combinations of wire and lipiodol marking. ct guided wire markings have been associated with uncontrollable pneumothorax and wire dislodgement, as well as high proportion of minor adverse events such as small pneumothorax and perifocal bleeding [7, 8, 11, 12 ]. the technique, using ct guided administered barium contrast balls to mark lung nodules, was found to cause local acute inflammations in all cases, and this can potentially hinder surgical resection. the use of stealthstation treon treatment guidance system (medtronic ; louisville, ky) has been described. results have been successful with no reported major complications ; however, the equipment can be costly and requires training for use. surgical techniques using methylene blue have been described with the surgeon palpating more superficial nodules with a finger through the port site incision and marking the palpated area before continuing with vats resection. a technique using radiological guided methylene blue for marking prior to breast microdochectomy has also been shown to have equal diagnostic accuracy compared to wire marking. the need for accurate localization particularly for deeper lung nodules is essential for the success of vats procedure for lung metastasectomy. the technique described is a cost effective, safe, and reliable method in marking lung nodules prior to resection. the method does not require expensive equipment described in other studies ; nor does it run the risk of wire dislodgement. as with the studies from lenglinger. in 1994 and mcconnell. in 2002, no patients experienced any adverse effects of the methylene blue - autologous blood mixture and no major complications occurred. small pneumothorax is a common minor complication in any procedure where a needle passes through the pleura and would usually resolve spontaneously without further management ; however, all these patients were undergoing immediate surgery. piercing the pleura multiple times increases the risk of causing pneumothorax and therefore good planning prior to the procedure with a safe route for the needle to pass is essential. one of the main risk factors for pneumothorax development is background chronic obstructive pulmonary disease and its associated complications, such as blebs and bullae. other risk factors for the development of pneumothorax include increasing patient age, increased depth of lesion, increased time of needle across the pleura, and traversing a fissure [16, 17 ]. consideration also needs to be made of the motion of the lung with breathing and intermittent breath holds may be required during acquisition of images and advancement of the needle. the incidence of pneumothorax is reported to range from 9% to 54% for lung nodule biopsy [16, 17 ], with an average of 20%, rising to 39% in patients with background emphysema. our pneumothorax rate of 7% with this technique is less than the aforementioned 9 studies and none lead to any complication during subsequent induction of general anaesthesia. none of the patients in this cohort had significant emphysema, bulla, or blebs. an issue that we discovered is that even though only a small volume is injected, the dye can spread over a much larger area in the adjacent lung than simply the parenchyma surrounding the nodule to be marked ; thus, as the injection is started, this needs to be at a slow and steady rate while withdrawing the needle back to the visceral pleural surface. clear communication with surgeons performing the subsequent metastasectomy is paramount and reconstructed multiplanar ct images of the nodule location aid their procedure planning. this and the discussion with the surgeon when there is the slightest concern when the marking is not exact will inevitably reduce failure rates and complications. | aim. we describe our experience of a simple, safe, and reproducible technique for lung nodule marking prethoracoscopic metastasectomy. thoracoscopic lung nodule resection reduces patient discomfort, complications, higher level of care, hospital stay, and cost ; however, small deeply placed lung nodules are difficult to locate and resect thoracoscopically. materials and methods. we describe and review the success of our novel technique, where nodules are identified on a low dose ct and marked with methylene blue using ct fluoroscopy guidance immediately prior to surgery. results. 30 nodules were marked with a mean size of 8 mm (418 mm) located at a mean depth of 17 mm, distributed through both lungs. dye was detected at the pleural surface in 97% of the patients and at the nodule in 93%. there were no major complications. thoracoscopic resection was possible in 90%. conclusion. this is a simple and safe method of lung nodule marking to facilitate thoracoscopic resection in cases where this may not be technically possible due to nodule location. |
various investigations have focused on it due to its biological activities, high tendency to recur and different growth mechanisms in comparison with other cystic lesions. many studies have been conducted on cell - cycle associated proteins such as antiapoptotic markers (tp53, bcl-2) and cell proliferation markers (ki67, proliferating cell nuclear antigen, epidermal growth factor receptor [egfr ]). these studies have shown different biological and proliferative activities for the lining epithelium of kcot. bcl-2, which is located in the external membrane of mitochondria, endoplasmic reticulum and nuclear membrane, play an important role in apoptosis. uncontrolled expression of bcl-2 has been found before histopathologic changes in the early stage of neoplasm. epidermal growth factor receptor is the most important growth factor ligand on the cell surface. overexpression of egfr - related genes is seen in many neoplasms, which causes the over sensitivity of cells to the normal level of growth factor. therefore, it causes specific phenotypes of cells, which can affect the cellular reaction to the treatment. kichi. have reported that bcl-2 is seen only in kcot and not in dentigerous cyst. sandra. have reported strong expression of bcl-2 in ameloblastoma and suggested that antiapoptotic proteins are expressed more than apoptotic proteins in ameloblastoma. in a study done by shear, expression of egfr marker the strongest reaction was related to kcot, and the weakest was in the radicular cyst. indeed, because of specific activity of kcot in comparison with other odontogenic cysts, it is possible to evaluate the expression of egfr and bcl-2 proteins in kcot compared with dentigerous cyst and ameloblastoma. expression of these proteins provides us with the knowledge about the biological activity of kcot. in this study, the expression of egfr and bcl-2 proteins in kcot was evaluated and compared with dentigerous cyst and ameloblastoma. in a cross - sectional study, paraffin - embedded tissues of 16 kcot, 16 dentigerous cysts, and 16 ameloblastoma samples were obtained from the archives in department of oral pathology, dental school, isfahan university of medical sciences. in order to detect the specific antigens of egfr and bcl-2, briefly, the main procedures were : serial sectioning (in 3 - 4 um sections), deparafinization, rehydration, and antigen retrieval. all specimens were then placed in the phosphate - buffered saline (pbs) and treated for 5 min in protein block solution to prevent any false staining. the specimens were then incubated for 30 min with primary antibody of egfr (ncl - egfr-384) clone egfr25, bcl-2 (ncl - bcl-2 - 486) clone 3.1 (novocastra / germany). furthermore, the sections were exposed to novolink polymer (re7112) or a secondary antibody for 30 min and washed with pbs. they were then incubated for 5 min with diaminobenzidine for visualization. after washing the slides finally, the slides were mounted after being dried. to quantify the percentage of positive cells within the lesions of egfr and bcl-2 markers, the sections were observed by two oral pathologists separately by olympus light microscope (cx21fs, tokyo, japan) at 400 magnification. the score of egfr staining and bcl-2 in each specimen / layer was calculated by the following formulas : score of egfr staining in each layer = d iscore of egfr staining in each specimen = td tidistribution (d) : mean percentage of stained cells by 1000 cells in each layerintensity of staining in each layer (i) : negative (0), weak (+ 1), moderate (+ 2), severe (+ 3)pattern of staining in each specimen (p) : membranous, cytoplasmic, membranous cytoplasmictotal distribution of each (td) : mean distribution of the total layertotal intensity (ti) : mean intensity of total layer. score of egfr staining in each layer = d i score of egfr staining in each specimen = td ti distribution (d) : mean percentage of stained cells by 1000 cells in each layer intensity of staining in each layer (i) : negative (0), weak (+ 1), moderate (+ 2), severe (+ 3) pattern of staining in each specimen (p) : membranous, cytoplasmic, membranous cytoplasmic total distribution of each (td) : mean distribution of the total layer total intensity (ti) : mean intensity of total layer. according to the distribution, the intensity of bcl-2 staining was classified as below : d / td intensity 50%:+3. whitney and kruskul wallis tests using spss software (spss inc., chicago, il, usa). the results of immunohistochemical staining of egfr marker showed that none of the kcot specimens expressed egfr with the score of 0. dentigerous cysts also indicated the score of 1.2 [figures 1 and 2, table 1 ]. there was no difference between the intensity of egfr expression in ameloblastoma and dentigerous cyst specimens. on the other hand, 91.8% of cells in ameloblastoma and 74.8% of cells in dentigerous cyst expressed this protein. the pattern of expression in 94% of ameloblastoma specimens was membranous, and it was membranous - cytoplasmic in 100% of dentigerous cysts specimens. there was a significant difference between egfr expression in peripheral and internal layers of ameloblastoma. 98.4% of the peripheral layer cells and 85.3% of the internal layer cells expressed egfr protein. the intensity of egfr expression was also significantly different in peripheral and internal layers (p = 0.01). in the evaluation of different layers of dentigerous cysts, the scores of egfr expression in basal cells versus supra - basal and superficial layers were 1.85 and 0.47, respectively. (p < 0.01) the results of immunohistochemical evaluation of bcl-2 marker revealed that the intensity of bcl-2 expression was not significantly different in ameloblastoma and kcot. the intensity of bcl-2 expression in ameloblastoma showed statistically significant differences compared with dentigerous cyst [figures 3 and 4, table 2 ]. expression of bcl-2 protein with high intensity in all of the ameloblastoma specimens (as a neoplastic lesion) and kcot (as a cystic lesion with neoplastic features) indicated the abnormal control of cell - cycle in these two lesions. this result can be due to the increase of cell vitality and invasive growth pattern of the lesion, which is similar to those reported by lo muzio. strong expression of bcl-2 in the peripheral layers and minimal expression in the central layers of ameloblastoma indicated the high potential of cell vitality in peripheral layers. it can be argued that bcl-2 has an important role in the survival of main cells in the peripheral layers of ameloblastoma. higher expression of bcl-2 in the basal layer of kcot is related to the physiological potency of the basal layer as a cell source for epithelium. according to the present results, no expression of egfr (as the most important protein in neoplastic proliferation) no expression of this protein and high level of bcl-2 protein expression show that aggressive features of kcot are related to apoptotic proteins. however, the aggressive potential of kcot is not as severe as that of a neoplasm such as ameloblastoma. indeed, the significant expression of antiapoptotic proteins with lack of proliferative proteins can cause a neoplastic condition with less aggressive features. high and severe expression of egfr and bcl-2 in ameloblastoma shows the neoplastic nature of this lesion., egfr expression causes an increase in the proliferation and aggressive activity of this lesion. therefore, its treatment needs aggressive surgical techniques. in aggressive ameloblastoma cases, control of proliferation and cell vitality the goal of investigations is to find less aggressive methods such as a monoclonal antibody of anti - egfr protein to prevent functional damage of the chewing system and deformity caused by surgical procedures. anti - egfr drugs and radiotherapy have synergistic effects in controlling the neoplasm and decreasing the recurrence. in physiological conditions all cases of dentigerous cyst indicated a membranous - cytoplasmic expression of egfr with lower value and distribution than ameloblastoma. kichi. have reported the high distribution of tdt - mediated dutp - biotin nick end labeling positive cells in the superficial layers and lack of these cells in the basal and middle layers of dentigerous cyst. however, these findings show apoptosis in the superficial layer of dentigerous cyst. furthermore, in ameloblastoma, more severe expression and higher distribution of egfr protein in the peripheral layer of ameloblastoma have the same pattern as bcl-2 expression compared with the central layer. li. have reported that the proliferative nature of cells decreases from the peripheral layer to the central layer of tumoral mass. biological activities, high tendency to recur and growth mechanisms of kcot are different in comparison with other cystic lesions that are related to apoptotic proteins. however, the aggressive potential of kcot is not as severe as that of the neoplasms such as ameloblastoma. | background : keratocystic odontogenic tumor (kcot) is a developmental odontogenic cyst on which various investigations have been focused due to its biological activities, high tendency to recur and different growth mechanisms in comparison with other cystic lesions. previous studies have shown different biological and proliferative activities for the lining epithelium of kcot. the aim of this study was immunohistochemical evaluation of bcl-2 and epidermal growth factor receptor (egfr) expression in kcot compared with dentigerous cyst and ameloblastoma.materials and methods : formalin - fixed and paraffin - embedded tissue sections of 16 cases of kcot, 16 cases of dentigerous cyst and 16 cases of ameloblastoma were immunohistochemically analyzed to determine bcl-2 and egfr proteins expression. biotin - stereotavidin method was used. it was observed by two oral pathologists separately, and the data were analyzed by mann whitney and kruskul wallis. p < 0.05 was considered as significant.results:regardless of staining intensity, all cases of ameloblastoma and kcot except dentigerous cases were positively stained for bcl-2. expression of bcl-2 was higher in the peripheral layer of ameloblastoma and basal layer of kcot. furthermore, all cases of ameloblastoma and dentigerous cysts except kcot samples were positively stained for egfr. expression of egfr was higher in the peripheral layer of ameloblastoma and basal layer of dentigerous cysts.conclusion:according to the expression of bcl-2 in ameloblastoma and kcot, and no expression of egfr in kcot, it can be concluded that the biological activity and growth mechanisms of kcot are different compared with other cystic lesions. however, the aggressive potential of kcot is not as severe as that of a neoplasm such as ameloblastoma. |
. the natural history of this disease entity has not been well established, but it has been postulated that the prognosis is generally good. in contrast to carcinoid tumors arising in the jejunum and ileum, the clinical and laboratory findings of carcinoid syndrome are absent in patients with a carcinoid tumor of the ampulla of vater, such as duodenal carcinoid tumors. however, in small numbers of carcinoid tumors of the ampulla of vater can show more aggressive behaviors, such as distant metastasis.. there have been several reports of endoscopic treatment of small carcinoid tumors arising from the duodenum, but there has been no report of pretreatment diagnosis and intentional endoscopic treatment of carcinoid tumors of the ampulla of vater in korea. the ampulla of vater is a complex structure, and is the confluent portion of common bile duct, pancreatic duct and contains the sphincter of oddi. this may explain the reason why an ampullary carcinoid often clinically manifests as obstructive jaundice or acute pancreatitis, and an attempt to remove the tumor may result in more frequent procedure - related complications. here, a case of a carcinoid tumor of the ampulla of vater, diagnosed before treatment and intentionally treated by endoscopic snare papillectomy, is reported. a 62-year - old male was admitted with an incidental finding of a carcinoid tumor of the ampulla of vater. the patient had undergone an endoscopic mucosal resection (emr) of early gastric cancer 1 year earlier. every 3 month following the emr, he has undergone surveillance gastroscopic examination with forward - viewing endoscopy. during the latest endoscopic examination, biopsies were taken from the lesion, which was histologically confirmed as a carcinoid tumor. he denied weight loss, diarrhea, flushing, or any respiratory difficulties or obstructive biliary symptoms. he underwent ercp for a more detailed evaluation of the ampulla of vater. an examination with a side - viewing duodenoscope showed prominent, but a preserved configuration of the ampulla, but depressed erosion with hyperemia on the covering mucosa (figure 1). contrast agent was injected into common bile and pancreatic ducts, but no abnormality within the lumen of either the bile or pancreatic ducts were seen. following the ercp, this revealed a hypoechoic, ovoid shaped lesion less than 1 cm in diameter arising from the mucosal layer (figure 2). the patient then underwent endoscopic snare papillectomy for the removal of the carcinoid tumor of the ampulla of vater (figure 3). after elevation of the tumor by submucosal injection of saline solution, the ampulla was grasped by snare and a papillectomy performed using blended electrosurgical current (cut to coagulation ratio 4:1) at a setting of 50 watts employing a electrosurgical unit (ues-20, olympus optical co., tokyo, japan). then, plastic biliary (7 fr, 3 cm) and pancreatic (5 fr, 3 cm) stents were inserted to prevent possible cholangitis and pancreatitis. there was no complication after endoscopic snare papillectomy, i.e., cholangitis, pancreatitis, bleeding or perforation. pathologic analysis of the resected specimen revealed a 0.70.50.1 cm sized carcinoid tumor of the ampulla of vater within the mucosal layer (figure 4). the patient had an uneventful hospital course and was discharged on the 5 post - papillectomy day following the removal of the biliary and pancreatic stents. two months after the endoscopic snare papillectomy, a follow - up duodenoscopic examination was performed, which revealed no evidence of tumor recurrence, either macroscopically or microscopically. six months after the endoscopic snare papillectomy, there was no evidence of local recurrence and metastasis of the tumor, either endoscopically or radiologically. carcinoid tumors of the ampulla of vater are very rare. according to a review of 90 reported ampullary carcinoid cases, the mean patient age was 52 years, and affected patients were more often male. jaundice was the most common presenting symptom of a carcinoid tumor of the ampulla of vater and was present in 59% of patients. it is often apparent at an early stage because of the location ; obstructive symptoms may arise early when the tumor is relatively small. despite frequent regional lymph node metastasis, the prognosis of carcinoid tumors of the ampulla of vater has generally been considered good. in one study, the 5 year survival was 90%, with only 4 (6%) patients dying of a metastatic disease or progressive tumor. even though there are reports on the benign nature of this tumor, they have malignant potential. unlike carcinoid tumors of the duodenum, the size of the tumor seems to have no prognostic implications for carcinoid tumor of the ampulla of vater. in one review of 73 cases of carcinoid tumors of the ampulla of vater, thirty one were larger than 2 cm in diameter and 48% (15/31) of these had metastasized. interestingly, 40% (17/42) of patients with tumors less than 2 cm also had a metastatic disease. hence, the tumor size was not a reliable predictor of aggressiveness ; therefore, complete resection of the tumor is mandatory regardless of the size. in our case, careful examinations, including eus and abdominal ct scan, were performed before determining the therapeutic options. the size of the tumor was small (less than 1 cm) and there was no evidence of muscularis propria invasion and regional lymph node metastasis on eus. an in-111 octreotide scan performed to evaluate occult metastases showed negative findings. since the majority of carcinoid tumors express receptors for somatostatin, the detection rate using an in-111 octreotide scan ranges from 80 to 90%, and most investigators agree that this radionuclide scan and conventional imaging, such as ct, are complementary. the treatment of choice for carcinoid tumors of the ampulla of vater is complete resection, and the standard treatment modality for the tumor has been surgery, such as pancreaticoduodenectomy or local excision of the tumor. in one review of 90 patients with an ampullary carcinoid, local excision was generally performed in patients with tumors less than 2 cm in diameter, while a pancreaticoduodenectomy was performed in patients with tumor larger than 2 cm. on the other hand, 21 of the 22 patients who underwent local excision are alive, with no evidence of recurrence after long - term follow - up, and only one patient died of local recurrence 20 months following the local excision. either a pancreaticoduodenectomy or local excision, via a duodenotomy, may be selected depending on the size of the tumor. although a pancreaticoduodenectomy enables complete resection of the tumor, this procedure has the disadvantage of relatively higher morbidity., local excision may be an option for the treatment of a carcinoid tumor of the ampulla of vater if the size of the tumor is small and there is no evidence of regional lymph node or distant metastasis. compared to local surgical excision, endoscopic snare papillectomy may be much less harmful to the patient, since it does not require a laparotomy and duodenotomy. in the management of an ampullary adenoma, endoscopic therapy seems to be successful and may be a reasonable alternative to surgical resection. a carcinoid tumor of the ampulla of vater originates from the deep mucosa. most reports describe it as a round or oval mass, with intact overlying duodenal mucosa, with negative biopsies. only a few reports present it as a prominent papilla, with a shallow ulcer on the surface. however, a carcinoid tumor of the ampulla of vater can show mucosal change, theoretically because it originated from the deep mucosa, not the submucosa. in our case, depressed erosion, which may be misinterpreted as papillitis, was found. without a biopsy, thorough examination, including the ampulla, is essential in all patients undergoing esophagogastroduodenoscopy, and biopsy of suspicious mucosal changes should always be carried out to diagnose the tumor in early stage. in conclusion, endoscopic snare papillectomy may be one therapeutic option in the management of carcinoid tumors of the ampulla of vater in selected patients, such as those with a small sized and localized in the mucosa or submucosal layer, without regionally or distant metastasis. further experience with more cases will be needed to establish the exact indication for endoscopic snare papillectomy. | here, a case of a patient with incidental finding of a carcinoid tumor of the ampulla of vater, who was treated with endoscopic snare papillectomy, is reported. a 62-year - old male was admitted to our hospital due to a carcinoid tumor of the ampulla of vater, which was found during follow - up endoscopy after an endoscopic mucosal resection of early gastric cancer. no lymphadenopathy or visceral metastasis was found on an abdominal ct scan, in-111 octerotide scan and eus. the ampulla was then en bloc removed by endoscopic snare papillectomy. the resected specimen revealed a 0.70.50.1 cm sized carcinoid tumor. all margins of resection were negative for tumor. after six months of follow - up, there was no evidence of recurrence and metastasis, either endoscopically or radiologically. to our knowledge, this case is the first report of an ampullary carcinoid tumor treated by endoscopic snare papillectomy in korea. |
modern cataract surgery is entering the era of refractive surgery, allowing people not only to be able to see but also to be able to see clearly at all distances. it is well known that the presence of astigmatism in the eye in which the multifocal intraocular lens (iol) is implanted can affect vision [1, 2 ]. the prevalence of preoperative astigmatism in patients undergoing cataract surgery has been estimated to be between 15 and 29%. there are many techniques to correct corneal astigmatism, including corneal limbal relaxing incision [6, 7, 8, 9, 10 ]. recent studies have shown that the implantation of an astigmatism - corrected iol is more effective. restor toric has the advantages of the multifocal astigmatism correction of an artificial lens, and it can correct astigmatism. children with cataracts who have lost vision not only need to see clearly at both near and far distances without the use of spectacles to meet the needs when studying and doing sports, but also to reconstruct the binocular visual function. in this case report, the effects and complications of restor toric in the eye of a child with traumatic cataract and astigmatism were observed. his left eye had been hit inadvertently by plastic pellets 2 years prior to surgery. an ocular examination revealed right - eye visual acuity of 20/20 and left - eye visual acuity of 3/20 ; intraocular pressure was 15 mm hg (left eye), the cornea was transparent, the anterior chamber was normal in depth, the iris was clear, and the pupil diameter was 3 mm and sensitive to light reflection. the lens was opaque (upper temporal significant t - shaped porcelain white opacity) with posterior capsular opacity. corneal endothelial cell count was 2,332.8/mm (left eye) ; b - scan ultrasonography detected no obvious abnormalities in the eyes. axial length (al) was 23.55 mm, k1/k2 was 41.82 d/43.95 d, and cylinder was 2.13 d at 174 (left - eye corneal topography). the child 's parents wanted the patient to have a chance to see near and far clearly without glasses to meet the needs when studying and doing sports. restor toric combines the advantages of an artificial lens with the astigmatism correction of an iol and the advantages of a multifocal iol. the diffractive aspect of the iol optical part is a step height of the diffraction pattern from the centre to the periphery of the optical part. the optical surface is non - spherical, which can improve the contrast sensitivity and compensate for the physiological spherical aberration of the cornea. in cataract surgery, the benefits of using a non - spherical iol have been widely described in the literature. in most patients the patient 's eye records were as follows : k1 : 41.82 d 174 (corneal curvature), k2 : 43.95 d 84 (steep corneal curvature), 0.50 d (sia), 135 (design incision position), and the iol power was calculated as 21.0 d (snd1t5). the astigmatism axial angle was 77. the addition of snd1t5 to iol in the plane is + 3.00 d, which corresponds to + 2.25 d near vision and corrects + 2.0 d astigmatism. because the child had a history of trauma, a multifocal iol would not have been implanted if the capsule bag had been torn. the restor toric iol axial position was labelled with a marking pen after topical anaesthesia, and then the pupil was dilated with mydrin - p. the corneal incision was made manually using a 2.2-mm keratome and a 0.75-mm side - port blade. phacoemulsification suction was 350 mm hg, flow was 35 ml / min, and emulsifying time was 17.8 s. after successful removal of the lens cortex, the posterior capsule and anterior capsules were polished. lens astigmatism axial angle was adjusted to 77. after successful removal of the viscoelastic agent, the anterior chamber was formatted. the eye was coated with tobramycin dexamethasone oculentum and covered with a sterile gauze mask. follow - up was performed at 1 day, 3 days, 1 week, 1 month, 2, 3 and 7 months after surgery. at each visit, the eye was scanned using a dioptre corneal analyser system (nidek co., ltd.) for automatic optometry. questions included do you wear glasses? and whether there were halo glare, astral glare, ghosting and diplopia, or discomfort phenomena. each question had 4 options for the answer : never ; in some cases ; in most cases, and always. the patient reported no halo glare, astral glare, ghosting and diplopia, or discomfort phenomena. the far visual acuity remained between 16/20 and 24/20, and the near visual acuity remained between j1 and j3 without the use of spectacles at any distance during 7 months of follow - up. in the face of paediatric cataract, many doctors only pay attention to achieving superb surgical techniques and good surgical results and overlook the fact that children who have lost binocular vision also need reconstruction of binocular visual function. to rebuild binocular visual function, the most important problem is how to choose the artificial crystal with the best optical effect, specifically the effect most consistent with human physiology. in most cases, school - age children receive treatments restoring vision in the near distance because their demand for near vision is higher than that of adults. due to the lack of multifocal capability, single - focus iols are far from meeting patients needs for learning and life. multi - focal iols can make far and near objects appear on the retina, thereby meeting the different requirements of vision at the same time. this approach can solve the problem of the lack of adjustment of a single - focus iol to achieve normal or close to normal vision. multifocal iols have been reported as a good alternative to a monofocal iol in cooperative children over 4 years of age with unilateral cataract. however, most cataract patients also suffer from astigmatism. in a study of 4,540 patients, 87% patients had various degrees of astigmatism before cataract surgery, 64% of patients were between 0.2 and 1.25 d, 22% of patients were above 1.5 d, and 5 or 6% of patients with astigmatism were even above 2 d. it is well known that the astigmatism of the eye of the diffractive multifocal iol can impair vision. many studies have demonstrated the safety and effectiveness of various methods of astigmatism correction after implantation of a multifocal iol, including laser - assisted in situ keratomileusis and limbal relaxing incision [16, 17 ]. the multifocal astigmatism - correcting type of iol has the ability to correct astigmatism in a single operation. acrysof astigmatism correction [18, 19 ] has been tested in randomized trials to confirm its safety, effectiveness, rotational stability, optical quality, and subjective patient satisfaction. a clinical study of the 3.00- and 4.00-d iol confirmed their safety and effectiveness, optical quality, and patient satisfaction [20, 21 ]. a diffractive multifocal toric iol reduces astigmatism more than a monofocal iol and provides more satisfactory near vision. the astigmatism correction was safe and effective and can be expected to remain satisfactory, and it also decreased the rate of spectacle use. because the child 's al was 23.76 mm, greater than 23 mm, we used the srk / t formula. negative optical phenomena (halo, glare, astral, ghost, and diplopia) are inherent in a diffractive multifocal iol design because the different annular zones of the step edges produce these phenomena. however, there were no glare, halo, or discomfort phenomena in our study. our results show that the implantation of an acrysof iq restor toric iol provided excellent far and near vision in a patient over 6 years of age, offering the pseudophakic patient the possibility of satisfactory vision for both distant and near conditions while reconstructing binocular visual function without the use of spectacles to meet children 's needs when studying and doing sports. in this case, the iol master showed good repeatability in terms of the corneal topography, ab ultra, and the optometric tests that could be performed. the development of abnormal children with astigmatism in cataract patients with multifocal light iols can produce better visual acuity, better stereo vision, and no obvious visual symptoms and complications. this approach allows the pseudophakic eye to function with its fellow eye, which has active accommodation. it may be a good alternative for children over 6 years of age with unilateral cataract | a child suffering from traumatic cataract and corneal astigmatism of 2.14 d had a phacoemulsification operation and implantation of a restor toric intraocular lens (iol) to correct the astigmatism. the primary outcome measurements were the uncorrected distance visual acuity (udva), uncorrected near vision at 40 cm, intraocular pressure, spherical equivalent refraction, residual astigmatism, corneal astigmatism, presence of unusual optical phenomena, and use of spectacles. at 7 months postoperatively, udva was maintained between 16/20 and 24/20, near vision was between j1 and j3, residual spherical refraction was 00.37 d, and residual refractive cylinder was between 0 and 0.67 d. a multifocal toric iol can provide the possibility of satisfactory vision for both distant and near conditions without the use of spectacles to meet children 's needs when studying and doing sports. additionally, binocular vision can be reconstructed. this intervention, therefore, seems to be a satisfactory alternative. |
cervical carcinoma is the second most common cause of cancer - related deaths in women worldwide, with a global annual incidence of approximately 470,000 new cases and an estimated 233,000 deaths per year. management for early cervical cancers generally involves radical abdominal hysterectomy and radiotherapy, with good results in terms of survival and quality of life. however, the 5-year survival rate of advanced patients is from 3.2% to 13%, which is due to their resistance to the traditional treatments. mounting evidence suggests that cancer may in fact arise from a transformed stem cell that is able to self - renew, differentiate into diverse progenies, and drive continuous growth. aldh is a detoxifying enzyme that oxidizes intracellular aldehydes and confers resistance to alkylating agents [47 ]. recent studies have shown that aldh1 is a cancer stem cell marker and that its presence strongly correlates with tumor malignancy and self - renewal properties of stem cells in different tumors, including breast cancer, hepatoma, colon and lung cancer [811 ] ]. however, whether aldh1 can be a useful marker of csc and/or aid cancer diagnosis in cervical carcinoma is still an open question. given this evidence, we investigated whether cervical carcinomas contain a tumorigenic stem cell - like population. the human cervical cancer cell lines c33a, caski, hela and siha were stored in our lab. the cells were cultured in rpmi 1640 media (invitrogen life technologies, carlsbad, ca) supplemented with 10% fetal bovine serum and incubated at 37c in an atmosphere containing 5% co2. tumors from the uterine cervix were collected from archives of the department of gynecology at the 2nd affiliated hospital of sun yat - sen university. a total of 112 specimens were analyzed, whose diagnoses were invasive squamous carcinoma (89 cases) and adenocarcinoma (20 cases), and normal cervical epithelium (3 cases) obtained from patients undergoing hysterectomy for various non - malignant causes served as normal controls. formalin - fixed paraffin - embedded specimens were cut into 3m sections and mounted on glass slides pre - treated with poly - l - lysine. sections were boiled for 15 minutes in citrate buffer (ph 6), cooled in the same buffer, and subsequently incubated for 10 minutes in 0.3% h2o2 to block the endogenous peroxidase activity. aldh1 antibody (santa cruz) was used at a 1/100 dilution stored overnight at 4c in a humid chamber, and rinsed in pbs. sections were washed and incubated with horseradish peroxidase (hrp)-conjugated anti - mouse secondary antibody (1:100) (zymed, san francisco, ca) for 60 minutes at room temperature, followed by abc vectastain kit (vector laboratories, burlingame, ca, usa) for 5 minutes. replacement of the monoclonal antibody with mouse igg1 protein of the same concentration was used as a negative control. immunohistochemical staining of aldh1 was classified as 3 + (50% positive tumor cells), 2 + (< 50% but 10%), 1 + (< 10%), or negative (0%). cd133 antibody (ab19898) and smad3 antibody (ab28379) were used at a 1/100 dilution with anti - rabbit secondary antibody (ab6721). the population with high aldh enzymatic activity was isolated by use of the aldefluor kit (stemcell technologies, durham, nc). cells were suspended in aldefluor assay buffer containing aldh substrate (baaa, 1 mol / l per 110 cells) and incubated for 45 minutes at 37c to allow the conversion of aldefluor substrate, a green fluorescent product retained within the cell due to its negative charge. for each experiment, an aliquot of aldefluor - stained cells was immediately quenched with 5 l 1.5 mm diethylaminobenzaldehyde (deab), a specific aldh inhibitor, to serve as a negative control. the sorting gates were established using as negative controls the cells stained with pi only ; for viability, the aldefluor - stained cells treated with deab ; and the staining with secondary antibody alone. cells were blocked with fcr blocking reagent and stained with cd133 mouse igg1 antibody conjugated to phycoerythrin (pe) (miltenyi biotec inc., auburn, ca). mouse igg1-pe (bd biosciences, san jose, ca) was the isotype control antibody. all reactions were done in a 20 l reaction volume in triplicate by sybr green real - time pcr universal reagent (genepharma co., ltd.) and mx-3000p real - time pcr machine (stratagen). standard curves were generated and the relative amount of aldh1 and cd133 were normalized to -actin (2). primers are aldh1 (forward, 5-ctgctggcgacaatggagt-3 ; reverse, 5-gtcagcccaacctgcacag-3), cd133 (forward, 5-caaccctgaactgag gcagc-3 ; reverse, 5-ttgatagccctgttggaccag-3) and -actin (forward, 5-gcatgggtcagaaggattcct-3 ; reverse, 5-tcgtcccagttggtgacgat-3) was used as endogenous control. protein was extracted from cervical cancer cell lines c33a, caski, hela and siha. briefly, 10 g protein of each sample was subjected to sds - page on 10% polyacrylamide gels, electrophoretically transferred to pvdf membranes (millipore, billerica, ma, usa). the membranes were blocked for 1 hour at room temperature, and then incubated overnight at 4c in tris buffered saline with 0.05% tween (tbst) containing 5% dry milk and detected with mouse monoclonal igg1 anti - aldh1(bd, usa), rabbit polyclonal igg anti - cd133 (abcam, usa) and commercial ecl kit (pierce, rockford, il, usa). protein loading was estimated using mouse anti--actin monoclonal antibody (sigma, usa) and peroxidase - conjugated goat anti - mouse igg (multisciences biotech co. ltd). the human cervical cancer cell lines c33a, caski, hela and siha were stored in our lab. the cells were cultured in rpmi 1640 media (invitrogen life technologies, carlsbad, ca) supplemented with 10% fetal bovine serum and incubated at 37c in an atmosphere containing 5% co2. tumors from the uterine cervix were collected from archives of the department of gynecology at the 2nd affiliated hospital of sun yat - sen university. a total of 112 specimens were analyzed, whose diagnoses were invasive squamous carcinoma (89 cases) and adenocarcinoma (20 cases), and normal cervical epithelium (3 cases) obtained from patients undergoing hysterectomy for various non - malignant causes served as normal controls. formalin - fixed paraffin - embedded specimens were cut into 3m sections and mounted on glass slides pre - treated with poly - l - lysine. sections were boiled for 15 minutes in citrate buffer (ph 6), cooled in the same buffer, and subsequently incubated for 10 minutes in 0.3% h2o2 to block the endogenous peroxidase activity. aldh1 antibody (santa cruz) was used at a 1/100 dilution stored overnight at 4c in a humid chamber, and rinsed in pbs. sections were washed and incubated with horseradish peroxidase (hrp)-conjugated anti - mouse secondary antibody (1:100) (zymed, san francisco, ca) for 60 minutes at room temperature, followed by abc vectastain kit (vector laboratories, burlingame, ca, usa) for 5 minutes. replacement of the monoclonal antibody with mouse igg1 protein of the same concentration was used as a negative control. immunohistochemical staining of aldh1 was classified as 3 + (50% positive tumor cells), 2 + (< 50% but 10%), 1 + (< 10%), or negative (0%). cd133 antibody (ab19898) and smad3 antibody (ab28379) were used at a 1/100 dilution with anti - rabbit secondary antibody (ab6721). the population with high aldh enzymatic activity was isolated by use of the aldefluor kit (stemcell technologies, durham, nc). cells were suspended in aldefluor assay buffer containing aldh substrate (baaa, 1 mol / l per 110 cells) and incubated for 45 minutes at 37c to allow the conversion of aldefluor substrate, a green fluorescent product retained within the cell due to its negative charge. for each experiment, an aliquot of aldefluor - stained cells was immediately quenched with 5 l 1.5 mm diethylaminobenzaldehyde (deab), a specific aldh inhibitor, to serve as a negative control. the sorting gates were established using as negative controls the cells stained with pi only ; for viability, the aldefluor - stained cells treated with deab ; and the staining with secondary antibody alone. cells were blocked with fcr blocking reagent and stained with cd133 mouse igg1 antibody conjugated to phycoerythrin (pe) (miltenyi biotec inc., auburn, ca). mouse igg1-pe (bd biosciences, san jose, ca) was the isotype control antibody. all reactions were done in a 20 l reaction volume in triplicate by sybr green real - time pcr universal reagent (genepharma co., ltd.) and mx-3000p real - time pcr machine (stratagen). standard curves were generated and the relative amount of aldh1 and cd133 were normalized to -actin (2). primers are aldh1 (forward, 5-ctgctggcgacaatggagt-3 ; reverse, 5-gtcagcccaacctgcacag-3), cd133 (forward, 5-caaccctgaactgag gcagc-3 ; reverse, 5-ttgatagccctgttggaccag-3) and -actin (forward, 5-gcatgggtcagaaggattcct-3 ; reverse, 5-tcgtcccagttggtgacgat-3) was used as endogenous control. protein was extracted from cervical cancer cell lines c33a, caski, hela and siha. briefly, 10 g protein of each sample was subjected to sds - page on 10% polyacrylamide gels, electrophoretically transferred to pvdf membranes (millipore, billerica, ma, usa). the membranes were blocked for 1 hour at room temperature, and then incubated overnight at 4c in tris buffered saline with 0.05% tween (tbst) containing 5% dry milk and detected with mouse monoclonal igg1 anti - aldh1(bd, usa), rabbit polyclonal igg anti - cd133 (abcam, usa) and commercial ecl kit (pierce, rockford, il, usa). protein loading was estimated using mouse anti--actin monoclonal antibody (sigma, usa) and peroxidase - conjugated goat anti - mouse igg (multisciences biotech co. ltd). aldh1 expression was observed in 23/89 (89 cases) cases of invasive squamous carcinoma and 4/20 cases of adenocarcinoma (20 cases) (figure 1a d). no aldh1 was expressed in normal cervical epithelium obtained from patients undergoing hysterectomy for various non - malignant causes. the majority of positive staining demonstrated focal moderate staining, and occasionally moderate diffuse staining with moderate intensity. among positive staining cases in primary cervical carcinomas and metastatic carcinomas, staining tended to be diffuse, with a spectrum ranging from weak to moderate - to - strong. the results of immunostaining of the tumor microarrays, organized according to clinicopathologic characteristics of the patients, are shown in table 1. correlation was found between the expression of the aldh1 and lymph nodal metastasis in cervical carcinoma, and patients with recurrent disease were more likely to have a higher aldh1 expression (table 1). we also found the expression of aldh1 has some relationship with smad3 (table 2). we utilized the aldefluor assay to assess the presence and size of the population with aldh enzymatic activity in normal human breast epithelium. analysis of c33a, caski, hela and siha cells showed an average of 3.10.26%, 22.072.63%, 11.633.54% and 27.533.96% aldefluor - positive population, respectively (figure 3a f). we also studied expression of cd133 by the cervical cancer cell lines c33a, caski, hela and siha. figures 4a d show the 0.220.01%, 0.670.03%, 0.430.06% and 3.781.02% cd133-positive populations, respectively (the control antibody background was not subtracted because it did not overlap cd133 fluorescence). aldh1 mrna expression was significantly higher in cervical cell lines than was cd133 mrna expression (figure 5). aldh1 protein was detected as a single band at 55 kda in all of the cervical cell lines tested by western blot (figure 6). aldh1 expression was observed in 23/89 (89 cases) cases of invasive squamous carcinoma and 4/20 cases of adenocarcinoma (20 cases) (figure 1a d). no aldh1 was expressed in normal cervical epithelium obtained from patients undergoing hysterectomy for various non - malignant causes. the majority of positive staining demonstrated focal moderate staining, and occasionally moderate diffuse staining with moderate intensity. among positive staining cases in primary cervical carcinomas and metastatic carcinomas, staining tended to be diffuse, with a spectrum ranging from weak to moderate - to - strong. the results of immunostaining of the tumor microarrays, organized according to clinicopathologic characteristics of the patients, are shown in table 1. correlation was found between the expression of the aldh1 and lymph nodal metastasis in cervical carcinoma, and patients with recurrent disease were more likely to have a higher aldh1 expression (table 1). we also found the expression of aldh1 has some relationship with smad3 (table 2). we utilized the aldefluor assay to assess the presence and size of the population with aldh enzymatic activity in normal human breast epithelium. analysis of c33a, caski, hela and siha cells showed an average of 3.10.26%, 22.072.63%, 11.633.54% and 27.533.96% aldefluor - positive population, respectively (figure 3a f). we also studied expression of cd133 by the cervical cancer cell lines c33a, caski, hela and siha. figures 4a d show the 0.220.01%, 0.670.03%, 0.430.06% and 3.781.02% cd133-positive populations, respectively (the control antibody background was not subtracted because it did not overlap cd133 fluorescence). aldh1 mrna expression was significantly higher in cervical cell lines than was cd133 mrna expression (figure 5). aldh1 protein was detected as a single band at 55 kda in all of the cervical cell lines tested by western blot (figure 6). the cancer stem cell hypothesis supposes that the cancer stem cells may relate to the cancer risk assessment, early detection, prognostication and prevention. the development of more effective cancer therapies may require targeting cancer stem cell populations. the success of these new approaches concentrates on the identification, isolation, and characterization of cancer stem cells. the study of cervical cancer sc would be greatly enhanced by availability of specific markers to identify and isolate these cells. the aldehyde dehydrogenase (aldh) families of enzymes are cytosolic isoenzymes that are responsible for oxidizing intracellular aldehydes and contributing to the oxidation of retinol to retinoic acid in early stem cell differentiation. visus. suggested that aldh1a1 is a marker for distinguishing premalignant cells in head and neck squamous cell carcinoma (hnscc), and the increased incidence of aldh1 expression correlated positively with the staging of patients. bin chang. analyzed the associations between the expression of the aldh1 and clinical factors (diagnosis, tumor grade, stage, and clinical response to chemotherapy), as well as overall and disease - free survival. they found aldh1 was a favorable prognostic factor of ovarian carcinoma. in breast carcinomas, the expression of aldh1 detected by immunostaining was correlated with poor prognosis. a key finding of our study was that immunohistochemistry identifies a small subpopulation of aldh1 + cells localized in the cervical carcinoma. in the current study, high levels of aldh1 expression were observed in patients with recurrent or metastatic disease, suggesting that aldh1 might be a potential independent prognosis predictor in cervical cancer. loss of responsiveness to tgf--induced cell growth inhibition during cancer development is a feature of many tumors, including cervical cancer. in this study, we found that increased smad3 protein expression in cervical cancers was associated with aldh1 expression. a second key finding of our study is that aldh - based markers can be used to track cervical sc during cervical tumorigenesis. utilizing in vitro assays, aldh activity in cells was measured by using dansyl aminoacetaldehyde, and the activity of aldh was used to isolate precursor cells. unlike the previously described breast cancer stem cell phenotype, which requires the use of a combination of 10 surface antigens, testing for aldh1 activity is a simple method for identifying normal and cancer stem cells. cells with high aldh activity contain the tumorigenic cell fraction able to self - renew and to recapitulate the heterogeneity of the parental tumor. these cells may also have the highest ability to grow in vivo in a xenotransplantation animal model. we observed high levels of aldh1 in primary cultures obtained from resected tumor tissue, confirming that aldh1 overexpression is not a phenomenon restricted to established cell lines. thus far cellular markers including cd133 have been used to identify cscs in several tumors. recently, the relevance of cd133 as a reliable csc marker has been doubted, since it was shown that even cd133-negative gbm cells may behave as brain cscs. however, in cervical carcinoma, we found the expression of cd133 was very limited. since aldh1 is also expressed in hematopoietic and neuronal stem cells, this marker may prove useful for the detection and isolation of cancer stem cells in other malignancies, thus facilitating the application of cancer stem cell biology to clinical practice. our study suggests that aldh1 expression may be used to detect cervical cancer stem cells. identification of aldh1 as a potential marker of cancer stem cells opens important new avenues of research in cervical carcinogenesis. | summarybackgroundaldh1 has been shown to play a role in the early differentiation of stem cells in some human malignancies. whether cancer stem cells occur in aldh1-associated cervical cancer is not known.material/methodswe tested the hypothesis that cervical carcinomas contain subpopulations of cells that express aldh1. the following sources of cervical carcinoma tissues were examined for the presence of stem cell marker aldh1 by immunohistochemistry. flow cytometric isolation of cancer cells was based on enzymatic activity of aldh (aldefluor assay). the mrna and protein levels of aldh1 were investigated by qrt - pcr and western blot, respectively. we also detected the expression of cd133 identified as a stem cell marker for several cancers.results23/89 samples of invasive squamous carcinoma and 4/20 samples of adenocarcinomas exhibited immunoreactivity to stem cell marker aldehyde dehydrogenase 1 (aldh1). expression of aldh1 was found in 24.77% of the samples. flow cytometric analysis, qrt - pcr and western blot also confirmed the presence of small subpopulations of aldh1-positive cells. in contrast, we found cervical carcinoma had low cd133 population.conclusionscervical carcinoma contains a small subpopulation of cells that may relate to a cancer stem cell - like phenotype, aldh1. |
a descriptive cross - sectional study was conducted from january 28, 2013 to february 8, 2013 in burka jato kebele, nekemte town, east wollega zone, west ethiopia, 330 km away from addis ababa, capital of ethiopia. sample interval was determined by dividing the total number of households by sample size and select the first study unit using lottery method. the sample size was determined using the formula : n = 384, n = minimum sample size where ; n = desired sample size from the population z = confidence interval, 95% = 1.96 p = population prevalence = 0.5 q = 1 p = 10.5 = 0.5 d = degree of accuracy desired at 0.05 since the number of the population was less than 10,000 (9,432) and number of households was calculated to be 1070. a convenience sample of 282 individuals (households) was selected from the community by systematic random sampling. data were collected by two persons, and the households were interviewed by using using both closed and open ended questionnaires. data collectors were briefed on the objectives, relevance of the study, on terms and how to collect the data. the collected data was first checked for completeness and analysis was performed both by computer and manually. chi square test was used to determine the statistical significance of association between some variables. results were analyzed, interpreted and presented in texts, tables and figures and they were discussed in comparison with some similar studies done before. letter of permission was written from jimma university, pharmacy department to the chairman of burka jato kebele community and explanation about the objectives and use of the study was given to the community. the interviewers were advised to be as polite as possible and respect the response of the person. a descriptive cross - sectional study was conducted from january 28, 2013 to february 8, 2013 in burka jato kebele, nekemte town, east wollega zone, west ethiopia, 330 km away from addis ababa, capital of ethiopia. sample interval was determined by dividing the total number of households by sample size and select the first study unit using lottery method. the sample size was determined using the formula : n = 384, n = minimum sample size where ; n = desired sample size from the population z = confidence interval, 95% = 1.96 p = population prevalence = 0.5 q = 1 p = 10.5 = 0.5 d = degree of accuracy desired at 0.05 since the number of the population was less than 10,000 (9,432) and number of households was calculated to be 1070. a convenience sample of 282 individuals (households) was selected from the community by systematic random sampling. data were collected by two persons, and the households were interviewed by using using both closed and open ended questionnaires. data collectors were briefed on the objectives, relevance of the study, on terms and how to collect the data. the collected data was first checked for completeness and analysis was performed both by computer and manually. chi square test was used to determine the statistical significance of association between some variables. results were analyzed, interpreted and presented in texts, tables and figures and they were discussed in comparison with some similar studies done before. letter of permission was written from jimma university, pharmacy department to the chairman of burka jato kebele community and explanation about the objectives and use of the study was given to the community. the interviewers were advised to be as polite as possible and respect the response of the person. a total of 282 respondents participated in the study. their mean age was 30 and the modal age group was 33 (3039). the sex ratio was predominated by male (63%) and the majority was found in the age group of 3039 (31.56%). oromo took the highest ethnic group (74%), followed by amhara (15%). a total of 52 medicinal plant species were identified among this 28 were documented [table 1 ]. distribution of the study population by socio demographic and economic characteristics of burka jato kebele, west ethiopia, february 2013 more than half of the burka jato people (56%) were married, and majority of their educational status (48.6%) were between grade 9 and 12. more than quarter (26.24%) live by daily labor and 67% of people earn more than 500 birr / month [table 2 ]. knowledge of burka jato people towards tm, west ethiopia, february 2013 majority of burka jato kebele people were aware of alternative treatment options apart from modern medicine (i.e., 98.22%). the type of tm known to the respondents were ; medicinal herbs (55.7%), traditional bone setting (18.8%), cauterization (12.23%), spiritual or faith healing (7%), animal or mineral product (4.8%) and traditional birth attendant (1.53%). the majority of the people got information from informal sources (family, friend, etc.,) 80%, followed by mass media (tv or radio) 17.85% and newspaper or magazine 1.23%. about half of the respondents, 40.79% were aware of the major side - effects of tm, such as diarrhea (36.64%), vomiting (20.5%), abdominal pain (14.91%), and the remaining 59.21% were not aware of the side effects of tm [table 3 ]. attitude to tm therapy by the respondents of burka jato kebele, west ethiopia, february 2013 based on the rate of curing illness ; percentage of people who selected modern medicine were 73.65%, both modern and tm, 21.7%, tm alone 3.97% and those who did not know 0.72%. the majority of the respondents did not believe that tmps could cure diseases better than (mm) 78.7% and the remaining 21.3% could believe that tm could cure illness better than mm : such as ; hemorrhoids 49.15%, asthma 16.95%, rabies 6.8%, jaundice 16.95%, bone fracture 8.47% and toothache 6.8%. about 67.87% of the respondents preferred a modern doctor, 26.71% both modern doctor and tmp and the remaining 5.41% prefer tmp. figure 1 shows the various reasons of respondents for preferring tm to mm and they were : cheap 31.85%, effective 26.3%, accessible 24.07%, acceptable 16.3% and delay in hospital 1.5%. percentage distribution of respondents on reasons for preferring traditional medicine in burka jato kebele, west ethiopia, february 2013 the percentage of respondents who suggest the tm should not be integrated with mm, but should be replaced by mm are ; 9.7% and 22.02%, respectively. the percentage of respondents who suggest the tm should not replace by mm, but should be integrated are, 77.98% and 90.3% respectively [table 4 ]. practice of tm by burka jato kebele, west ethiopia majority of burka jato people 94.22% had practiced tm at least in the last 2 years and the remaining 5.78% did not practice tm which is shown in figure 2 below. percentage distribution of respondents on practice of traditional medicine in burka jato kebele, west ethiopia, february 2013 as shown in table 4, tm was most commonly given to elder 32.62%, children 24.11%, adult 17.02%, pregnant women, 2.13% and the remaining 24.11% did not know for whom tm was most commonly given. half of those people who practiced tm in the last 2 years, advised sick person to go first for tm 49.82% and the remaining 50.18% did not advice sick person to go first for tm. as depicted in table 5 people who practiced tm in the last 2 years, those who manage acute or chronic illness by themselves (self - medication) were 50.57%, those who visited traditional healers 21.07% and those who practiced both (i.e. self - medication and visit tmp) were 28.35%. there were different type of illness that were managed by tm, among them cough 38.24% was the leading one. type of tm used ; medicinal herbs 70.57%, bone setting 16%, animal or mineral product 11.43% and spiritual faith healing 2%. management of tm by burka jato kebele, west ethiopia the majority of the people did not select time to collect or use tm accounts for 86.24% and the remaining 13.76% had time selection to collect or use tm. the containers where they store tm were : bottles 43.27%, pieces of cloth 16.98%, leaves 10.57%, paper 1.92%, cup of tea 0.96% and the remaining 26.3% do not store it (i.e. for single use only). most of the species (50%) were harvested for their leaves to prepare remedies. the principal methods of remedy preparation were reported through pounding (27.54%), crushing (18.84%), a concoction (15.95%) and squeezing (13.04%) of the various parts of medicinal plants. about 53.84% of the medicinal species were cited to be used in fresh forming remedy preparations. relatively 42.31% medicinal plant species were reported to be used in dried form and 3.84% in fresh or dried. remedies were reported to be administered through oral (53.85%), dermal or topical (36.54%), buccal (3.85%) and anal (5.77%) which are shown in table 5. among the cited medicinal plant species of the study area, the majority (65.35%) were wild or cultivated. there was no relationship or association between respondents age, educational status and the practice of tm (tables 6 and 7) respectively, as all tests of association were not statistically significant p > 0.05. however, there was association between income and practice of tm. as all tests of association was statistically significant p < 0.05 [table 8 ]. this is because the majority of the people who practice tm, prefer it due to its cost (cheap : 31.85%), since tm is very cheap compared to modern medicine. about twenty eight different medicinal plants, six animal products and a mineral product were found to be used traditionally for treatment and/or management of various ailments by the study subjects in burka jato kebele, west ethiopia [tables 9 and 10 ]. association between age and practice of tm association between educational status and practice of tm association between income and practice of tm herbal medicines used in traditional medicine among the people in burka jato kebele, west ethiopia, feburary 2013 some animal products and minerals used in traditional medicine among the people in burka jato kebele, west ethiopia, feburary 2013 due to poor access to health services, especially in the rural areas, the majority of the ethiopian people rely mainly on tm for the system of their primary health care needs. as there is no adequate study done in burka jato kebele and also in other regional kebeles in ethiopia, regarding tm knowledge, practice, management as well as their attitudes, it is hoped that findings from this study will also initiate different stake holders to get involved in this area and it will be helpful as a baseline for other studies that will be conducted in the future. it will be of great importance to plan for control measures against any health hazards from abuse of alternative medical therapy. therefore, it would be of important value to assess the knowledge, attitude, practice and management of tm among the community. tm and associated knowledge in the study area, one of the alternatives for the solution of health problem, rises in a large segment of the rural population are employing tm in general and medicinal plant in particular. however, documentation of this indigenous knowledge of healing system still remains at minimum level. the present study reported that the majority 98.22% of the respondents were aware of an alternative way of getting treatment for their ailments apart from modern medicine. the forms of alternative medical therapies (amt) respondents were aware of include ; medicinal herbs (55.7%), and traditional bone settings (18.8%) among others similar to the research conducted in nigeria ; 90.4% of the respondents were aware of alternative way of getting treatment, medicinal herbs and traditional bone settings were common among other forms of amt. the major source of information regarding tm were through : informal sources (friends, family) 80%, which was higher than the research done at arsi zone (56.2%), east ethiopia, this might be due to lack of documentation or written standards and information concerning tm as it was transmitted orally from generation to generation. about 40.79% of the respondents were aware of the side effects from tm and these include diarrhea (36.67%), vomiting (20.5%) and abdominal pain (14.91%), which is similar to the research conducted in nigeria, where 54.9% of the respondents were aware of side effects, diarrhea 69.7%, vomiting 40.2% and abdominal pain 42.2%. only 3.97% of all the respondents who were aware of amt believed that it could cure all forms of illness better than modern medicine (73.65%) and the remaining 21.7% respondents believed in both traditional and modern medicine. this is slightly lower than the research conducted in nigeria, about 42% of all respondents believed in amt. notwithstanding their knowledge of side effects and injuries from tm, about 21.3% of the respondents preferred tm to modern medicine which is similar to the research reported by elujoba.. the reasons for preference of tm might be it is ; cheap (31.85%), effective (26.3%), accessible (24.07%), acceptable (16.3%) and delay in hospital (1.5%). this is similar to the research conducted in nigeria ; cheap (21.4%), accessible (16.4%) and acceptable (13.4%). more than half (77.98%) of respondents do not prefer a replacement of tm by modern medicine. among this 90.3% prefer the integration of tm to modern medicine which correlates to the research done at arsi zone (east ethiopia), 83.8% of the people are advocating for integration of tm into the modern medicine. the percentage of respondents who practiced tm in the last 2 years was, 94.22%, which correlates to the research reported by who, where 80% ethiopian people practice or use tm. half of those respondents who practiced tm (49.82%), advised sick people to go to first for traditional medical practitioners which is higher than the research reported by elujoba. : this might be because tm is intricately interwoven with the culture, socioeconomic and social cultural heritages of the respondents. the ailments most commonly managed by the respondents were cough (38.24%) and diarrhea (16.86%) among others. this is similar to the research reported in bench ethnic group (south west ethiopia) by giday. the local community mostly used leaves (50%) for the preparation of remedies and the root takes the second proportion 13.46%. certain ethno botanical research in another part of the country reported similar result leaves (64.52%) followed by roots (19.35%), to be mostly used in the treatment of various health problems. the majority of the harvested medicinal plants (65.38%) were wild, which is correlated to the research reported in bench ethnic group by giday. about 53.84% of the medicinal plant species were cited to be used in fresh form for remedy preparations and followed by dried (42.31%), which correlates to the research reported by yineger and yewhalaw, where 64.52% of the medicinal plant species were in fresh form. oral and topical (dermal) were the major routes of administration of plant remedies. accordingly 53.85% of the preparations were taken orally followed by topical or dermal (36.54%). similarly yirga and zeraburk, have reported oral route (67.3%) and topical (30.6%) of application. a study conducted in bench ethnic group indicated that some tm were used against the following eight human ailments : gastro - intestinal complaints (14%), ear diseases (11%), deformation of fingers (9%), inflammation (6%), toothache (6%) rheumatic pain (3%), rabies (3%) and jaundice (3%). the majority (86%) of medicinal plants used by bench ethnic group were uncultivated species, most of them were weeds and abundantly growing in disturbed habitats, mainly in crop fields, fallow lands and along hedgerows. a cross - sectional study conducted in arsi zone indicated that the local community acquired knowledge about tm from relatives accounted for (56.2%), traditional healers (30%), by themselves (3.7%), religious books (2.5%).those who obtained knowledge from their relatives, expressed willingness to convey their knowledge, and preferred modern medicine accounted for 56.2%, 78.7% and 77.5%, respectively. according to this study, 83.8% of respondents supported the integration of tm with modern health service system and the remaining 12.3% did not support and few (3.8%) had no opinion. this study revealed that the use of tm is quite popular among the respondents and a large proportion of them not only preferred but also used tm not withstanding that they live around urban communities with better access to modern medical care and medical practitioners. since safety and efficacy of the remedies used in tm remain largely unknown and untested to have met the gold standard therapy, advising patients and the general public who use or seek tm presents a professional challenge. furthermore, local leaders, government and other stake holders in the sector should regulate the activities of the alternative medical therapists or practitioners and find a way of fully integrating their practices into the modern medicine. regulations should be made concerning the advertisement of alternative medicine and practices since appropriate diagnosis were usually not made for the use of these drugs and respondents were usually not aware of the requirements. | background : traditional medicine (tm) has maintained its popularity in all regions of the developing world. even though, the wide acceptance of tm is a well - established fact, its status in a population with access to modern health is not well clear in the whole country. this study was carried out to assess the knowledge, attitudes, practice and management of tm among the community of burka jato kebele, west ethiopia.methodology:a descriptive cross - sectional study was conducted on a total of 282 sampled individuals selected using systematic random sampling from january 28, 2013 to february 8, 2013 in burka jato kebele, nekemte town, east wollega zone, west of ethiopia.results:the majority (94.22%) of people in the study area relied on tm. most of them were aware of medicinal herbs (55.7%). about half (40.79%) of the respondents were aware of the major side - effects of tm such as diarrhea (36.64%). about 31.85% of them prefer traditional medical practices (tmp) because they are cheap. most (50%) of the species were harvested for their leaves to prepare remedies, followed by seed (21.15%) and root (13.46%) and the methods of preparation were pounding (27.54%), crushing (18.84%), a concoction (15.95%) and squeezing (13.04%). about 53.84% of them were used as fresh preparations. remedies were reported to be administered through oral (53.85%), dermal or topical (36.54%), buccal (3.85%) and anal (5.77%).conclusion : the study revealed that the use of tms were quite popular among the population and a large proportion of the respondents not only preferred, but also used tms notwithstanding that they lived in the urban communities with better access to modern medical care and medical practitioners. to use tm as a valuable alternative to conventional western medicine, further investigation must be undertaken to determine the validity, efficacy of the plants to make it available as an alternative medicine to human beings. |
consecutive patients > 2 years of age with fever (> 38c tympanic) who came to teaching hospital karapitiya were enrolled (8). standardized epidemiologic and clinical data and blood were obtained during acute illness and 24 weeks later. during the study (march october 2007), the atmospheric temperature ranged from 27.5c32c (high) to 24c26c (low), and rainfall was variable (mean 301 mm / mo, range 36657 mm / mo). because rickettsial species broadly cross - react within groups (9,10), paired serum samples were tested by using an igg indirect immunofluorescence assay (ifa) and rickettsia rickettsii and r. typhi antigens (focus diagnostics, cypress, ca, usa) to identify infections with spotted fever group (sfgr) and typhus group (tgr) rickettsial infections. serum samples reactive at a titer of 80 were considered potentially positive and were titered. to identify scrub typhus (st) infections, we tested paired serum samples using igg elisa as described (11), except for use of recombinant antigens (0.2 g each of r56 chimeric1, gilliam, and kato strains) to detect antibodies to orientia tsutsugamushi. comparative blind testing of 200 serum samples with an established (pooled - antigen) quantitative assay enabled validation (12). acute rickettsioses (sfgr, tgr, and st) required a > 4-fold rise in specific igg titer or its equivalent ; patients with equal sfgr and tgr convalescent - phase titers were sfgr / tgr group - indeterminate. igg (titer > 160) in acute - phase serum defined rickettsial exposure (seroprevalence). stata ic version 11.0 (statacorp lp, college station, tx, usa) was used for analyses. we analyzed paired serum samples for rickettsioses for 883 (81.9%) of 1,079 patients. median acute convalescent phase follow - up was 21 days (intraquartile range 1533 days). acute rickettsioses were documented in 156 (17.7%) patients (table 1). the increase in convalescent - phase geometric mean titer was 14-fold (845) for sfgr, 17-fold (920) for tgr, and 11-fold (951) for sfgr / tgr rickettsiae. sfgr, spotted fever group rickettsiae ; tgr, typhus group rickettsiae ; iqr, intraquartile range ; anc, absolute neutrophil count ; alc, absolute lymphocyte count. n = 309 ; 6/6 with rickettsioses and rash had eschars, compared with 11/22 of those without rickettsioses (p 18 years of age and 10.5% of patients 160 at either sampling were used to denote rickettsial exposure (preexisting or acute), 342 (38.7%) patients had exposure to sfgr and/or tgr rickettsiae. if a lower titer (> 80) were used, 398 (45.1%) had such exposure. similarly, 121 (13.7%) patients had either acute- or convalescent - phase igg against o. tsutsugamushi. sfgr, spotted fever group rickettsiae ; tgr, typhus group rickettsiae ; iqr, interquartile range. we documented endemic rickettsioses as a major cause of acute febrile illness in southern sri lanka. underrecognition of rickettsioses could reflect nonspecific clinical features, limited diagnostic tools, lack of awareness that rickettsioses occur or cause severe illness, and absence of evidence - based local algorithms for acute febrile illness. studies in neighboring regions have used flawed methods, including spectrum bias, small sample size, selective enrollment, and testing acute - phase serum only. in many instances, clinical features were not predictive (5,13) ; however, older age was reported with rickettsioses (14) and lymphadenopathy and rapid respiratory rate with st compared with tgr rickettsiosis (4). although part of the rickettsial triad, rash is often absent initially (10) and among unselected febrile patients (5,14). sfgr / tgr cross - reactions and apparent co - infections could also impair clinical differentiation of specific rickettsioses. laboratory abnormalities associated with st (3) could reflect disease severity, not etiology, and such testing is often unavailable. divergent conclusions might reflect different study populations, diagnostic criteria, reference groups, features evaluated, or real geographic differences. confirmation required follow - up (return or home visit) ; the us centers for disease control and prevention no longer accepts a single high titer to confirm r. rickettsii infection (10). we required a 4-fold rise in titer even for seroconversions because ifa results are subjective, even among experts. we used r. rickettsii as a surrogate antigen, which could be less sensitive for detecting local sfgr. we chose elisa for st because a commercial ifa was not available and ifa as a standard for st has been questioned (15). optimal clinical management of acute rickettsioses requires development of locally tested, evidence - based algorithms for acute febrile illness. | we studied rickettsioses in southern sri lanka. of 883 febrile patients with paired serum samples, 156 (17.7%) had acute rickettsioses ; rickettsioses were unsuspected at presentation. additionally, 342 (38.7%) had exposure to spotted fever and/or typhus group rickettsioses and 121 (13.7%) scrub typhus. increased awareness of rickettsioses and better tests are needed. |
neoplasms that arise in salivary gland are rare and yet they represent a wide variety of benign and malignant histological subtypes. salivary gland neoplasms make up 1% of all head and neck tumors. among these, 80% arise in parotid gland, 10 - 15% in submandibular gland and the remainder in sublingual and minor salivary glands. oncocytes are most abundant in the parotid gland ; they are also found in other major and minor salivary glands as well as in the larynx, trachea, bronchi, oesophagus, nasal mucous membranes, thyroid, parathyroid, pancreas, liver, and stomach. although focal oncocytic features are seen in a wide variety of salivary neoplasms, pure oncocytic carcinoma of salivary gland origin is rare. most cases of oncocytic carcinoma have occurred in the parotid glands, but recent reports have described tumors that involved the submandibular gland and minor glands of the palate, nasal cavity, and ethmoid and maxillary sinuses. we describe a new case of oncocytic carcinoma that arose in the minor salivary gland and metastasized to lymph nodes. a 43-year - old female presented with progressively increasing painless swelling on the lower lip for 25 days. physical examination revealed a fixed hard 3 3 cm mass on the lower lip. these cells had well - defined cell borders, round to oval, central to eccentrically located moderately pleomorphic nuclei with fine chromatin, prominent nucleoli and abundant eosinophilic granular cytoplasm. surgical excision of the swelling was done and the specimen was submitted for histopathological examination. gross examination revealed a well - circumscribed, firm, grey white tumor measuring 3 cm in diameter. microscopically, a neoplasm with infiltrative growth pattern consisting of large, round to polyhedral cells in solid sheets, islands and cords with abundant eosinophilic granular cytoplasm was seen. biotin immunohistochemical study was performed on paraffin - block sections by the standard labelled method. the primary antibody was a mouse monoclonal antihuman mitochondrial antibody (dako - lsab kit, k0675). after 5 months, the patient again presented with bilateral submandibular and right axillary lymphadenopathy. fna was done from these lymph nodes and the smears prepared revealed similar morphology suggesting metastatic deposits from oncocytic carcinoma [figure 1a d ]. (a) fna from swelling lip showing pleomorphic oncocytic cells (mgg, 200). (b) cytoplasmic positivity revealed by the oncocytic cells using antimitochondrial antibody (ihc, 400). (c) oncocytic carcinoma : photomicrograph showing mitosis (h and e, 400). (d) fna from submandibular lymph node showing metastatic deposits from oncocytic carcinoma (mgg, 400) gross examination revealed a well - circumscribed, firm, grey white tumor measuring 3 cm in diameter. microscopically, a neoplasm with infiltrative growth pattern consisting of large, round to polyhedral cells in solid sheets, islands and cords with abundant eosinophilic granular cytoplasm was seen. biotin immunohistochemical study was performed on paraffin - block sections by the standard labelled method. the primary antibody was a mouse monoclonal antihuman mitochondrial antibody (dako - lsab kit, k0675). after 5 months, the patient again presented with bilateral submandibular and right axillary lymphadenopathy. fna was done from these lymph nodes and the smears prepared revealed similar morphology suggesting metastatic deposits from oncocytic carcinoma [figure 1a d ]. (a) fna from swelling lip showing pleomorphic oncocytic cells (mgg, 200). (b) cytoplasmic positivity revealed by the oncocytic cells using antimitochondrial antibody (ihc, 400). (c) oncocytic carcinoma : photomicrograph showing mitosis (h and e, 400). (d) fna from submandibular lymph node showing metastatic deposits from oncocytic carcinoma (mgg, 400) oncocytic carcinoma of salivary gland origin is rare and rarest when it arises in minor salivary gland. most cases of oncocytic carcinoma have occurred in parotid glands, and some have been described in submandibular gland and minor salivary glands. the terms oncocytic carcinoma, malignant oncocytoma, oncocytic adenocarcinoma and malignant oxyphilic adenoma are synonymous. they have been estimated to represent only 5% of all oncocytic neoplasms and 0.0005% of all salivary gland tumors. they are characterized by oncocytes with marked cellular atypia, frequent mitosis, destruction of adjacent organs, perineural or vascular invasion and distant or regional lymph node metastasis. various benign and malignant salivary gland neoplasms may have foci of oncocytic cells, but the oncocytic component usually comprises such a small portion that is unlikely to be confused with oncocytic carcinoma. oncocytosis is non - neoplastic proliferation of oncocytes that can be differentiated from oncocytic carcinoma by the presence of multiple foci of oncocytic cells within salivary gland lobules without altering the normal architecture. other neoplasms that arise from salivary gland with a granular cytoplasm are oncocytoma, acinic cell carcinoma and salivary duct carcinoma. oncocytoma can be differentiated by the presence of a well - differentiated connective tissue capsule. moreover, compared to oncocytoma, oncocytic carcinoma shows greater nuclear pleomorphism and mitotic activity. malignant nature of the neoplasm can be recognized by its morphological features and infiltrative growth pattern. acinic cell carcinoma can be differentiated from oncocytic carcinoma by the presence of amphophilic or basophilic granules in the cytoplasm of tumor cells and their predominant microcystic and papillary growth pattern. salivary duct carcinoma, in contrast, forms duct like spaces with papillary and cribriform growth pattern and also shows comedo necrosis. in addition, immunohistochemically, oncocytic cells show positivity for mitochondrial antigen, keratin and alpha-1-antichymotrypsin. shintaku and honda reported that immunohistochemistry with antimitochondrial antibody is highly sensitive and specific for light microscopic identification of mitochondria and superior to routine hande staining, especially for the detection of isolated oncocytic cells. in this patient, the immunohistochemical study with antimitochondrial antibody revealed intense immunoreactivity of the cytoplasm in the tumor cells. despite being described five decades ago the biological behavior can not be evaluated fully because of the paucity of cases reported and the lack of follow - up information. however, the main treatment modality is surgery with or without adjuvant radiotherapy. goode and corio reported that tumors smaller than 2 cm in diameter appeared to be associated with better prognosis than those that were larger. presence of distant rather than local lymph node metastasis is the most important prognostic indicator. however, clinical course and prognosis of patients with oncocytic carcinoma remains to be evaluated as more cases are being reported. | oncocytic carcinoma is an extremely rare neoplasm of the salivary gland, with only a few cases reported in literature till date. we report the occurrence of this rare lesion in lip in a 43-year - old female presenting with a progressively increasing swelling for which excision was done. fine needle aspiration was done and the smears revealed tumor cells with well - defined cell borders, round to oval, central to eccentrically located moderately pleomorphic nuclei with fine chromatin, prominent nucleoli and abundant eosinophilic granular cytoplasm. microscopic examination of the resected tumor showed solid sheets, nests, islands and cords of oncocytic cells diffusely infiltrating the surrounding tissues. after 5 months, the patient again presented with bilateral submandibular and right axillary lymphadenopathy revealing metastatic deposits from oncocytic carcinoma. we report this case of oncocytic carcinoma because of its unusual location, the minor salivary gland of lip being a rare site for the tumor. |
fasciola hepatica is the causative agent of liver fluke disease in sheep and cattle and has worldwide distribution especially in the temperate areas (1 - 4). furthermore, fasciolosis is a foodborne zoonotic disease in rural areas of developing countries such as bolivia, peru, equador, egypt and iran (5). f. hepatica larvae penetrate and traverse the intestinal wall, enter the liver capsule, migrate through the parenchyma and cause extensive tissue damage. protease enzymes have an important role in the infectivity and adaptation of fasciola in a wide variety of their hosts (7, 8). these enzymes are stored in secretary vesicles of the gastrodermal epithelial cells as inactive proenzyme. activation of the proenzyme takes place following secretion into the acidic environment of parasite gut lumen where are needed for food digestion (9). protease enzymes are secreted by all stages of the developing parasites and are essentially required for various mechanisms necessary for parasitism (10). release a number of cysteine proteases during their life cycle, including cathepsin l and b proteases (11). cathepsin protease secreted by the parasite plays a critical role in invasion, migration and survival by cleaving interstitial matrix proteins such as fibronectin, laminin and native collagen, catabolism of host proteins to absorbable peptides and amino acids, and modulation of the host immune response by cleaving immunoglobulin or by altering the activity of immune effecter cells. liver fluke cathepsin l cytosine proteinases are reported as sensitive and specific markers for the diagnosis of human and animal fasciolosis. a number of fasciolicides including closantel, brotianid, oxyclozanide, clorsulon, rafoxanide, nitroxynil, diamphenethide, mebendazole, albendazole and triclabendazole have been available for the treatment of animal fasciolosis. tcbz is a benzimidazole derivative, which have high efficacy against both mature and juvenile stages of fasciola spp., so it is considered as a drug of choice for treatment of liver fluke infections and human fascioliasis. resistance to tcbz could severely compromise its future use (13, 14). in the present study, we have examined the effect of tcbz on protease enzymes activities of esp of f. hepatica in vitro. f. hepatica parasites were obtained from liver of cattle at a local abattoir (ehsane - rey, tehran, iran). for removing host materials and emptying of the parasite gut, flukes were washed 6 times (1 h) with washing buffer [phosphate buffer saline (pbs) : 0.15 m, ph 7.2 ]. then, parasites were cultivated for 6 hours in fresh rpmi 1640 (gibco 51800 - 019) with additional 10 m penestrep. tcbz (egaten 250 mg tablet ; novartis ; switzerland) was initially prepared as a stock solution in dimethyl sulphoxide (dmso) and 0.6 l added to the treated culture medium (containing 15 g / ml concentration tcbz). in addition, control flukes culture medium was treated with 0.6 l dmso. following incubation, so, suspensions were centrifuged at 4 c, 13000g for 10 min and were stored at -20c until required (15 - 18). the concentration of total proteins of esp samples were measured according to bradford assay method which involves reacting the esp sample with a dye that binds proteins. to measure of protein concentration, standard solutions (bovine serum albumin, merk germany) and the absorbance of esp samples and standard solutions were measured at 595 nm after 10 min incubation at room temperature. a standard curve was prepared using the standard solutions absorbance and the protein concentrations of samples were estimated (18). proteases activity of esp samples was determined by using sigma s non - specific protease activity assay method. when the protease digests casein as substrate, the amino acid tyrosine is released along with other amino acids. briefly, casein solution prepared and incubated in 37 c for 5 min, then esp sample was added to treated tube and incubated for 10 min. trichloroacetic acid (tca) added to the treated and control tubes and the reaction was stopped. esp sample were added to control tubes and incubated for 30min, and then centrifuged for 5 min at 13000g at 25 c. finally, the supernatant was poured into the tube and added sodium carbonate solution and ciocalteus phenol, and was incubated for 30 min at 37 c. then, tubes were centrifuged (as above) and tyrosine residues were measured spectrophotometer at 660 nm. the protease activities were compared to a standard curve and reported as units /ml (19). sodium doddery sulfate polyacrylamide gel electrophoresis (sds - page) and coomassie blue staining were used to separate and stain the components of esp sample proteins. esp samples (30 g / ml protein concentrations) were mixed with sample buffer and were run on 10% acrylamide gels. molecular weight of sample proteins in treated samples and controls were compared with respect to the marker (18). independent t - test was performed to compare mean protein concentration and protease enzyme activity between test and control groups (with the confidence interval of 95 %). f. hepatica parasites were obtained from liver of cattle at a local abattoir (ehsane - rey, tehran, iran). for removing host materials and emptying of the parasite gut, flukes were washed 6 times (1 h) with washing buffer [phosphate buffer saline (pbs) : 0.15 m, ph 7.2 ]. then, parasites were cultivated for 6 hours in fresh rpmi 1640 (gibco 51800 - 019) with additional 10 m penestrep. tcbz (egaten 250 mg tablet ; novartis ; switzerland) was initially prepared as a stock solution in dimethyl sulphoxide (dmso) and 0.6 l added to the treated culture medium (containing 15 g / ml concentration tcbz). in addition, control flukes culture medium was treated with 0.6 l dmso. following incubation, so, suspensions were centrifuged at 4 c, 13000g for 10 min and were stored at -20c until required (15 - 18). the concentration of total proteins of esp samples were measured according to bradford assay method which involves reacting the esp sample with a dye that binds proteins. to measure of protein concentration, standard solutions (bovine serum albumin, merk germany) and the absorbance of esp samples and standard solutions were measured at 595 nm after 10 min incubation at room temperature. a standard curve was prepared using the standard solutions absorbance and the protein concentrations of samples were estimated (18). proteases activity of esp samples was determined by using sigma s non - specific protease activity assay method. when the protease digests casein as substrate, the amino acid tyrosine is released along with other amino acids. briefly, casein solution prepared and incubated in 37 c for 5 min, then esp sample was added to treated tube and incubated for 10 min. trichloroacetic acid (tca) added to the treated and control tubes and the reaction was stopped. esp sample were added to control tubes and incubated for 30min, and then centrifuged for 5 min at 13000g at 25 c. finally, the supernatant was poured into the tube and added sodium carbonate solution and ciocalteus phenol, and was incubated for 30 min at 37 c. then, tubes were centrifuged (as above) and tyrosine residues were measured spectrophotometer at 660 nm. the protease activities were compared to a standard curve and reported as units /ml (19). sodium doddery sulfate polyacrylamide gel electrophoresis (sds - page) and coomassie blue staining were used to separate and stain the components of esp sample proteins. esp samples (30 g / ml protein concentrations) were mixed with sample buffer and were run on 10% acrylamide gels. molecular weight of sample proteins in treated samples and controls were compared with respect to the marker (18). independent t - test was performed to compare mean protein concentration and protease enzyme activity between test and control groups (with the confidence interval of 95 %). the concentrations of protein in the esp samples are presented in table 1 and 2. mean protein concentrations in control and treated group of f. hepatica esp samples were 196.1 (se = 14.52) and 376.4 (se = 28.20) g / ml respectively. protease activity in the f. hepatica esp samples are presented in table 1 and 2. mean proteases enzyme activities level in control and treated group were 0.370 (se = 0.1) and 0.089 (se = 0.03) u / ml, respectively. according to obtained results, significant difference between control group in comparison to the treated esp samples in protein concentrations [t (18) = 5.84, t - value = 2.1 ] and proteases activities [t (18) = 2.52, t - value = 2.1 ] was observed (p<0.05). protein of esp samples were analyzed by sds - page electrophoresis and are shown in fig 1. there are qualitatively differences in protein bands between sds - page results of treated and control esp samples. protein bands 146, 96 and 77 were observed in treated samples but are not seen in control samples. the concentrations of protein in the esp samples are presented in table 1 and 2. mean protein concentrations in control and treated group of f. hepatica esp samples were 196.1 (se = 14.52) and 376.4 (se = 28.20) g / ml respectively. protease activity in the f. hepatica esp samples are presented in table 1 and 2. mean proteases enzyme activities level in control and treated group were 0.370 (se = 0.1) and 0.089 (se = 0.03) u / ml, respectively. according to obtained results, significant difference between control group in comparison to the treated esp samples in protein concentrations [t (18) = 5.84, t - value = 2.1 ] and proteases activities [t (18) = 2.52, t - value = 2.1 ] was observed (p<0.05). protein of esp samples were analyzed by sds - page electrophoresis and are shown in fig 1. there are qualitatively differences in protein bands between sds - page results of treated and control esp samples. protein bands 146, 96 and 77 were observed in treated samples but are not seen in control samples. in the present study, the effect of tcbz on esp protein of f. hepatica was examined. protein concentrations in treated esp samples were more than control esp samples and show the tegument and viteline cells were probably disrupted by tcbz (20 - 22). since protease enzymes are proteins, reduction prote enzymes activity could be was due to interference of tcbz with protein synthesis (23). in s3, s6 and s8 treated samples, enzyme activity reduction were less than other samples because primary enzyme level of individually parasite was probably higher than others. bennett and kohler showed that the tcbz reduces the secretory protease enzyme and stated that likely tcbz s obvious ability to strongly bind to various kinds of proteins including microtubules and leads to decrease enzyme activity (24). proteases play an important role in the evasion from host immune system. on the one hand, suppresses the th1 lymphocytes and propels the immune system toward th2 cause to prevent the acute phase of illness. on the other hand, cleaving immunoglobulin (25, 26), prevents antibody - mediated eosinophil attachment to newly hatched juveniles (24) and eosinophil apoptosis (27). due to the reduction of protease enzymes by tcbz and with attention to roles of proteases on the immune system can be concluded that one of the effects of tcbz is probably restoring the immune system. hence, proteases can inhibit the immune system and controversially the tcbz reduces the activity of protease enzymes, so resulting reduction in immunosuppression and staying active that can be an important step in eliminating the parasite. then, tcbz treatment has direct effect on the f. hepatica, by destruction of parasite tegument, also reduce of proteases activity and recover immune system indirectly. the results of sds - page gel of f. hepatica esp show protein bands with different molecular weights in the control and treated samples. in the treated samples protein bands with different molecular weights (146, 96 and 77 kda) can be seen without existing in control group. the mentioned protein bands were probably released from parasites due to destruction of tegument, but need to verify by qualitative methods in more researches. tcbz increases protein concentrations, protein bands and reduces the proteases enzyme activities in esp of f. hepatica. the tcbz reduced secretory protease enzymes activities and possibly effects on invasion, migration, nutrition and particularly survival of the parasite in the host tissues. | backgroundfasciola hepatica is one of the most important helminthes parasites and triclabendazole (tcbz) is routinely used for treatment of infected people and animals. secreted protease enzymes by the f. hepatica plays a critical role in the invasion, migration, nutrition and the survival of parasite and are key targets for novel drugs and vaccines. the aim of study was to determine the protease activity of excretory- secretory products (esp) of f. hepatica in the presence of tcbz anthelmintic.methodsf. hepatica helminthes were collected and cultured within rpmi 1640 [tcbz treated (test) and untreated (control) ] for 6 h at 37 c. esp of treated and control were collected, centrifuged and supernatants were stored at -20c. protein concentrations were measured according to bradford method. protease enzymes activities of esp samples were estimated by using sigma s non - specific protease activity assay. esp protein bands were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds - page).resultsmean protein concentrations in control and treated of esp samples were determined 196.1 14.52 and 376.4 28.20 g / ml, respectively. mean protease enzymes activities in control and treated were 0.37 0.1 and 0.089 0.03 u / ml, respectively. significant difference between proteins concentrations and protease enzymes activities of two groups was observed (p<0.05). sds - page showed different patterns of protein bands between treated and control samples.conclusionthe tcbz reduced secreted protease enzymes activities and possibly effects on invasion, migration, nutrition and particularly survival of the parasite in the host tissues. |
environmental, genetic, and immunologic factors are involved in the pathogenesis of celiac disease (cd), a chronic inflammatory disease characterised by flattened villi on the small bowel mucosa. cd is induced by the ingestion of cereals containing proline - rich and glutamine - rich proteins (such as wheat, rye, and barley) in genetically susceptible individuals [2, 3 ]. from a clinical point of view, cd is characterised by a clinical heterogeneity that ranges from asymptomatic to severely symptomatic and by increased morbidity and mortality. at present, the only proven treatment for cd is strict and life - long adherence to a gluten - free diet (gfd) [14 ]. an increase of markers of oxidative damage of lipids (thiobarbituric acid - reactive substances and lipid hydroperoxides) and proteins (carbonyl groups) and a decrease of antioxidant enzymes have been demonstrated in plasma, in circulating cells and in intestinal cells of patients with cd with respect to controls [58 ]. higher levels of nitric oxide (no) and biochemical markers of oxidative damage of dna has been demonstrated in leukocytes and in urine samples of celiac children [9, 10 ], suggesting that oxidative stress could be involved in the pathogenesis of cd. paraoxonase-1 (pon1) is an enzyme associated with the high - density lipoproteins (hdl) that plays both an antioxidant and an anti - inflammatory role [1113 ]. a decrease in pon1 has been reported in several human diseases associated with inflammation and alteration of lipoprotein metabolism [1417 ]. interest in the study of pon1 in celiac disease is supported by recent studies that have suggested a possible role of pon1 in inflammatory intestinal diseases [1820 ]. furthermore it has been hypothesised that in the gastrointestinal tract, pon1 could behave as a local detoxifier, antioxidant, immunomodulator, and/or quorum - quenching factor. to further investigate the relationship between oxidative damage and cd, we studied the levels of lipid peroxides, total antioxidant capacity, the susceptibility to copper - induced lipid peroxidation, and the activity of the enzyme paraoxonase-1 (pon1) in serum of three groups of subjects : untreated patients with a new diagnosis of celiac disease (cd patients), cd patients on a gluten - free diet (gfd patients), and healthy subjects. the study included 27 consecutive patients who were referred to the celiac disease clinic of the department of gastroenterology of the polytechnic university of marche, and the study protocol was in accordance with the ethical standards formulated in helsinki declaration as revised in 2000. eleven patients (3 males and 8 females, mean age 31.2 11.7 years) had a new diagnosis of cd (cd patients), whereas 16 (gfd patients ; 7 males and 9 females, mean age 35.8 12.1 years) were celiac patients on a gluten - free diet (mean duration : 19.2 5.7 months). twenty - five subjects (12 males and 13 females, mean age 39.6 8.1 years) who underwent esophagogastroduodenoscopy for the presence of dyspeptic syndrome and who did not result to be affected by cd were used as controls. subjects with diabetes and clinical evidence of cardiovascular diseases or receiving antacids, lipid - lowering drugs, or antioxidant supplements were excluded from the study to avoid possible interferences on pon1 activity and plasma lipids. all subjects included in the study underwent upper gastrointestinal endoscopy and at least 46 well - oriented duodenal specimens were taken for the histological evaluation. thereafter, each stage was given a score from 0 (normal mucosa) to 5 (total villous atrophy) for statistical analysis. serum levels of antitransglutaminase(t - tg) antibodies were assessed by the laboratory of our institution by using a commercial fluoroenzymeimmuno assay kit (phadia, sweden) and the results were expressed as unit (u)/ml of serum. a value higher than 10 u / ml was considered positive. in the same day of endoscopy, blood drawn by venous puncture by fasted subjects was collected into serum - separator tubes. after clotting, blood was centrifuged for 15 min at 3500 rpm to separate serum that was stored at 80c until examination. pon1 activity has been evaluated using three different substrates : paraoxon for paraoxonase activity, phenylacetate for arylesterase activity, and dihydrocoumarin for lactonase activity. all assays were performed in a 96-well plate, in a total reaction volume of 200 l. the basal assay mixture included 5 mm tris - hcl, ph 7.4 containing 0.15 m nacl, 4 mm mgcl2, 2 mm cacl2, and 1.0 mmol / l paraoxon. paraoxon hydrolysis was spectrophotometrically monitored for 8 min (every 15 s) at 412 nm. one unit of pon1 paraoxonase activity was equivalent to 1 nmol of paraoxon hydrolyzed / min / ml. the serum samples were diluted 1 : 10 with 1 mmol / l cacl2 in 50 mmol / l tris hcl, ph 8.0 and then, 5 l was taken for a total reaction volume of 200 l. after addition of the substrate phenyl acetate (1.0 mmol / l), the hydrolysis was monitored at 270 nm for 3 min (every 15 s). one unit of arylesterase activity was equivalent to 1 mol of phenyl acetate hydrolyzed / min / ml. the serum samples were diluted 1 : 10 with 1 mmol / l cacl2 in 50 mmol / l tris hcl, ph 8.0 and 3 l was then taken for the assay. after addition of the substrate dhc (1.0 mmol / l), the hydrolysis was monitored at 270 nm for 10 min (every 15 s). one unit of lactonase activity was equivalent to 1 mol of dhc hydrolyzed / min / ml. basal serum oxidative status was determined by the evaluation of the levels of lipid hydroperoxides using fox2 assay. briefly, an aliquot (200 l) of serum was mixed with 1800 l of fox2-reagent (250 mol / l ammonium ferrous sulphate, 100 mol / l xylenol - orange, 25 mmol / l h2so4 and 4 mmol / l bht in 90% methanol (v / v) in 100 ml). after incubation at room temperature for 30 minutes, samples were centrifuged at 4000 rpm for 10 minutes. the supernatant was carefully decanted into a cuvette and the absorbance was determined at 560 nm. the levels of lipid hydroperoxides were quantified using a stock solution of t - butyl hydroperoxide. serum samples were diluted 4x with phosphate - buffered saline (pbs - citrate) and were incubated with 50 serum lipid peroxidation was monitored by following the kinetic of conjugated dienes formation at 234 nm. a kc4 software was used to calculate the lag times of kinetic of conjugated dienes formation. the lag time (t - lag, min), defined as the intercept of the straight lines derived from the lag phase and the propagation phase, is an index for resistance of serum to lipid peroxidation. as previously demonstrated longer lag time indicates a lower susceptibility of serum to lipid peroxidation. serum total antioxidant capacity (tac) was assessed using the oxygen radical absorbance capacity (orac) assay described by ou.. the oxygen radical absorbance capacity of serum employs the oxidative loss of the intrinsic fluorescence of fluorescein induced by the free radical initiator 2,2-azobis(2-amidinopropane)hydrochloride (aaph). pon1 activities were presented as medians. for the comparison of normally distributed variables between groups, student 's t - test was used. paraoxonase-1 activity showed a non - gaussian distribution ; therefore we used a nonparametric test (wilcoxon rank sum test). spearman 's correlation coefficient was used to test the strength of any association between different variables. all tests were 2-tailed and a p gfd patients > cd patients. the differences between both groups of cd patients compared with controls were statistically significant (table 2). the median values of lactonase and arylesterase activities of pon1 in serum of cd and gfd patients were significantly lower with respect to controls (table 2). pon1 paraoxonase activity was significantly correlated with arylesterase (r = 0.871, n = 52, p < 0.0001) and lactonase (r = 0.875, n = 52, p < 0.0001) activities. the levels of lipid hydroperoxides in control subjects were 2.02 0.76 mol / l. higher levels of lipid hydroperoxides were observed in serum of cd and gfd patients with respect to controls. in serum of gfd patients, the values were significantly lower compared with untreated cd patients (table 2). to investigate serum susceptibility to lipid peroxidation in celiac patients, we evaluated the formation the conjugated dienes in serum oxidized in vitro with copper ions. the mean value of the lag time of the kinetic of lipid peroxidation in serum of controls was 66.4 16.2 minutes (table 2). in gfd patients the lag time was significantly higher with respect to cd patients (table 2). as summarized in table 2, in gfd patients serum tac was lower than controls and significantly higher than that of cd patients. to study whether the activity of paraoxonase-1 and markers of oxidative stress were related to severity of cd, we compared the mean values of serum levels of anti - t - tg and the degree of mucosal damage (following marsh index) in two subgroups of celiac patients. the first group included 14 cd patients having pon1 paraoxonase activity below the median value of pon1 observed in all celiac patients (124.6 u / ml) (10 untreated cd and 4 gfd patients). the second group included 13 patients with pon1 activity above the median value (1 untreated cd patient and 12 gfd patients). as shown in figure 1, the mean levels of serum anti - t - tg and the degree of mucosal damage were significantly higher in celiac patients with serum pon1 activity below the median value with respect to patients with pon1 activity above the median value. similar results were observed also for pon1 arylesterase and lactonase activities (data not shown). the individual values of pon1-paraoxonase activity and the levels of serum hydroperoxides were negatively correlated (r = 0.735, n = 52, p < 0.0001), in agreement with our previous studies (figure 2(a)). in the whole group of subjects included in the study, the enzyme activity was positively correlated with the lag time of kinetic of lipid peroxidation (r = 0.811, n = 52, p < 0.0001) (figure 2(b)) and with the values of total antioxidant capacity (r = 0.74, n = 52, p < 0.0001) (figure 2(c)). levels of lipid hydroperoxides were negatively correlated with total antioxidant capacity (r = 0.52, n = 52, p < 0.0001) and lag - time values (r = 0.59, n = 52, p < 0.0001). no significant correlation was observed between pon1 activity and hdl cholesterol levels (data not shown). the clinical interest in human pon1 has been mainly focused on atherosclerosis and cardiovascular disease [14, 26 ], since pon1 exerts antioxidant and anti - inflammatory roles [1416 ]. however, more recently a possible role of pon1 in the human small intestine in normal and pathological conditions has been suggested [1820 ]. although pon1 paraoxonase activity has been manly investigated in normal and pathological conditions using paraoxon as substrate [1517 ], we evaluated also pon1 arylesterase and lactonase activities because previous studies have suggested that serum arylesterase activity could reflect serum enzyme concentration and lactonase activity could represent a more physiological activity of the enzyme. our results demonstrated, for the first time, a significant decrease of pon1 activities (paraoxonase, arylesterase, and lactonase) in serum of celiac patients. the modifications of pon1 activities in celiac patients were related with the severity of the disease and were associated with an increase of lipid hydroperoxide levels and with a higher susceptibility of serum to lipid peroxidation induced in vitro. moreover, a lower serum total antioxidant capacity (tac) has been shown in celiac patients. the values of pon1 activity, markers of lipid peroxidation, and total antioxidant capacity in gfd patients were significantly different compared with healthy subjects and untreated cd patients. our results confirm that cd is associated with oxidative damage as suggested in previous studies mainly carried out in cd children [6, 810 ]. some hypotheses can be made to explain the molecular mechanisms involved in the lower pon1 activity and the higher oxidative stress in cd patients (figure 3). previous studies have demonstrated that, in cd patients, gluten ingestion induces an increased oxidative stress due to overproduction of free radicals (ros and rns) [2931 ]. the higher levels of ros and the decrease of dietary antioxidant can trigger lipid peroxidation (looh) in intestinal mucosa and blood lipoproteins. pon1 is a lipid - dependent enzyme associated with high - density lipoprotein (hdl). previous studies demonstrated that a decrease of serum pon1 activity could be related to a lower hepatic synthesis and secretion or to inhibition exerted by lipid hydroperoxides. no significant correlation has been established between pon1 activity and levels of hdl cholesterol, suggesting that the lower enzyme activity in cd patients is not associated with alterations of hdl levels. although pon1 is mainly synthesised by the liver, it has been demonstrated that it is expressed also in human gastrointestinal tract and caco-2 cell lines, suggesting that the intestine, as well as the liver, could represent a source for circulatory pon1. since a decreased expression of pon1 mrna has been reported by rothem l. in intestinal biopsies of celiac patients, we could speculate that the lower pon1 activity could be related to changes of the enzyme synthesis from the gastrointestinal tract. however, contrasting results have been reported on the synthesis of pon1 in intestinal cells [18, 3436 ]. the lower activity of pon1 in cd patients could be also mediated by inhibition exerted by lipid peroxidation products, since previous studies have shown that pon1 paraoxonase, arylesterase, and lactonase activities are inhibited by lipid peroxidation products [37, 38 ]. this hypothesis is supported by the significant negative correlation found between pon1 paraoxonase activity and levels of lipid hydroperoxides in serum of controls and celiac patients. the higher susceptibility of serum of celiac patients to cu - triggered lipid peroxidation, demonstrated in our experimental conditions, may be related to the lower pon1 activity and/or to the lower serum total antioxidant capacity, as shown by the significant correlations observed between the lag time of kinetic of lipid peroxidation and pon1 paraoxonase values and tac values. the lower serum total antioxidant capacity in cd patients could be likely the result of a malabsorption of dietary antioxidants due to the mucosal damage. in agreement with this hypothesis other authors have reported lower levels of antioxidants (retinol, -tocopherol, and ascorbic acid) in plasma of cd patients [5, 9 ]. the mean level of serum total antioxidant capacity in gfd patients was significantly higher compared with untreated cd patients, although it did not reach the values observed in controls ; these results suggest that gluten - free diet is able to only partially improve the functionality of intestinal mucosa. whatever are the causes of the decrease in the activity of the antioxidant and anti - inflammatory enzyme pon1, our results might have a physiopathological relevance. in fact, the enzyme pon1 exerts several physiological roles. in the gastrointestinal tract pons proteins and could behave as detoxifier against food antigens, antioxidant against exogenous and endogenous lipid peroxides, immunomodulators and/or inhibitors of bacterial quorum - quenching factor [18, 35 ]. moreover, pon1 exerts a protective effect against lipid peroxidation of lipoproteins and biological membranes [1113 ]. oxidative stress is one of the main causes of damage to cell membranes and lipoproteins. end products of lipid peroxidation (aldehydes, lipid peroxides) react with biological macromolecules such as dna and proteins and can cause changes in intestinal cell membrane structure and properties leading to loss of its integrity. moreover, lipid peroxidation products exert biological effects, modulate signal transduction and it has been proposed that they may have a role in inflammatory processes. in conclusion, our results confirm that celiac disease is associated with oxidative damage and with significant decrease of pon1 activities. although a decrease of paraoxonase-1 activity and an increase of lipid peroxidation products are not specific of celiac disease [1417 ], they could exert a proinflammatory and cytotoxic effect and could therefore contribute to gastrointestinal cell injury in cd. | paraoxonase-1 (pon1) plays an antioxidant and anti - inflammatory role. aim of the study was to investigate the alteration of paraoxonase-1 activity in celiac disease (cd), an intestinal disorder characterized by toxic injury exerted by gluten peptides. activities of pon1, levels of biochemical markers of lipid peroxidation and total antioxidant capacity were evaluated in serum obtained from 27 celiac patients (11 at diagnosis, 16 treated with gluten free diet) and 25 healthy subjects. moreover, the serum susceptibility of cu2 + -induced lipid peroxidation was investigated in controls and patients. the results showed a lower pon1 activity in serum of both groups of celiac patients with respect to control subjects. pon1 activity in cd was related with markers of disease severity and was negatively correlated with the levels of lipid hydroperoxide and with the susceptibility of serum to lipid peroxidation induced in vitro by metal ions. the alteration of pon1 activity and markers of lipid peroxidation realized at lower extent in patients who were on a gluten - free diet. |
cardiovascular diseases have become a ubiquitous cause of morbidity and a leading contributor to mortality in most countries and this fact is well documented in developed countries. it is projected that the circulatory system disease in india would rise by 103% in man and 90% in women during the period 1985 to 2015. the treatment of cardiovascular diseases with allopathic drugs causes moderate to severe adverse events which could cause death. ayurvadic system of medicine is practised in india, but the scientific evidence for treating diseases with herbs and fixed herbal formulations are poorly documented. the marketed polyherbal formulation triglize claims that it can be used for the treatment of obesity, hypertension, palpitation, myocardial necrosis, coronary atherosclerosis, hypercholesterol, ischemic heart diseases and peripheral vascular diseases. diuretics and antioxidants play a role in the treatment of hypertension and ischemic heart disease. so, the present study was planned to determine the free radical scavenging property of triglize, a polyherbal formulation (phf), and its diuretic effect using in vitro and in vivo models, respectively. triglize (batch no. : 13335002, mfg date : jun, 2005, apex laboratories ltd., chennai) is a marketed soft gelatin capsule and it was formulated using aqueous extracts of terminalia arjuna, cissus quadrangularis, boerhaavia diffusa, commiphora mukul, phyllanthus embilica, terminalia bellirica, terminalia chebula, tribulus terrestris, allium sativum and trigonella foenumgraecum. healthy, adult, male albino rats of wistar strain, weighing 170 200 g were obtained from animal house, j.s.s. the rats were housed under 22 2 c temperature, 40 - 60 % humidity and 12:12 1 h light dark cycle. the study was approved by the institute animal ethics committee and all the animal experiments were carried out as per cpcsea guidelines. the antioxidant activity of test compounds was assessed on the basis of the radical scavenging effects of greiss ilosvay reaction. the aqueous extract of ployherbal formulation (phf) and the known antioxidant ascorbic acid were dissolved in dmso (dimethyl sulfoxide) (sigma - aldrich) separately and used for the in vitro antioxidant testing by dpph (2, 2-diphenyl-1-picrylhydrazyl) (sigma - aldrich) method. the assay was carried out in a 96 well microtiter plate. to 200 l of dpph solution, 10 l of each of the test and standard solutions were added separately in wells of the 96 well microtiter plates. the final concentration of the test, standard solution used was 15.62 to 1000.00 g/ ml. the plates were incubated at 37 c for 30 min and the absorbance of each was measured at 490 nm using elisa reader against the corresponding test and standard blanks and the remaining dpph was calculated. ic50 the concentration of the sample to scavenge 50 % of dpph free radicals was calculated and recorded. male wistar rat (200 g) peritoneal macrophages cells were induced using 3% w / v sodium thioglycolate (i.p.). after 3 days, the macrophages cells were collected, washed with phosphate buffer saline and resuspended with rpmi 1640 medium (fisher chemicals), supplemented with nahco 3 (2 g / i), penicillin (100 iu / l), streptomycin (100 g / ml), gentamicin (20 g / ml) and fetal calf serum (10%) and plated in 24 wells microtiter plate. equal amounts of the macrophages cells (2.6 10/well) were incubated in humidified 5% co2 at 37 c for 24 h to allow macrophages adherence and the plate was washed 3 - 4 times with serum free fresh medium to dislodge the non - adherent cells. the macrophages were cultured for 3 h and incubated with lps 25 ng / ml along with five different concentrations of phf at the concentration of 3 g - 1000 g. the aqueous extract of phf (dissolved in dmso) was added 2 - 5 min to the cells before incubation of cell with lps 25 ng / ml. finally free nitrites were estimated using greiss reagents and the cell viability was calculated using the mtp (microculture tetrazolium) assay method. thirty - six male wistar rats (170 - 190 g) were divided into six groups containing six animals and grouped as follows : group i - control (0.5% w / v cmc) group ii - standard drug treatment (furosemide- 5mg / kg) group iii - phf- 12.5 mg / kg group iv - phf- 50 mg / kg group v - phf- 200 mg / kg group vi - phf- 400 mg / kg seventeen to twenty - four hours prior to the experiment, food and water were withdrawn and animals were placed individually in a metabolic cage. the aqueous extract of phf at various concentrations like 12.5, 50, 200, and 400 mg / kg and reference standard furosemide (5 mg / kg) made as suspension with 0.5% w / v carboxymethyl cellulose (cmc) and administered orally as a single dose. additionally, 5 ml of 0.9% w / v nacl solution per 100 g body weight was given by oral gauge. urine was collected 0- 5 and 5 - 24 h of dosing and urine volume, acidity (ph) was measured immediately. the sodium, potassium content of urine was determined by flame photometry and concentration of chloride was determined by argentometric titration. significant difference between groups was determined using one - way anova followed by tukey 's multiple comparison test. a p value less than 0.05 was considered to be significant. triglize (batch no. : 13335002, mfg date : jun, 2005, apex laboratories ltd., chennai) is a marketed soft gelatin capsule and it was formulated using aqueous extracts of terminalia arjuna, cissus quadrangularis, boerhaavia diffusa, commiphora mukul, phyllanthus embilica, terminalia bellirica, terminalia chebula, tribulus terrestris, allium sativum and trigonella foenumgraecum. healthy, adult, male albino rats of wistar strain, weighing 170 200 g were obtained from animal house, j.s.s. the rats were housed under 22 2 c temperature, 40 - 60 % humidity and 12:12 1 h light dark cycle. the study was approved by the institute animal ethics committee and all the animal experiments were carried out as per cpcsea guidelines. the antioxidant activity of test compounds was assessed on the basis of the radical scavenging effects of greiss ilosvay reaction. the aqueous extract of ployherbal formulation (phf) and the known antioxidant ascorbic acid were dissolved in dmso (dimethyl sulfoxide) (sigma - aldrich) separately and used for the in vitro antioxidant testing by dpph (2, 2-diphenyl-1-picrylhydrazyl) (sigma - aldrich) method. the assay was carried out in a 96 well microtiter plate. to 200 l of dpph solution, 10 l of each of the test and standard solutions were added separately in wells of the 96 well microtiter plates. the final concentration of the test, standard solution used was 15.62 to 1000.00 g/ ml. the plates were incubated at 37 c for 30 min and the absorbance of each was measured at 490 nm using elisa reader against the corresponding test and standard blanks and the remaining dpph was calculated. ic50 the concentration of the sample to scavenge 50 % of dpph free radicals was calculated and recorded. male wistar rat (200 g) peritoneal macrophages cells were induced using 3% w / v sodium thioglycolate (i.p.). after 3 days, the macrophages cells were collected, washed with phosphate buffer saline and resuspended with rpmi 1640 medium (fisher chemicals), supplemented with nahco 3 (2 g / i), penicillin (100 iu / l), streptomycin (100 g / ml), gentamicin (20 g / ml) and fetal calf serum (10%) and plated in 24 wells microtiter plate. equal amounts of the macrophages cells (2.6 10/well) were incubated in humidified 5% co2 at 37 c for 24 h to allow macrophages adherence and the plate was washed 3 - 4 times with serum free fresh medium to dislodge the non - adherent cells. the macrophages were cultured for 3 h and incubated with lps 25 ng / ml along with five different concentrations of phf at the concentration of 3 g - 1000 g. the aqueous extract of phf (dissolved in dmso) was added 2 - 5 min to the cells before incubation of cell with lps 25 ng / ml. finally free nitrites were estimated using greiss reagents and the cell viability was calculated using the mtp (microculture tetrazolium) assay method. thirty - six male wistar rats (170 - 190 g) were divided into six groups containing six animals and grouped as follows : group i - control (0.5% w / v cmc) group ii - standard drug treatment (furosemide- 5mg / kg) group iii - phf- 12.5 mg / kg group iv - phf- 50 mg / kg group v - phf- 200 mg / kg group vi - phf- 400 mg / kg seventeen to twenty - four hours prior to the experiment, food and water were withdrawn and animals were placed individually in a metabolic cage. the aqueous extract of phf at various concentrations like 12.5, 50, 200, and 400 mg / kg and reference standard furosemide (5 mg / kg) made as suspension with 0.5% w / v carboxymethyl cellulose (cmc) and administered orally as a single dose. additionally, 5 ml of 0.9% w / v nacl solution per 100 g body weight was given by oral gauge. urine was collected 0- 5 and 5 - 24 h of dosing and urine volume, acidity (ph) was measured immediately. the sodium, potassium content of urine was determined by flame photometry and concentration of chloride was determined by argentometric titration. the aqueous extract of phf showed a moderate antioxidant activity by dpph method with an ic50 value of 219.22 1.64 mg / ml when compared to standard ascorbic acid (ic50 = 64.33 0.91). in vitro results indicated that thioglycolate activated macrophages were hypersensitive to lps and produced no between 55 - 59 m/ 2.610 cells, whereas normal cells produced no between 17 - 22 m/2.610 cells under similar conditions. no production was significantly inhibited by simultaneous and pre - incubation with phf aqueous extract in a dose dependent manner [table 1 ]. effect of phf on lps induced no production and cell viability on thioglycolate induced rat macrophages a summary of the diuretic activity is presented in tables 2 and 3. the results showed significant increase in urine volume in 0 - 5 h and 5 - 24 h. the percentage differences in urine volume in treatment group at the dose levels of 50, 200 and 400 mg / kg b.wt were 115.87%, 129.03% and 151.61% for 0 - 5 h and 106.4%, 108.0% and 146.4% for 5 - 24 h when compared to control. effect of phf on urine volume, ph and electrolytes (0 - 5 hour urine biochemical estimation) effect of phf on urine volume, ph and electrolytes (5 - 24 hour urine biochemical estimation) excretion of potassium ion in urine significantly increased with furosemide 5 mg / kg and phf 50, 200 and 400 mg / kg (p < 0.01). the percentage changes in excretion of potassium ion with phf 50, 200 and 400 mg / kg were 52.63%, 47.36% and 57.87% for 0 - 5 h urine sample and 40.90%, 45.45%, and 54.45% for 5 - 24 h urine sample as compared to control. phf showed 15% significant reduction in chloride ion excretion at dose levels of 50 and 400 mg / kg at 0 - 5 h urine sample (p < 0.05), but there is no significant reduction of chloride ion concentration in phf treated animals at 5 - 24 h urine sample. the results of present study suggest that phf has free radical scavenging property and diuretic activity. however, the free radical scavenging property or antioxidant property of phf is moderate when compared to ascorbic acid while the diuretic activity is comparable to frusemide. free radicals are formed in the cells as a consequence of both oxidative biochemical reaction and have been implicated in the pathogenesis of wide variety of clinical disorders, resulting from deficient natural antioxidant defences. phf possesses a series of substituted phenolic compounds and variety of flavones which have known antioxidant property. this is the reason why it significantly inhibited no production in a dose - dependent manner in rat peritoneal macrophage cells in our study. phf exhibits diuretic activity at the dose levels of 50, 200 and 400 mg / kg and the effect is comparable to furosemide (10 mg / kg). since phf exhibits significant diuretic activity, it may be useful in the management of vascular and cardiac diseases. though the in vitro results can be extrapolated to in vivo system, in the present study, the free radical scavenging property of phf was studied only for no free radical. in a living system, the oxidative reaction can occur through many mediators/ toxins and their inducing superoxide and hydroxyl free radicals. hence phf should be ideally tested in an in vivo system to confirm its free radical scavenging property and what mediators are involved. as there are no published reports on phf, the results of our study could not be compared. it has been shown that some of the ingredients in the phf have antioxidant and diuretic properties.[1315 ] phf exhibits significant antioxidant effect in vitro and diuretic effect in vivo. since it is currently approved for use in humans (as an ayurvedic medicine), it is worthwhile to explore the usefulness of the phf in the treatment of diseases of cardiovascular system such as hypertension and cardiac failure. | objective : to determine the in vitro free radical scavenging property and in vivo diuretic effect of triglize, a marketed polyherbal formulation in experimental models.materials and methods : the aqueous extract of polyherbal formulation (phf) triglize was used for the experiment. the free radical scavenging property and antioxidant effect of phf were studied by lps - induced free radicals in rat macrophages cells and dpph (2, 2-diphenyl-1-picrylhydrazyl) methods, respectively. the diuretic effect of a phf was studied with lipschitz model using male wistar rats.results:phf significantly inhibited lipopolysaccharide -induced free radicals in rat macrophages and it showed moderate antioxidant potential in dpph model. polyherbal formulation at 50, 200 and 400 mg/ kg significantly increased potassium excretion in urine at 0 - 5 h and 5 - 24 h. the diuretic effect of phf was as similar as furosemide.conclusion : the phf has significant diuretic effect and free radical scavenging properties. |
pain is a common and disabling symptom in persons with multiple sclerosis (ms), with a prevalence ranging widely between 29% and 86% [13 ]. several different pain conditions are associated with ms and may be defined by location, by presumed mechanism, and by duration, ranging from paroxysmal to chronic. restricting our focus to chronic pain, approximately 75% of ms patients one broad category of ms - related pain is central neuropathic pain, which includes dysesthetic extremity pain, a chronic form of pain described as the most common. other chronic pain conditions are musculoskeletal pain (e.g., back pain), painful tonic spasms, and headache. fibromyalgia (fm), originally described as a psychogenic disorder or a variant of depression, has recently been considered a model of central neuropathic pain. neurophysiologic methods able to explore the nociceptive afferent system suggest that in fm patients weak painful stimuli delivered at both tender and non - tender points induce cortical hyperactivation and lead to generalized hyperalgesia. these phenomena have been confirmed by functional magnetic resonance imaging, whose findings showed a significantly greater activation in the anterior insula and the cingulate cortex in response to painful stimuli in fm patients compared to controls. a study investigating the intensity - dependence of auditory - evoked cortical potentials found that fm patients are hypervigilant to acoustic stimuli and showed a reduced inhibition response to noxious and intense auditory stimuli. this pattern of cortical hypervigilance to multimodal stimuli is well known in fm patients [810 ]. therefore, on the basis of both neurophysiological and functional magnetic resonance imaging methods, it has been supposed that fm is related to cns malfunction and altered pain transmission and perception, as supported by the augmented stimulus responses transmitted by primary afferent fibers, the impairment of descending inhibitory pain pathways, and the altered release of neurotransmitters involved in pain modulation. fm may appear alone, with a prevalence of 2.94.7% in the general population, or associated with other rheumatic diseases, such as up to 25.3% in systemic lupus erythematosus (sle) and up to 19.8% in rheumatoid arthritis (ra). the prevalence of fm among ms patients and its impact on health - related quality of life (hrqol) has not yet been clearly determined. the few studies available on this topic exclusively collected data from administrative records or self - reported questionnaires. however, fm was the only co - morbidity analyzed in ms that was proved to exert a moderate impact on physical hrqol, being generally milder than the effect of other diseases. in our own experience, we have observed a high incidence of fm associated with ms, as ascribable to various factors. first, some of the pathophysiological abnormalities of each of these disorders may be shared. a previous study by our group, which evaluated the thermal and discomfort thresholds in ms patients, yielded lower values in patients than in healthy controls. similarly, fm is characterized by central sensitization, which is a disordered sensory processing in the central nervous system and is responsible for an increased perception of pain in response to low stimuli of different types., fm may result from a cervical myelopathy due to various causes, such as a motor vehicle accident, surgery, arnold - chiari malformation, spinal canal stenosis, positional cervical compression, or trauma. third, some of the predisposing risk factors to these 2 diseases, such as female sex, may be shared. for all these reasons, we believe that the frequency of fm in ms patients is higher than in the general population and similar to that observed in other rheumatic diseases. the primary aim of this study was to evaluate the frequency of fm in a continuous series of italian ms patients by means of specific validated diagnostic criteria, regardless of the various types of chronic pain referred. the secondary aim of the study was to detect any demographic and clinical characteristics associated with fm in ms. moreover, when associated with a highly disabling disease, such as ms, it clearly exacerbates the patients already compromised quality of life. the third objective of this study was to evaluate the possible impact of fm on ms patients hrqol, regardless of the other types of chronic pain. we recruited, between october 2012 and august 2013, a continuous series of patients with definite ms according to the mcdonald criteria, from a total of 205 in- and out - patients undergoing rehabilitation for ms at the santa lucia foundation, a large, free - standing, university - affiliated rehabilitation institute not dependent on an acute - care general hospital. the exclusion criteria were age < 18 years, other known neurological or rheumatic diseases, other disorders of the spine, cancer, renal diseases, diabetes mellitus, severe psychiatric diseases, relapses within 1 month prior to enrollment, and a mini - mental state examination score < 23. the study was approved by the ethics committee of our foundation and informed consent was given in writing by all the patients. all the patients underwent a structured interview to collect their clinical history, as well as ms preventive treatment intake. a neurological examination was performed by means of the kurtzke expanded disability status scale (edss), with its functional systems. the presence of chronic pain, which had been constant or nearly constant in the previous month, was investigated at the time of evaluation. the referred pain was further rated as neuropathic or not according to the dn4 questionnaire, consisting of both sensory descriptors and signs related to bedside sensory examination, and providing very good sensitivity (83%) and specificity (90%) for identification of chronic pain associated with a lesion in either the peripheral or central nervous system ; a score of at least 4 out 10 is suggestive for neuropathic pain. the patients with a score < 4 were diagnosed with musculoskeletal pain to simplify the classification, even if it may include pain of mixed type caused by the complex interplay of nociceptive, neuropathic, and pathogenic mechanisms. depression was assessed by means of the beck depression inventory ii (bdi ii). the cut - off scores used were : 013 : minimal depression ; 1419 : mild depression ; 2028 : moderate depression ; and 2963 : severe depression. a deep rheumatologic examination, as well as a wide panel of auto - antibodies, was performed by a ward rheumatologist to exclude any co - morbidity with other rheumatic diseases. to investigate the fm co - morbidity, all the patients were assessed according to the 1990 american college of rheumatology (acr) diagnostic criteria. for a diagnosis of fm to be made, the 1990 acr diagnostic criteria require tenderness on pressure (tender points) in at least 11/18 specified sites, as well as the presence of widespread pain for at least 3 months. hrqol was assessed by means of the medical outcome 36-item short form health survey (sf-36), which analyzes physical and mental health status (phs and mhs, respectively). the item scores range from 0 (poor health) to 100 (optimal health). according to published reports, the frequency of fm is approximately 19.8% in ra and 3.8% in the general population. for this pilot study, a sample size of at least 75 patients would provide 80% power for detecting a difference in fm frequency not below 16% between the ms group and the general population, at the 5% level of significance. both the epidemiological and clinical characteristics of the patients enrolled were summarized by mean and standard deviation for numerical variables, and by frequencies and percentages for categorical variables. the shapiro - wilk test was used to check the normality distribution for each variable used in the study. a p<0.05 was sufficient to reject the null hypothesis that the variable was normally distributed. patients were divided into 3 sub - groups (1 without pain, 1 with pain not fm - type, and 1 with pain fm - type) according to the modified acr 1990 criteria. the statistical analysis between the 2 groups was performed by means of the mann - whitney rank sum test or the chi - square test, as appropriate. the nonparametric kruskal - wallis one - way analysis of variance followed by the dunn s post test was performed to compare more than 2 unmatched sub - groups. we recruited, between october 2012 and august 2013, a continuous series of patients with definite ms according to the mcdonald criteria, from a total of 205 in- and out - patients undergoing rehabilitation for ms at the santa lucia foundation, a large, free - standing, university - affiliated rehabilitation institute not dependent on an acute - care general hospital. the exclusion criteria were age < 18 years, other known neurological or rheumatic diseases, other disorders of the spine, cancer, renal diseases, diabetes mellitus, severe psychiatric diseases, relapses within 1 month prior to enrollment, and a mini - mental state examination score < 23. the study was approved by the ethics committee of our foundation and informed consent was given in writing by all the patients. all the patients underwent a structured interview to collect their clinical history, as well as ms preventive treatment intake. a neurological examination was performed by means of the kurtzke expanded disability status scale (edss), with its functional systems. the presence of chronic pain, which had been constant or nearly constant in the previous month, was investigated at the time of evaluation. the referred pain was further rated as neuropathic or not according to the dn4 questionnaire, consisting of both sensory descriptors and signs related to bedside sensory examination, and providing very good sensitivity (83%) and specificity (90%) for identification of chronic pain associated with a lesion in either the peripheral or central nervous system ; a score of at least 4 out 10 is suggestive for neuropathic pain. the patients with a score < 4 were diagnosed with musculoskeletal pain to simplify the classification, even if it may include pain of mixed type caused by the complex interplay of nociceptive, neuropathic, and pathogenic mechanisms. depression was assessed by means of the beck depression inventory ii (bdi ii). the cut - off scores used were : 013 : minimal depression ; 1419 : mild depression ; 2028 : moderate depression ; and 2963 : severe depression. a deep rheumatologic examination, as well as a wide panel of auto - antibodies, was performed by a ward rheumatologist to exclude any co - morbidity with other rheumatic diseases. to investigate the fm co - morbidity, all the patients were assessed according to the 1990 american college of rheumatology (acr) diagnostic criteria. for a diagnosis of fm to be made, the 1990 acr diagnostic criteria require tenderness on pressure (tender points) in at least 11/18 specified sites, as well as the presence of widespread pain for at least 3 months. hrqol was assessed by means of the medical outcome 36-item short form health survey (sf-36), which analyzes physical and mental health status (phs and mhs, respectively). the item scores range from 0 (poor health) to 100 (optimal health). according to published reports, the frequency of fm is approximately 19.8% in ra and 3.8% in the general population. for this pilot study, a sample size of at least 75 patients would provide 80% power for detecting a difference in fm frequency not below 16% between the ms group and the general population, at the 5% level of significance. both the epidemiological and clinical characteristics of the patients enrolled were summarized by mean and standard deviation for numerical variables, and by frequencies and percentages for categorical variables. the shapiro - wilk test was used to check the normality distribution for each variable used in the study. a p<0.05 was sufficient to reject the null hypothesis that the variable was normally distributed. patients were divided into 3 sub - groups (1 without pain, 1 with pain not fm - type, and 1 with pain fm - type) according to the modified acr 1990 criteria. the statistical analysis between the 2 groups was performed by means of the mann - whitney rank sum test or the chi - square test, as appropriate. the nonparametric kruskal - wallis one - way analysis of variance followed by the dunn s post test was performed to compare more than 2 unmatched sub - groups. from october 2012 to august 2013, 205 ms in- and out - patients were admitted at our hospital. out of them, 72 (35.1%) fulfilled the exclusion criteria, such as diabetes mellitus (4.8%), relapses in the last month prior to enrollment (3.9%), cancer (2.4%), age < 18 years (0.9%), disorders of the spine (5.8%), other rheumatic disease (7.3%), patients with acute or occasional pain (6%), as well as severe cognitive impairment (9.7%), and were excluded. the remaining 133/205 (64.8%) ms patients were enrolled in the study ; the patients demographic and clinical characteristics are shown in table 1. the f / m ratio was 1.8, which is in keeping with previous reports. the patients enrolled had a mean age of 51.812.9 years, a high disability (mean edss 5.52.6) and mean disease duration of 19.311.7 years. most of the patients (67.7%) were in the secondary progressive phase of disease. all the patients enrolled were investigated for the occurrence of chronic pain, which was referred by 88/133 (66.2%) and absent in the remaining 45/133 (33.8%) (patients with no pain [nop ]) (table 1). according to the dn4 questionnaire, the pain was neuropathic in 12/88 (13.7%) patients and musculoskeletal or mixed in 76/88 (86.3%) patients. the demographic and clinical characteristics of these sub - groups did not differ significantly (table 2). among all the patients enrolled, 23/133 (17.3%) also fulfilled the 1990 acr criteria for fm (pfm+ patients) ; out of them, 5 (21.7%) had neuropathic pain, while in the remaining 18 (78.3%) the pain was musculoskeletal (p=0.03), as determined by the dn4 questionnaire. these 2 pfm+ subgroups did not differ in any demographic and clinical characteristics, and were grouped together for further statistical analysis (table 3). the other 65/133 (48.9%) ms patients with chronic pain, not fulfilling the 1990 acr criteria for fm, were considered to have chronic pain not fm type (pfm patients). out of them, 7 (10.8%) had neuropathic pain and 58 (89.2%) had musculoskeletal pain (p=0.01), as determined by means of the dn4 questionnaire. these 2 pfm subgroups did not differ in any demographic and clinical characteristics, and were grouped together for further statistical analysis (table 4). the distribution of our sm patients in the 3 sub - groups (nop, pfm+ and pfm) is shown in figure 1. the frequency of neuropathic and musculoskeletal pain did not differ between the pfm+ and pfm sub - groups (p=0.18) the pfm+ patients were predominantly female (p=0.03) and had a greater edss than the nop group (p=0.01), but not greater than the pfm patients ; no significant differences among pfm+, pfm, and nop groups emerged as regards the other demographic and clinical characteristics (table 5). depression was present in 54.1% of the patients enrolled, generally displaying a mild depression (mean dbi - ii 17.0111.7). out of the patients with chronic pain, 42 (66.7%) were diagnosed with depression, compared to 11 (31.4%) patients in the nop group, by means of the dbi - ii (p=0.007). when the subgroups were considered separately, the pfm+ patients had a more pronounced depression than the nop group (p=0.002), mainly ranging in the moderate / severe grade (p=0.01), but did not differ from the pfm patients (table 6). fatigue, as assessed by means of the fss, was present in 102/133 (76.7%) of our sample, but did not differ among the 3 sub - groups (table 6). the pfm+ patients scored the worst in both the phs and the mhs when compared to the pfm and the nop groups (p<0.01). the prevalence of chronic pain in ms is estimated at 5379% in studies based on mailed survey, in - person survey, or in - person assessment, and evaluating the prevalence of pain during the month prior to assessment [3335 ]. despite the slightly different values reported in the literature, it may vary because of the heterogeneous methods and definition of pain adopted, the 66.2% prevalence of chronic pain we found in our sample may be considered in keeping with the literature. this frequency is higher than the 3.8% reported in the general population, and similar to those reported in other rheumatic diseases, such as ra and sle. few studies have investigated the co - morbidity of fm in ms. the fm prevalence in one such study, which investigated different types of chronic pain and was based on administrative claim records, was 14.06% in the ms cohort. in another study, which investigated various co - morbidities by means of a self - reported validated questionnaire in an american ms population, fm was reported by 4.9% of the patients. it is known that the validity of self - reported diagnoses varies depending on the co - morbidity investigated. indeed, it is very accurate for common disorders such as hypertension and diabetes, but is less accurate for uncommon disorders such as arthritis and fm. in our study, a ward rheumatologist performed an examination to assess the presence of co - morbid fm in our ms patients, and the differences in the diagnostic methods adopted may explain the high frequency of fm detected in our ms sample. of the patients with chronic pain, only 13.7% had a neuropathic pain, without significant difference between the pfm+ and pfm sub - groups. this frequency is low if compared with other data on neuropathic pain derived from systematic review and meta - analysis, which estimate a prevalence of 28.5% in ms. we identified some methodological variables in the studies included, such as deficient blinding, different study design, and varying diagnostic criteria. in our study we focused on pain lasting for no less than 1 month, excluding forms of acute and paroxysmal pain, and used the dn4 questionnaire. these 2 factors may justify the low prevalence of neuropathic pain found in our sample. when considering the pfm+ subgroup, we found a higher prevalence of females with respect to the nop sub - group only. female sex appears to play a critical role in the development of ms, and women are diagnosed as having fm approximately 7 times more often than men. the fact that female sex appears to be a major risk factor for the development of both ms and fm may, at least in part, explain this co - morbidity. moreover, women are about 2 times more likely to report chronic and widespread pain, because of higher pain sensitivity, and this may explain the lack of difference between the 2 different sub - groups of ms patients with chronic pain (pfm+ and pfm). the other difference between pfm+ and nop patients was a higher degree of disability in the former, although the edss was high in both groups. this finding seems to suggest that pain may have a negative effect on disability and is in line with those reported in the literature. the impact of pain on disability was found to be modest in patients with a low edss and high in those with a high edss. the association between pain and disability may be inter - related, with these 2 factors exacerbating each other, especially in patients with a higher degree of impairment. our data confirm the high prevalence of depression in ms, which is estimated at around 50%. in addition, we found a more pronounced depression in pfm+ patients than in nop patients, but similar to the pfm patients. this finding seems to show a negative impact of fm, but not of other types of chronic pain, on patient mood. findings from studies that examined the relationship between pain and depression in ms are mixed, with roughly equal numbers of studies showing a positive versus a null relationship [4548 ]. instead, the high rate of co - morbidity between fm and depression is well known. moreover, fm and depression share a similar pathophysiology, with a more pronounced deficit in pain inhibition in fm patients with depressive symptoms. in our study, fatigue was detected in 75% of the patients, which is in keeping with reports in the literature, with no difference between the 3 sub - groups. however, fatigue is also very frequent in fm, with figures ranging between 78% and 94%. fatigue in both fm and ms may arise from a range of mechanisms that have yet to be fully clarified, and the complexity of its pathophysiology may thus explain why we could not find any associations with the presence of chronic pain or fm. the impact of fm on family, social, and professional life is well known. patients with fm were found to have a worse qol even than ra patients and ms has a significant impact on health - related qol at all stages of disease. thus, the co - morbidity of fm in ms clearly exacerbates the already compromised qol. in our sample, the pfm+ group scored the worst in both phs and mhs, which points to a marked effect of fm co - morbidity on ms patient qol. our findings are consistent with those of previous studies, which have shown that, out of various co - morbidities, fm has a relevant impact on physical and mental hrqol in ms patients. in our series we found a prevalence of 66.2% of chronic pain, with those patients displaying mainly musculoskeletal - type pain, greater disability, and more severe depression. in our sample, almost 1/5 of ms patients had a co - morbidity with fm, which is a significantly higher prevalence than that observed in the general population, but similar to those reported in sle and ra. even if pfm+ patients were similar to pfm patients, according to the clinical and demographic characteristics, fm occurrence was associated with a stronger impact on patient qol. fm co - morbidity should be further investigated in ms patients, given its frequency and impact on patient quality of life. one limitation of this study is the lack of a wider range of edss scores. indeed, a larger study, including patients with lower edss scores, might shed more light on how frequent fm is, even in the early stage of disease. | backgroundchronic pain is common in persons with multiple sclerosis (ms), but the co - morbidity of fibromyalgia (fm) has yet to be investigated in ms. objectives of the study were to evaluate, among the various types of chronic pain, the frequency of fm in ms and its impact on ms patients health - related quality of life (hrqol).material / methods133 ms patients were investigated for the presence and characterization of chronic pain within 1 month of assessment. a rheumatologist assessed the presence fm according to the 1990 acr diagnostic criteria. depression, fatigue, and hrqol were also assessed by means of specific scales.resultschronic pain was present in 66.2% of patients (musculoskeletal in 86.3% ; neuropathic in 13.7% ; absent in 33.8% [called nop ]). pain was diagnosed with fm (pfm+) in 17.3% of our ms patients, while 48.9% of them had chronic pain not fm type (pfm) ; the prevalence of neuropathic pain in these 2 sub - groups was the same. pfm+ patients were prevalently females and had a higher edss than nop. the pfm+ patients had a more pronounced depression than in the nop group, and scored the worst in both physical and mental qol.conclusionsin our sample of ms patients we found a high prevalence of chronic pain, with those patients displaying a higher disability and a more severe depression. moreover, fm frequency, significantly higher than that observed in the general population, was detected among the ms patients with chronic pain. fm occurrence was associated with a stronger impact on patients qol. |
the diagnosis is usually made by demonstration of antigen in the urine, serum, relevant body fluids or pus and by fungal culture. herein we are presenting a rare case of genitourinary histoplasmosis where the diagnostic dilemma was resolved by fungal culture of prostatic abscess and histopathological examination of epidydimal tissue. a 37-year - old male, a renal transplant recipient, with the mother as donor, presented with complaints of pain and swelling in the scrotum and fever of one week duration, three years after the transplant. post transplantation, he was on triple immunosuppressive therapy with tacrolimus, azathioprim, and prednisolone, and his allograft function was stable. one - and - a - half years after his renal transplant, he had dysphagia and malena, and was diagnosed with esophageal candidiasis and colonic histoplasmosis on the basis of upper gastrointestinal endoscopy and colonoscopy, respectively. histoplasmosis was confirmed by demonstrating histoplasma capsulatum on histopathology of the biopsy from colonic ulcers and stool culture. he responded to itraconazole, which was continued for nine months and was asymptomatic on follow - up. at the time of this presentation, three years after the transplant, blood investigations revealed severe anemia, leucopenia, thrombocytopenia, and serum creatinine of 3.0 mg / dl. urinalysis showed proteinuria (300 mg / dl) with numerous red blood cells (rbcs) and pus cells. digital rectal findings suggested bogginess on the anterior rectal wall with prostatic landmarks not well appreciable. the ultrasound showed enlarged, heterogenous epididymis, with fluid collection and a thick - walled collection in the prostate and retrotrigonal area. computerized tomography of the pelvis confirmed the ultrasound findings [figures 1a, b ]. ct scan pelvis showing retrotrigonal (a) and prostatic location (b) of collection the epididymal swelling was explored and tissue biopsy was sent for polymerase chain reaction (pcr) for mycobacterium tuberculosis and histopathology. the prostatic and retrotrigonal collection was drained via the per - rectal route and sent for culture studies. histopathology diagnosed histoplasmosis [figure 2 ] and culture of the pus (obtained from the prostatic / retrotrigonal abscess) confirmed the growth of histoplasma capsulatum [figure 3 ]. a repeat colonoscopy was done, which did not reveal any ulcers or polyps or any other finding suggestive of intestinal histoplasmosis. the patient was restarted on itraconazole for nine months, to which he responded successfully. photomicrograph showing sheets of histiocytes with intracytoplasmic pas - positive yeast forms, suggestive of histoplasmosis : pas stain 400 magnification photomicrograph showing saprophytic phase of histoplasma capsulatum with spherical, tuberculate, macroconida (8 14 m) : lactophenol cotton blue stain 40 magnification the risk of genitourinary tuberculosis is increased in transplant recipients, who are either born or live in areas where tuberculosis is endemic. the frequency of mycobacterium tuberculosis disease among the recipients of solid organ transplants in most developed countries is 1.2 6.4%, but among transplant recipients living in areas of high endemicity, it can reach 15%. it is worth noting that diverse, unsuspected, and elusive sites of tuberculosis infection have been described. in this patient, with enlargement of epididymis coupled with findings of an abscess in the prostatic and retrotrigonal region, tuberculosis was a strong probability. however, tissue pcr and culture for mycobacterium tuberculosis was negative and findings of histopathology and culture were conclusive for histoplasmosis. even as most cases of histoplasmosis were asymptomatic or self - limiting, they could rarely lead to severe and progressive disseminated infections. diagnosis of disseminated histoplasmosis required a high index of suspicion.[24 ] only four cases of epididymal histoplasmosis have been reported in literature so far. two cases were diagnosed from the united states in 1981, one was presented by kahn dg,., at a conference in the united states in 1992 (international conference on acquired immunodeficiency syndrome (aids) 1992 jul 19 - 24 ; 8 : 93), and the fourth case was reported from india in 1995. three cases were diagnosed by histopathological examinations and pus cultures, and the fourth case was diagnosed with the help of a seminal culture. all the cases had a similar clinical presentation : fever, weight loss, and testicular pain and / or swelling. involvement of the prostate is very rare and only nine cases have been previously reported. reinfection can occur in patients who have had histoplasmosis in the past, like our patient who had colonic histoplasmosis. when there is a decline in immunity in such a host, residual host defenses can not combat invasion by histoplasma. diagnosis of histoplasmosis is made by the demonstration of antigen in the urine, serum, relevant body fluids or pus. histopathology of the suspected lesion can reveal yeast structures of h. capsulatum via the hematoxylin and eosin stain ; however, the organism is better visualized using methenamine silver or periodic acid schiff stains (pas). confirmation can be obtained by setting up appropriate cultures, demonstrating the growth and dimorphic properties of the fungus. a high index of suspicion is needed for the diagnosis of histoplasmosis in immunosuppressed patients. the tissue, pus, and body fluid available for diagnosis must be subjected to investigations for uncommon infections, too. | genitourinary histoplasmosis is very rare and to our knowledge only four cases of epididymal histoplasmosis and nine cases of prostatic histoplasmosis have been reported in literature. we hereby report a case of a middle - aged male, who presented three years after renal transplant, with complaints of fever, pain, and swelling in the scrotum. imaging disclosed an enlarged right epididymis with prostatic and retrotrigonal abscess, suggesting tuberculosis infection. however, histopathology of the epididymal biopsy revealed histoplasmosis, and the drained pus on culture confirmed infection with histoplasma capsulatum. |
simulated datasets with known underlying disease mechanisms have been widely used to develop efficient statistical methods for deciphering the complex interplay between the genetic and environmental factors responsible for complex human diseases, such as hypertension, diabetes and cancer [1 - 3 ]. although genetic and environmental risk factors have been identified for various human diseases, little is currently known about how genes interact with environmental factors in these diseases. because the number of possible interactions between and within genetic and environmental factors is large, it is difficult to specify and simulate samples for a disease caused by multiple interacting genetic and environmental factors. consequently, existing studies have focused on simple models with low - order interactions between a few genetic and environmental factors, using specialized simulation programs. here, i discuss a recent article by amato and colleagues in bmc bioinformatics, which describes a mathematical model to characterize gene - environment interactions (gxe) and a computer program that simulates them using biologically meaningful inputs. i evaluate the usefulness of the authors ' method for simulating samples with gxe for future studies. if the number of genetic factors that cause a disease is g, we can denote each genetic factor by gi (where i = 1,..., g), and each of these will have three diploid genotypes. similarly, with e environmental factors, we can denote these xj, and each would have bj possible discrete values (where j = 1,..., e). a complete gxe model would then have possible items for each combination of genetic and environmental factors. in addition, the model would require this same number of parameters to specify the risk associated with each item. although such models can be used to specify arbitrary gene - gene and gene - environment interactions, estimating a large number of parameters from empirical data is challenging and usually not feasible. propose a statistical model, called the multi - logistic model (mlm), that is designed to describe disease risk in datasets that simulate case - control samples. mlm, which is a natural extension of logistic models used by others, allows the specification of disease risks caused by all genetic factors and by interactions between genotype and all environmental factors. it reduces the required number of parameters to 3 (1+e) by making the following assumptions : that the log odds ratios of environmental factors are additive ; and that the different environmental factors are independent and additive. the latter assumption means that only 1+e parameters are required for each combination of genotypes because the impact of bj levels of exposure for each environmental factor is represented by one parameter and no interaction between environmental factors is allowed. these assumptions limit the application of mlm in studies with correlated environmental factors (for example, smoking and drinking). the simplified model therefore can not be used to model complex gxe structures, such as the development of lung cancer caused by smoking and genetic factors because the impact of smoking is highly correlated with age, which is a common covariate in such models. despite this limitation, the mlm approach could be made more generally applicable by making adjustments, such as by applying principal component analysis, to the environmental factors to ensure their independence. even with the reduction of parameters afforded by the assumption of independence, the number of parameters in an mlm is still large if multiple genetic and environmental factors are involved. for example, an mlm requires 18 parameters when there are two genetic factors and one environmental factor. this is why the authors focused on a version of mlm with only one genetic factor and one environmental factor (giving only six parameters), which they implemented in matlab in their program gene - environment interaction simulator (gens). furthermore, users of gens can choose from four simpler models of gxe (figure 1) : a genetic model (no environmental factors, three parameters), an environmental model (no genetic factors, two parameters), a gene - environment interaction model (genotypes do not directly affect disease risk, four parameters), and an additive model (environmental factors have the same effect in all genotypes, four parameters). these models, although incomplete, should be sufficient for most theoretical studies of gxe models with one genetic factor and one environmental factor. four gxe interaction models provided by the knowledge - aided parameterization system (kaps) : (a) a genetic model, (b) an environmental model, (c) a gene - environment interaction model and (d) an additive model. each curve represents the relationship between disease risk (y - axis) and an environmental factor (x - axis) for individuals with a particular genotype (aa, aa or aa) at a disease - predisposing locus (dpl). the environmental model has only one curve because the relationship between environmental exposure and disease risk is identical for all genotypes. adapted from amato.. because changing an interaction item might change many properties (such as the marginal effects of a model) in an unpredictable way, it is difficult for users to adjust parameters in a gxe model to control for key epidemiological features of a disease such as population incidence.. used an innovative system, the knowledge - aided parameterization system (kaps), to translate user input in familiar epidemiological terminologies, such as model of inheritance, into the parameters used in mlm, which makes it easy for users to specify model parameters that are epidemiologically sensible. other constraints, such as relative risk between homozygotes and heterozygotes, are added to facilitate the search for suitable parameters. kaps works well for models with one genetic and one environmental factor because the number of epidemiological variables that users need to input is similar to the number of model parameters. for a general gxe model with multiple genetic and environmental factors, the number of epidemiological features of the disease and individual genetic and environmental factors will be far less than the number of model parameters because of the large number of interaction terms in mlm. because multiple models with different interaction terms could have the same epidemiological features, additional constraints are required to limit the number of plausible models, and a complex search algorithm might be needed to parameterize mlm with sensible interaction parameters., who used a genetic algorithm to discover, among a large number of plausible theoretical models, a special set of high - order gene - gene interaction models in which genes influence disease risk only through interactions with other genes, without any main effects. various different methods have been used to simulate case - control samples based on penetrance models. before applying their gxe model, amato. simulated a population to determine the affection status of each individual (that is, whether or not that individual is affected by the disease). the first method is to simulate a large population and then select case - control or other types of samples, such as pedigrees, from it. this approach allows maximum flexibility in the specification of a penetrance model and is usually used in a forward - time approach in which a population is simulated by evolving from a founder population forward in time under the influence of multiple genetic and demographic forces. this method can be inefficient if the disease is so rare that a large population needs to be simulated to obtain enough cases. if, alternatively, a disease model is simple enough, pr(gi, xj | affection status) (that is, the probability that the genotype is gi and the environmental factor value is xj given the affection status) can be determined from pr(affection status | gi, xj) together with other parameters, such as frequencies of these factors and disease prevalence. if this is the case, genotype and environmental factors can be simulated directly and only the required number of cases and controls needs to be simulated. this second approach has been used by many simulation programs, such as hapsample, hapgen and gwasimulator. as a compromise between these two approaches, a rejection - sampling algorithm can be used to simulate samples without simulating a large population (for example, genomesimla). this method repeatedly simulates individuals, assigns affection status, and collects cases and controls until enough samples have been simulated. this approach is suitable for situations in which environmental factors can be independently simulated for each individual, and could be used to improve the efficiency of gens. because genotypes at the disease - predisposing loci (dpl) of a genetic disease might not be available, many statistical methods rely on linkage disequilibrium (ld) between dpl and their surrounding markers to indirectly map the dpl. gens does not consider ld between dpl and surrounding genetic markers, so more sophisticated simulation methods are needed to simulate linked markers using genetically related individuals. existing approaches include : resampling from existing data (for example, hapsample or hapgen) ; reconstructing from statistical properties obtained from existing sequences (gwasimulator) ; simulating a complete genealogy (coalescent tree) of a sample or population (for example, cosi, genome) ; and evolving forward in time from a population. the power, flexibility, performance and quality of simulated samples vary greatly from program to program. for example, forward - time methods are most flexible because they can follow the evolution of a real population closely, but they are inefficient because they simulate all ancestors, including those who do not have offspring in the simulated population. gwasimulator retains the short - range ld structure of the human population (or more specifically, the hapmap sample) but discards long - range ld because the method simulates haplotypes according to short - range ld patterns obtained from the hapmap sample using a sliding - window approach. although it is generally possible to simulate large populations using these methods and then apply the gxe disease model proposed by amato. for example, many coalescent - based simulation methods simulate markers with varying location and allele frequency, so it is difficult to apply a fixed - disease model to replicate simulations. if a forward - time approach is used to simulate samples with the same set of markers, sample frequencies of the dpl will vary because of the impact of random genetic drift, unless special algorithms are used to control allele frequencies. even if samples with the same allele frequencies are simulated, the individuals generated may not have enough genetic variations to allow adequate modeling with a gxe model because of insufficient combinations of genetic and environmental factors. for example, from a sample of 20,000 sequences of 40 tightly linked markers over a 100 kb region on chromosome 17 simulated using hapgen, there were only 74 unique haplotypes because all the haplotypes were derived from the 63 unique haplotypes in the hapmap ceu sample using an imputation approach. amato. have provided a mathematical model for the specification of interactions between genetic and environmental risk factors. their simulation program gens can be used to generate simple, independent case - control samples with clear epidemiological interpretations and can be used to validate a statistical method or compare the performance of several statistical methods under specific assumptions. however, because real - world studies usually involve a large number of linked markers, a useful statistical method should be able to identify informative variables (dpl and environmental factors) from a large number of markers and covariates, or be efficient enough to be used to search for gxe signals exhaustively. the performance of statistical methods that detect gxe in complex human diseases, including sensitivity, specificity and ability to handle linked loci, should be tested against simulated samples of long genome sequences with realistic disease models and ld patterns. although progress has been made in both the simulation of long genome sequences and gxe disease models, the combination of these two approaches would produce realistic samples that could greatly aid the study of gxe in complex human diseases. dpl : disease - predisposing locus ; gens : gene - environment interaction simulator ; gxe : gene - environment interaction ; kaps : knowledge - aided parameterization system ; ld : linkage disequilibrium ; mlm : multi - logistic model. this work was supported by grant r01 ca133996 from the national cancer institute and by md anderson 's cancer center support grant ca016672 from the national institutes of health. | because little is currently known about how genes interact with environmental factors in human diseases, and because of the large number of possible interactions between and within genetic and environmental factors, it is difficult to simulate samples for a disease caused by multiple interacting genetic and environmental factors. a recent article by amato and colleagues in bmc bioinformatics describes a mathematical model to characterize gene - environment interactions and a computer program that simulates them using biologically meaningful inputs. here, i evaluate the advantages and limitations of the authors ' approach in terms of its usefulness for simulating genetic samples for real - world studies of gene - environment interactions in complex human diseases. |
although many effective chemically synthesized drugs exist for the treatment of hypertension, there is a great deal of interest in using natural plant extracts to attenuate the increased blood pressure levels in hypertensive patients and to prevent the cardiovascular disorders associated with this disease [13 ]. the beneficial properties of many plants have been frequently attributed to the presence of phenolic compounds. among them, iridoids, a particularly widespread group of monoterpenes, retain their chemical composition even when exposed to stressful environments or after processing. therefore, iridoids may be considered more stable and more bioavailable than other well - known phenolic compounds, as flavonoids. in addition, several biological properties have been attributed to iridoids which have been recognized as important components of medicine and healthy diet [5, 6 ]. despite the many known biological properties of these compounds, their effects on blood pressure have been poorly investigated [79 ]. a fraxinus excelsior l. seed extract (fese) the analytical study of fese revealed that it was rich in nuzhenide and gi3, two phenolic compounds belonging to the secoiridoid type. fese lowered the incremental postprandial plasma glucose concentration in nondiabetic volunteers and limited weight gain and hyperglycemia in high - fat induced obese mice. recently, we have demonstrated the short - term antihypertensive effect of this extract in spontaneously hypertensive rats (shr), a model for essential hypertension in humans. fese also showed diuretic and antioxidant properties that could contribute to the decreased arterial blood pressure in the shr. in addition, the safety and tolerability of this extract for consumption in healthy subjects have also been demonstrated. the aim of this study was to evaluate the effect of the long - term intake of fese on the development of hypertension of shr. weight gain and food and liquid intake were evaluated in the animals throughout the experimental period. we also investigate the possible antioxidant properties of fese by analysing plasma and liver tissue samples from the different groups of rats. moreover, we carried out complementary trials in aorta ring preparations obtained from these rats in order to investigate the possible modification of endothelial function by this extract. after being weaned at 3 weeks, 60 male shr (charles river laboratories espaa s.a.) were caged in groups of five rats at a temperature of 23c with 12 h light / dark cycles. they were in turn randomly divided into six groups of 10 animals with ad libitum intake. during the experimental period, the shr of the established groups were fed on a food standard diet for rats (a04 panlab, barcelona, spain). the rats drank different fluids from weaning until the 20th week of life (treatment period) and received therefore different treatments : tap water (negative control), fese 20 mg / kg / day, fese 40 mg / kg / day, fese 60 mg / kg / day, or captopril (sigma, usa) 50 the quantities of the different products, to be daily administered during this period of life, were selected according to the results obtained when we established the short - term antihypertensive effect of fese in shr. from the 20th to the 24th week of life all the animals were given tap water (follow - up period). we measured the antioxidant capacity of the plasma samples and determined malondialdehyde (mda) plasma levels. the thoraxes of the sacrificed rats were opened and the aorta was quickly extracted to evaluate the endothelial function in accordance with the studies published by furchgott in 1980 and in 1999 [15, 16 ]. systolic blood pressure (sbp) was measured weekly in the rats from the 6th to 24th week of life, by the tail cuff method. before the measurements, the rats were kept at 38c for 1015 minutes to make the pulsations of the tail artery detectable. arterial blood pressure measurements were performed at the same time of day (between 9 a.m. and 13 p.m.) in order to avoid the influence of the circadian cycle, and the values of sbp were obtained by estimating the average reading of five measurements. moreover, the person who measured the arterial blood pressure in the animals did not know the drinking fluid that had been administered to each group. the body weight of the rats was recorded weekly up to the 24th week of life. daily intake of drinking fluids and freely accessible feed was also estimated weekly in the shr from the different groups throughout the experimental period. at the end of the experimental period, the same determinations and procedures took place in these animals as for the sacrificed 20-week - old rats. all the above - mentioned experiments were performed as authorized for scientific research (european directive 86/609/cee and royal decree 223/1988 of the spanish ministry of agriculture, fisheries and food). the blood samples were collected into tubes containing lithium heparin as anticoagulant and centrifuged at 3500 g for 20 min to obtain the plasma. livers were homogenized at 4c in a potter with phosphate buffered saline (pbs) (0.01 m pbs, 0.15 m nacl, ph 7.4). the homogenates were centrifuged at 5000 g for 15 min at 4c and the supernatant was recovered. the plasma and the supernatants of the centrifuged samples were kept frozen at 80c until analysis. the liver protein content was determined by the bicinchoninic acid assay (pierce, rockford, ill, usa), using bovine serum albumin as standard. we measured the antioxidant capacity of the plasma samples by using the oxygen radical absorbance capacity (orac) assay, as previously described in manso. briefly, the final assay mixture contained fluorescein as oxidable substrate, 2,2-azobis(2-amidinopropane) dihydrochloride (aaph) as oxygen radical generator, and the antioxidant trolox either as standard or plasma samples. samples and standards were dissolved in pbs (ph 7.4). a polarstar galaxy plate reader with a 485-p excitation and a 520-p emission filter was used. fluorescence measurement was carried out at 37c and was recorded every minute for 80 min. orac values were calculated and expressed as mol of trolox equivalent / ml of plasma. plasma and liver mda levels were measured by the thiobarbituric (tba) assay previously described also in manso. briefly, plasma and homogenates of tissues were mixed with trichloroacetic acid, and the tubes were kept in ice for 20 min. samples were centrifuged at 1500 g for 15 min. before adding tba to the supernatant ; then, the mixture was boiled at 97c for 30 min. plasma mda values were expressed as nmol mda / ml and, for the different tissues, as nmol mda / mg tissue protein. the thoracic aorta was carefully cleaned of fat and connective tissue and cut into rings (approximately 4 mm in length). the aorta rings were mounted between two steel hooks in isolated tissue chambers containing krebs - henseleit solution with the following compositions (in mmol / l) : nacl, 118 ; kcl, 4.7 ; cacl2, 2.5 ; kh2po4, 1.2 ; mgso4, 1.2 ; nahco3, 25 ; and glucose, 10.0. all rings were allowed to equilibrate for 1 h at a resting tension of 2 g before adding drugs. isometric tension was recorded by using an isometric force displacement transducer connected to an acquisition system (protos 5, panlab, spain). after the equilibration period, the rings were first contracted by 80 mm kcl to assess their functionality, and when the contraction had reached the steady state (about 15 min) the preparations were washed until the basal tension was recovered. then the vessels were exposed to methoxamine (10 m), and the presence of functional endothelium was assessed by the ability of acetylcholine (ach, 10 to 10 m) to induce relaxation. relaxant responses to ach were expressed as a percentage of the precontraction induced by methoxamine. sem for 510 experiments and were analysed by a one- or two - way analysis of variance (anova), by using the graphpad prism software. for the analysis, we considered, on the one hand, the treatment period (from weaning until the 20th week of life) and, on the other hand, the follow - up period (from the 20th to the 24th week of life). differences between the groups were assessed by the bonferroni test and were considered to be significant when p < 0.05. the antihypertensive effect was more accentuated in the group treated with captopril, and it was quite similar in all the fese groups. nevertheless, it was paradoxically more accentuated in the groups treated with 20 mg / kg / day or 40 mg / kg / day of fese than in the group treated with the highest dose of this extract (60 mg / kg / day) (figure 1). in addition, food intake increased somewhat in the fese groups, and all the rats treated with this extract showed an increase in body weight gain. on the contrary, the animals treated with captopril showed less body weight than the rats of the water group, even if the food intake in all these animals was similar (figure 2). the arterial blood pressure increased in the treated shr nevertheless, at 24 weeks of life, systolic blood pressure remained lower in the captopril group than in the other groups. body weight of the fese treated animals returned to control values along the follow - up period. liquid intake was variable in all animals along the experimental period, but we could appreciate a clear increase in this variable in the captopril treated rats (figure 3). plasma orac values in the 20-week old rats that had been long - term treated with 20 or 40 mg / kg / day fese were significantly higher than the corresponding values in the control group. when these treatments were removed, plasma orac values returned to control values, and during the follow - up period (from the 20th to the 24th week of life) no significant differences were observed between the plasma orac values of these groups of rats and the corresponding values in the control rats that received tap water along all the experimental period. long - term treatment with 50 mg / kg / day captopril did not modify plasma orac values in the rats and the animals that had been treated with this drug showed also similar orac values to the control animals along the follow - up period (figure 4). long - term treatment with all the used doses of fese and also 50 mg / kg / day captopril significantly lowered plasma mda equivalents in the shr. long - term treatment with 20 mg / kg / day fese has the most accentuated effect, and when the corresponding treatments were removed, only mda values of the rats that had been treated with this dose of the extract remained significantly lower when compared with the control group. in addition, long - term treatment with all the used doses of fese and also 50 mg / kg / day captopril, significantly lowered liver mda equivalents in the shr when compared to control group. when the corresponding treatments were removed, all treated groups showed similar values compared to control group (figure 5). acetylcholine relaxation improved in the aorta rings from the rats that had been treated with captopril or fese. in particular, acetylcholine relaxation improved in the aorta rings from the rats that had been treated with captopril, but no significant differences were observed between the relaxations caused by this agonist in these preparations and the corresponding relaxations in aorta rings from the groups that had been treated with 40 mg / kg / day or 60 mg / kg / day of fese. in fact, acetylcholine relaxation improved more in the aorta rings from the rats that had been treated with these doses of fese than in the aorta preparations obtained from the 20 mg / kg / day fese treated animals (figure 6). after the follow - up period, acetylcholine relaxation was also more marked in the aorta rings from the rats that had been treated with captopril or fese than in the aorta rings from the rats that drank water along all the experimental period. in a previous study, we demonstrated the short - term antihypertensive effect of fese in shr. nevertheless, this study clearly demonstrated that this extract can also control arterial blood pressure in these animals when it is long - term administered. shr are considered nowadays one of the best experimental models to evaluate antihypertensive functional food ingredients. the progression of hypertension in these animals is in fact similar to that in humans. several researchers, including our own research group, have reported that, before arterial blood pressure stabilizes in shr, there is an initial period in the life of these animals in which this variable clearly increases [2123 ]. an initial gradual increase in sbp was in fact observed in the present study in the shr of the control group, and we also noted the stabilized arterial blood pressure period in these animals. both, fese and captopril, attenuated the development of hypertension in the shr and also clearly decreased their sbp in the period with stabilized arterial blood pressure values. nevertheless, the decreases in sbp caused by all the used doses of fese were always less accentuated than the decrease of this variable caused by 50 mg / kg / day captopril. in this context, it is important to notice that our research group characterized this dose of captopril as a very high dose, that caused the maximum effect when it was short - term administered to the shr. captopril is in fact a well - known antihypertensive drug with clinical use in hypertensive patients and, as was expected, during the experimental period, the lowest values of sbp were observed in the group of rats treated with 50 mg / kg / day of this drug, but the antihypertensive effect of fese was also maintained and could be clearly observed in the shr stabilized arterial blood pressure period. the highest dose of fese (60 mg / kg / day) caused even less effect than the other doses of this extract (20 mg / kg / day and 40 mg / kg / day), but neither in the short - term study with fese, we obtained the most pronounced antihypertensive effect with the highest dose of this product. moreover, it is important to state that this paradox also happened with different polyphenols and polyphenol - rich compounds that we have evaluated in shr. after removing fese treatments, in fact, from the 22nd week of life, sbp values were very similar in the rats that had drunk the solutions of fese and those that had drunk water. different researchers have similarly described a gradual rise in sbp when the intake of other antihypertensive food derived products was stopped in shr [22, 25, 26 ]. we also observed an increase in the arterial blood pressure of the rats that had been treated with captopril when this treatment was removed. nevertheless, the reversion was less noticeable, because this drug is a potent antihypertensive agent, and the rats that had drunk the solution of captopril had minor values of sbp than the other groups also during the follow - up period. (2000) also reported a slight increase in arterial blood pressure in shr following the withdrawal of captopril treatment. shr receiving captopril gained weight at a lower rate than the other animals even if the shr treated with this drug did not show an increase in food intake. this finding is in agreement with previous studies showing that blockade of renin angiotensin system slows body weight gain in this rat strain. it is nevertheless important to state that the shr treated with fese showed higher values of body weight than the control rats and slightly higher values of dry food intake. the mechanism implicated in this orexigenic effect of fese is unknown and should be studied in the future. nevertheless, liquid consumption increased markedly in the rats that drank the captopril solution, but this is not surprising because it has been clearly demonstrated that angiotensin converting enzyme inhibitors induce thirst and increase water intake and urine output in rats [29, 30 ]. many plants with antihypertensive properties, that are having a marked impact on the food sector, are rich in phenolic antioxidant compounds. the relationship between natural phenol content in natural products and health is nevertheless still controversial, and there are problems to draw general conclusions regarding a health effect common to all phenolic compounds, as their structures are extremely diverse. in spite of these difficulties, it is important to have in mind that the secoiridoids nuzhenide and gi3 (in particular gi3), which are present in fese, act as antioxidants and have proved to have preventive effects on oxidative stress in rats. moreover, our research group has already demonstrated that the acute administration of fese improves arterial blood pressure and the antioxidant state in shr. there exist a large number of methodologies aimed at evaluating the antioxidant capacity of complex mixtures. orac type methodologies stand among the most employed ones to measure the total antioxidant potency of foods and nutritional supplements. in addition, mda is a biomarker that enabled us to estimate lipid peroxidation in the rats. in this study, we have clearly demonstrated that the administration of 20 mg / kg / day or 40 mg / kg / day of fese, the most effective antihypertensive doses of this extract, significantly increased plasma orac values in the shr and significantly decreased plasma and liver mda levels in these animals. captopril failed nevertheless to increase the total antioxidant capacity of the shr plasma samples, even if this drug enhances the antioxidant capacity in hypertensive patients and has been postulated as a free radical scavenger [36, 37 ]. moreover, fese caused a more marked decrease in liver mda than captopril in the shr. the effects of fese on the orac and mda values did not totally disappear after removing the treatment. in particular, the plasma mda values of the 24-week - old rats that had been treated with 20 mg / kg / day fese remain significantly lower than those of the control rats of the same age. we should therefore assume that the antioxidant properties of fese could justify, at least in part, the antihypertensive effects of this extract. in this study, we could also assume that fese improves endothelial function, since the relaxation to acetylcholine increased in the aorta rings from the shr that had been treated with fese as compared to the control group. the improvement was dose - dependent and was still observed in the aorta from the rats after the follow - up period. nevertheless, the relaxation to acetylcholine increased always more in the aorta from the captopril positive control group than in the aorta from the fese groups. these results suggest that the effect on endothelial function could also justify in part the antihypertensive effect of fese. in summary, long - term intake of fese improves arterial blood pressure, oxidative stress, and endothelial function in shr. the slight and maintained effect produced by fese in the shr suggests that this extract could be used as a functional food ingredient with potential therapeutic benefits in the prevention and treatment of hypertension and other associated disorders. nevertheless, we are aware that before any clinical use of fese, it would be necessary to carry out clinical studies to demonstrate its long - term antihypertensive efficiency in humans. having in mind the results presented in this study, the possible orexigenic effect of fese should also be studied in the future. | the effect of long - term intake of different doses (20, 40, and 60 mg / kg / day) of a fraxinus excelsior l. seed extract (fese) on spontaneously hypertensive rats (shr) was evaluated. water was used as control and captopril (50 mg / kg / day) was used as positive control. systolic blood pressure, body weight, and food and liquid intake were registered weekly in shr. the antioxidant and vascular relaxing properties of fese were also studied in these animals. the development of hypertension was attenuated in the groups treated with captopril or fese. the antihypertensive effect was more accentuated in the captopril group than in the fese groups, and it was paradoxically more accentuated in the groups treated with 20 mg / kg / day or 40 mg / kg / day of fese than in the group treated with the highest dose of this extract. body weight gain and food intake increased in the fese groups. after removing the corresponding antihypertensive treatment, the arterial blood pressure and the body weight of the fese treated animals returned to control values. in addition, fese increased plasma antioxidant capacity and decreased plasma and liver malondialdehyde levels. moreover, acetylcholine relaxation improved in the aorta rings from the fese treated rats. |
bedding can be effectively used as a floor covering to absorb moisture and provide a soft surface. some studies have used the preference test to investigate the bedding preference in horses and ponies [3, 4, 6 ]. these studies have reported that the animals preferred straw over shavings and paper as the material for bedding. in japan, rice straw has frequently been used as bedding, and in some cases, used straw bedding is often reused after drying in the sunshine. (2004) reported that drying the bedding in sunshine was not sufficient to completely clean it from urine ammonia. moreover, rice straw has been unobtainable in japan and imported from foreign countries, and the law relating to excreta of livestock was proclaimed. therefore, it is required to use a new reusable material for bedding. in this study, the husk and fiber of coconuts, which are wasted during the production of coconut milk, oil, etc. from coconut, were used as bedding materials. we investigated the effect of these bedding materials and that of the commonly used bedding materials, namely, straw and sawdust, on the welfare of stable horses by observing their resting behavior. twenty horses ranging from 3 to 21 years of age were used at the equine research institute of the japan racing association, utsunomiya, japan. the breeds of horses were thoroughbred (n=8), anglo - arab (n=3), selle francais (n=3), thoroughbred mixed breed (n=2), and dutch warmblood, haflinger, hannover, and westfalen (n=1 each). the horses were kept individually in box stall which floor was concrete (2.6 m 3.6 m). they were ridden from 7:00 to 9:00 or were turned out into a pasture or a paddock from 09:00 to 13:00. each horse received a pelleted concentrate and oat mixture and cut alfalfa hay at 05:30 and 15:30, and long timothy hay at 12:00 as a maintenance ration. the following 4 types of bedding materials were used : straw, sawdust, coconut husk (husk), and coconut fiber (fiber). the husk was made of crushed coconut husk and the fiber bedding was prepared with husk bedding covered with fiber from coconut husk. three weeks before the behavioral observation, the bedding was changed to the experimental bedding. five horses were allocated to each bedding condition. in every bedding condition, the box stalls were cleaned and their faeces were removed once daily. dirty straw was replaced with new straw daily, and the other beddings were replaced with new bedding once a week. the behavior of each horse was recorded by video camera for 3 days and was continuously sampled from 17:00 to 05:00. the box stalls were illuminated during the observation period. the total duration (minutes per a day), the number of bouts (times per a day), the mean and the maximum duration of bouts (minutes) in standing rest, sternal lying, and lateral lying were calculated. the data in each behavioral category were analysed with the kruskal - wallis test, and a post hoc steel - dwass test was used to investigate the differences among beddings. twenty horses ranging from 3 to 21 years of age were used at the equine research institute of the japan racing association, utsunomiya, japan. the breeds of horses were thoroughbred (n=8), anglo - arab (n=3), selle francais (n=3), thoroughbred mixed breed (n=2), and dutch warmblood, haflinger, hannover, and westfalen (n=1 each). the horses were kept individually in box stall which floor was concrete (2.6 m 3.6 m). they were ridden from 7:00 to 9:00 or were turned out into a pasture or a paddock from 09:00 to 13:00. each horse received a pelleted concentrate and oat mixture and cut alfalfa hay at 05:30 and 15:30, and long timothy hay at 12:00 as a maintenance ration. the following 4 types of bedding materials were used : straw, sawdust, coconut husk (husk), and coconut fiber (fiber). the husk was made of crushed coconut husk and the fiber bedding was prepared with husk bedding covered with fiber from coconut husk. three weeks before the behavioral observation, the bedding was changed to the experimental bedding. five horses were allocated to each bedding condition. in every bedding condition, the box stalls were cleaned and their faeces were removed once daily. dirty straw was replaced with new straw daily, and the other beddings were replaced with new bedding once a week. the behavior of each horse was recorded by video camera for 3 days and was continuously sampled from 17:00 to 05:00. the box stalls were illuminated during the observation period. the total duration (minutes per a day), the number of bouts (times per a day), the mean and the maximum duration of bouts (minutes) in standing rest, sternal lying, and lateral lying were calculated. the data in each behavioral category were analysed with the kruskal - wallis test, and a post hoc steel - dwass test was used to investigate the differences among beddings. there was no significant difference among beddings in the standing rest and the sternal lying (tables 1 and 2table 1.effects of bedding materials on the standing rest in stabled horsebedding materialsstrawsawdusthuskfibertotal duration (minutes)381.6 88.7437.8 75.7409.8 75.7443.8 151.2number of bout20.7 4.824.1 4.926.8 9.816.7 5.8mean duration of bout (minutes)18.4 11.818.2 3.715.3 7.526.6 8.5maximum duration (minutes)93.0 59.0116.4 78.5102.8 43.393.9 husk was made of crushed coconut husk and fiber was prepared with husk bedding covered with fiber from coconut husk.table 2.effects of bedding materials on the sternal lying in stabled horsebedding materialsstrawsawdusthuskfibertotal duration (minutes)38.6 33.119.4 20.169.6 39.419.1 28.4number of bout2.8 2.61.7 1.56.1 3.61.7 1.4mean duration of bout (minutes)13.8 7.911.6 5.811.3 4.811.0 8.3maximum duration (minutes)31.5 27.913.6 9.828.7 10.213.4 husk was made of crushed coconut husk and fiber was prepared with husk bedding covered with fiber from coconut husk.). significant differences in the total duration, the number of bouts, and the mean and the maximum duration of bouts in the lateral lying were observed among beddings (p<0.05). the values of the means of the total duration, the number of bouts, and the mean and the maximum duration of bouts in the lateral lying when husk was used as bedding were greater than those when sawdust were used as bedding (p<0.05, table 3table 3.effects of bedding materials on the lateral lying in stabled horsebedding materialsstrawsawdusthuskfibertotal duration (minutes)4.0 5.60.02 0.0510.1 10.91.5 2.5number of bout1.6 1.80.1 0.153.4 0.90.3 0.5mean duration of bout (minutes)2.5 1.10.3 0.013.0 0.94.4 2.5maximum duration (minutes)4.4 3.90.07 0.155.1 2.23.8 husk was made of crushed coconut husk and fiber was prepared with husk bedding covered with fiber from coconut husk. numbers with different letters are statistically different (a, b : p<0.05).). husk was made of crushed coconut husk and fiber was prepared with husk bedding covered with fiber from coconut husk. husk was made of crushed coconut husk and fiber was prepared with husk bedding covered with fiber from coconut husk. husk was made of crushed coconut husk and fiber was prepared with husk bedding covered with fiber from coconut husk. numbers with different letters are statistically different (a, b : p<0.05). resting behavior when husk and fiber bedding were used was not different from that in straw treatment. the resting behavior indicates that these new bedding materials would be as usable as straw. however, lateral lying was observed less frequently with sawdust, which is a commonly used bedding material, than when husk was used as bedding. the lateral lying is said to be related to sleeping, which is a positive indicator of welfare in stabled horses. it is also reported that horses preferred straw bedding over shavings in the preference tests. the less frequent observation of the lateral lying with sawdust indicates that sawdust used as bedding material decrease the welfare of stabled horses. in this study, replacing dirty bedding with new bedding of sawdust was performed once a week although faeces in stalls were removed once a day. moreover, it is said that the sleeping behavior was related to learning and memory (review,). therefore, further investigations on the effect of bedding material and the method of cleaning the bedding on the horses welfare and learning are required. however, in animal welfare, we focus on the physical and mental health of the animals with reference to the 5 freedoms. the concept of 5 freedoms are (1) freedom from hunger and thirst ; (2) freedom from discomfort ; (3) freedom from pain, injury, or disease ; (4) freedom to express normal behavior ; and (5) freedom from fear and distress. bedding material is related to the freedom from discomfort in terms of moisture, soft surface, and air ammonia, freedom from pain, injury, or disease in terms of hoof disease and skin conditions, and the effect of bedding materials on the welfare of stabled horses should be evaluated not only based on the resting behavior but also based on the water content in bedding, airborne ammonia (e.g.), and hoof condition. moreover, the use of beddings should be evaluated in terms of management and costs for the applied purpose. | the objective of this study was to investigate the effect of straw, sawdust, coconut husk (husk), and coconut fiber (fiber) on the welfare of stable horses by observing their resting behavior. twenty horses with ages ranging from 3 to 21 years were used at the equine research institute of the japan racing association, utsunomiya, japan. five horses were allocated to each bedding condition. the behavior of each horse was recorded by video camera for 3 days and was continuously sampled from 17:00 to 05:00. the total duration, the number of bouts, and the mean and the maximum duration of bouts in standing rest, sternal lying, and lateral lying were calculated and analysed by the kruskal - wallis test and post hoc steel - dwass test. there was no difference in the standing rest and the sternal lying among beddings. significant differences were observed in these values in the lateral lying among the different beddings (p<0.05). the values of the means of the total duration, the number of bouts, and the mean and the maximum duration of bout in the lateral lying were greater when husk was used as the bedding material than when sawdust were used (p<0.05). the results of the observations show that the new bedding materials would be as usable as straw. however, lateral lying was observed less frequently when sawdust were used as bedding ; this indicates that use of sawdust as bedding material will decrease the welfare of stabled horses. |
obesity is associated with infertility and an increased risk of preterm birth in pregnant women. women who are unable to achieve this weight loss with conservative therapies can decide to undergo bariatric surgery, which has shown to improve fertility up to 58% and to reduce adverse maternal and neonatal outcomes [24 ]. bariatric surgery - related complications requiring acute abdominal surgery such as internal herniation after gastric bypass surgery might occur during pregnancy. additionally, cases of slippage of laparoscopic adjustable gastric banding (lagb) are described in the literature. banded laparoscopic roux - en - y gastric bypass (b - lrygb) is a relatively new technique and is believed to provide additional weight loss when compared with non - banded lrygb. however, no articles have been published regarding b - lrygb - related complications during pregnancy. we report a case of a 30-year - old woman who succeeded to become pregnant after weight loss due to b - lrygb, but presented with acute abdominal pain in week 33 of her pregnancy. a 30-year - old woman with a history of polycystic ovary syndrome (pcos) visited the obesity center amsterdam to opt for weight loss surgery. due to her body mass index (bmi) of 41 four years later, she visited our hospital with complaints of weight regain and persistent irregular menstruation, and a still unfulfilled pregnancy wish. she claimed that her eating pattern had not changed, she was no sweetener and she exercised by cycling 40 km / week. no abnormalities were found during esophagogastroduodenoscopy. during a multidisciplinary meeting, it was decided to revise the dilated pouch and apply a minimizer ring (bariatric solutions, stein an rhein, switzerland) around the gastric pouch. during surgery, the gastric pouch appeared not dilated, after which was decided not to revise the pouch and solely apply a minimizer ring (bariatric solutions, stein an rhein, switzerland) 2 cm above the gastrojejunostomy. the minimizer ring was locked at the first hole (8 cm ; diameter 2.55 cm) and fixated at the pouch with vicryl (ethicon inc. her excess weight loss was 28.1% ; total bmi loss was 4.5 points and total body weight loss 14 kg. in the third week of pregnancy, patient visited our emergency room with epigastric pain with radiation to her back since 2 days. laboratory tests showed a c - reactive protein 36 mg / l, leucocytes 9.1 10 9/l and hemoglobin 6.1 computed tomography (ct) of the abdomen suggested dislocation of the minimizer ring possible internal intestinal herniation and dilation of the intestines (fig. figure 1:ct - scan of the abdomen showing an ileus caused by a migrated minimizer ring. ct - scan of the abdomen showing an ileus caused by a migrated minimizer ring. therefore, patient received a nasogastric tube and was transferred to the operating room for emergency surgery. the fetus was monitored by a gynecologist with cardiotocography and the pediatrician was informed about the situation. inspection along the alimentary limb resulted in locating the minimizer ring at the transition of alimentary limb and common channel. after removal of this ring, the biliopancreatic limb and common channel were visible again (fig. since gastric retention was no longer present, the nasogastric tube was removed several hours post - surgery. no complications occurred. as patient desired a new minimizer, it was advised to discuss this when no further pregnancies were planned and only in case of weight regain. a recent systematic review and meta - analysis reported improved pregnancy outcomes such as less preeclampsia and less gestational diabetes mellitus after bariatric surgery. although the authors conclude that more research is needed comparing pregnancy outcomes between several bariatric procedures, they recommend lagb in women planning to become pregnant. in the netherlands, it is recommended to perform a laparoscopic sleeve gastrectomy in young women who want to become pregnant in future. this avoids the occurrence of gastric bypass - related complications such as internal herniation and band - related complications such as slippage. however, when a young woman is scheduled for b - lrygb, surgical precautions must be taken. during b - lrygb surgery, the usage of insoluble suture material such as mersilene instead of vicryl should be considered in order to decrease the risk of band migration due to increased abdominal pressure during pregnancy. in order to increase as much as perioperative safety as possible in pregnant women, several precautions could be taken : careful open introduction in the left upper quadrant, as high as possible, subcostal ; usage of three trocars ; left lateral lift to reduce vena cava pressure and increase venous backflow and perioperative cardiotocography. although the optimal time for pregnancy after bariatric surgery is unknown, it is recommended to delay pregnancy until the first postoperative year. furthermore, women who remain overweight or obese due to insufficient weight loss following bariatric surgery are still at increased risk of adverse maternal and neonatal outcomes and should be regarded as a risk group [1, 10 ]. nevertheless, bariatric surgery has shown to be successful in the improvement of fertility and should be considered in morbidly obese women with a pregnancy wish, but fail to conceive due to their obesity. | women desiring pregnancy might fail to conceive due to their obesity. bariatric surgery has shown to reduce this infertility up to 58% and is therefore considered a successful strategy for morbidly obese infertile women. nevertheless, when pregnancy has succeeded, surgery - related complications might occur.banded laparoscopic roux - en - y gastric bypass (b - lrygb) is a relatively new technique in which a band is placed around the small gastric pouch. we report a case of a 30-year - old woman who succeeded to become pregnant after weight loss due to b - lrygb, but presented with acute abdominal pain in week 33 of her pregnancy. |
cutaneous paraneoplastic syndromes include acanthosis nigricans, the sign of leser - trlat, eruptive acrochordons, acquired ichthyosis, erythroderma, palmoplantar hyperkeratosis, acquired hypertrichosis lanuginosa, erythema gyratum repens, necrolytic migratory erythema, bazex 's syndrome and dermatomyositis. the presence of either syndrome in patients warrants evaluation for a possible underlying malignancy. here we report the case of a 79-year - old man, who developed numerous several centimeter - large seborrheic keratoses on his chest and back within the time period of one year. workup led to the diagnosis of colonic adenocarcinoma, underscoring the paraneoplastic etiology of the seborrheic keratoses in this patient. a 79-year - old man with a previous diagnosis of chronic lymphocytic leukemia was seen for preoperative evaluation prior to elective right peripatellar bursectomy after having failed antibiotic treatment. on physical examination numerous seborrheic keratoses in a christmas tree pattern were noted on his chest and back (fig. the patient reported that the lesions had appeared and advanced within the past twelve months on his trunk. he was seen by a dermatologist, who suggested performing endoscopies of the upper and lower gastrointestinal tract. the patient had not noted clinical symptoms otherwise suggestive of colon cancer, such as unintentional weight loss or recent change of bowel habits. the clinical presentation of the seborrheic keratoses in this patient was found to be consistent with the sign of leser - trlat. leser - trlat is a rare dermatologic finding named after two surgeons, the german edmund leser (18281916) and the french ulysee trlat (18271890). however, the first descriptions of patients with characteristic signs of leser - trlat were published in 1900 and 1901 by the german dermatologist eugen hollnder. the leser - trlat sign is characterized by the sudden onset of multiple seborrheic keratoses or an unusually rapid increase in size and number of preexisting seborrheic keratoses, occasionally on an inflammatory base. it is commonly associated with malignancies, primarily gastrointestinal adenocarcinomas [5, 6 ], but also squamous cell carcinomas, leukemias and lymphomas. although patients with nonmalignant conditions, such as pregnancy, hiv infection or after having received heart transplantation, can also develop seborrheic keratoses of florid eruption, the term leser - trlat usually implies paraneoplasia. the significance of this sign has been debated in the medical literature, as leser - trlat is observed primarily in elderly individuals, who are statistically at higher risk for developing both seborrheic keratoses as well as various malignancies. therefore, accurate diagnosis is linked to the rapidity of onset of seborrheic keratoses, and to the growth behavior of previously noted seborrheic keratoses. only rarely has leser - trlat been reported in younger patients. the seborrheic keratoses seen in leser - trlat differ neither morphologically nor histologically from commonly seen seborrheic keratoses. they are found predominantly on back and chest of patients, and there is no predilection for patients gender or ethnic / ancestral background. the most prevalent clinical symptom is pruritus, seen in 43% of malignant cases [12, 13 ]. in approximately 20% of cases acanthosis nigricans is also present, either at the time of cancer diagnosis or it develops subsequently. while the pathogenesis of this condition is unclear and remains to be elucidated, due to the proliferative nature of cutaneous paraneoplasias, a possible pathogenic role of epidermal growth factor and alpha - transforming growth factor in the development of the seborrheic keratoses associated with malignancies has been hypothesized and reported. on rare occasions, skin manifestations similar to the leser - trlat sign can be mimicked by malignant melanoma, thus justifying excision biopsies and histologic examinations of suspicious lesions in distinct cases. florid eruption or abnormally fast growth of seborrheic keratoses especially in elderly patients should always raise the suspicion for an underlying malignant disease, in particular for gastrointestinal adenocarcinomas. performing an endoscopic evaluation of the gastrointestinal tract of affected patients | we report the case of a 79-year - old caucasian man, who developed numerous pruritic seborrheic keratoses on his chest and back within one year. an underlying malignant disease was suspected. upper and lower endoscopies were performed and the patient was diagnosed with adenocarcinoma of the ascending colon. we discuss the clinical significance of his cutaneous symptoms / signs, and review the medical literature on the paraneoplastic sign of leser - trlat. |
atherosclerotic renal artery stenosis (ras) is an important and frequently unrecognized contributor to refractory hypertension (htn), ischemic nephropathy, and cardiac destabilization syndromes (unstable angina, flash pulmonary edema, and decompensated heart failure).15 atherosclerotic ras is a progressive disease leading to renal atrophy over time and chronic kidney disease despite control of htn.613 presence and severity of incidental ras is an independent predictor of mortality in atherosclerotic patients regardless of the mode of treatment of underlying coronary artery disease.1315 the prevalence of ras has been reported to be in the range of 2030 percent in high risk populations including patients with known atherosclerotic vascular disease elsewhere.1618 in these patients invasive screening for ras is highly cost - effective especially when done at the time of another invasive diagnostic procedure like cardiac catheterization, and may affect treatment strategies.19 atherosclerosis is a diffuse process but affects certain regions of the vascular bed preferentially. the association between extent and severity of cad and ras has been well established in most previous studies,1214 but a few has been addressed the relationship between the distribution of lesions in coronary tree and ras.2,8,14 the main purpose of our study was to find out if any association exists. in parallel, we evaluated the relationship between demographic variables, atherosclerotic risk factors, renal function, and levels of intra - arterial pressures during catheterization and ras. in conclusion, we postulated that these findings might help decide in which group of patients screening renal angiography could be justified following coronary angiography. over a period of 12 months from november 2008, data collected prospectively on 500 consecutive patients who underwent simultaneous renal angiography following coronary angiography in shaheed rajaie cardiovascular medical, and research center. the indications for coronary and renal angiography were at the discretion of the attending cardiologist. demographic variables, laboratory data, and history of atherosclerotic risk factors obtained from the patients medical record. patients considered hypertensive, if they had a positive history of htn who were under life style modification and/or medical treatment. patients considered diabetic (dm) as either currently taking anti - diabetic medications or having more than one fasting plasma glucose (fpg) 126 mg / dl on laboratory record. dyslipidemia (dlp) defined if the patient was under medical treatment for known dyslipidemia or any of the following laboratory criteria existed : fasting low - density lipoprotein cholesterol (ldlc) 130 mg / dl, high - density lipoprotein cholesterol (hdlc) 40 mg / dl in men and 50 mg / dl in women or triglycerides 150 mg / dl. patients considered having a history of smoking if they were current or former smokers. a positive family history (fh) established based on a known history of cad in a first degree relative male or female less than 45 and 55 years respectively. glomerular filtration rate (gfr) estimated by the four variable modification of diet in renal disease (mdrd) equation using plasma creatinine concentration, age, and gender. according to the catheterization laboratory routine practice, all of these patients underwent abdominal aortography following coronary angiography using a pigtail catheter and 1020 degrees straight left anterior oblique (lao) projection with a pump injector at a rate of 15 ml / sec. in those patients for whom it was difficult to evaluate the degree of stenosis via aortography, selective renal angiography also performed using a right judkins catheter and 020 degrees straight lao projections with hand injection. by average, about 3040 ml additional contrast media were utilized which was either ultravist-300 (shering ag, germany) or visipaque-320 (ge healthcare, ireland). coronary disease considered ostio - proximal if it observed before first diagonal or septal branch in lad artery, before first sizable obtuse marginal (om) branch in lcx artery and before the first bend of the vessel in rca. all angiograms digitally recorded at 15 frames / sec speed and interpreted at a consensus of two interventional cardiology fellowships. all collected data entered into a database and subsequently analyzed using spss software version 15.0 for windows (spss). the relationship between ras and other variables was examined using student s t - test for normal continuous variables and chi - square test for categorical factors. continuous variables that revealed significant relationship with ras in univariate analysis categorized according to their distributions and entered into the multivariate model. numerical variables expressed as mean standard deviation (sd) and categorical factors as percentages. finally, independent predictors of ras derived by multivariate stepwise logistic regression analysis and expressed as odds ratios (or) with 95% confidence interval (ci). in these cohort of patients, 264 (52.8%) were male with mean age of 59.6 10.1 years and 236 (47.2%) were female with mean age of 60.8 8.7 years. as for major atherosclerotic risk factors, 373 patients (74.6%) had htn, 219 patients (43.8%) were diabetics, 445 individuals (89%) had dlp, 108 (21.6%) of them were smoker and 37 subjects (7.4%) had a positive fh (table 1). it was unilateral in 46 patients (9.2%) and bilateral in 24 (4.8%). in 26 (56%) of patients with unilateral disease, ras was detected on left renal artery and in 20 patients (44%) right renal artery was involved. high - grades stenosis (70% luminal narrowing) was present in 45 (9%) of them [37 (7.4%) unilateral, 81 (1.6%) bilateral, 18 (49%) on the left side and 19 (51%) on the right ] (fig. patients with high - grade stenosis underwent renal artery stenting at the same setting or differed intervention for another session. patients with moderate (50%70%) stenosis referred to nephrologists for further assessment about intervention or medical therapy. in 346 individuals, significant cad was present (69.2%). significant ras was more common (18.4%, p 100 mmhg was shown a powerful predictor of poor outcome following renal angioplasty and stenting. we found highly significant relationships between atherosclerotic involvement of lad, lcx, and rca and ras, which remained significant in their ostio - proximal segments ; this was not true for significant lm disease. as for number of coronaries involved, patients with three vessels cad showed strong relationship with ras and those with normal coronaries or insignificant cad had a potent negative association with ras. interestingly in multivariate model, three vessels cad no longer seen as an independent predictor of ras while rca involvement was a strong independent predictor of ras and lad artery disease showed borderline relationship. as far as we know this independent relationship between atherosclerotic disease of rca and significant ras not been reported before. the predilections of certain sites in vascular system to develop atheroma are clear.1 atherosclerotic ras predominantly affects the aorto - ostial segment,1 but relationship between distribution of coronary artery lesions and segments involved has not been addressed extensively. in a study by weber - mzell,8 lad, lcx, and rca stenosis were more frequent in patients with significant ras but in multivariate analysis, having > 2 significant coronary lesions recognized as an independent predictor of ras. in another study by conlon,14 lad disease there was no relationship between anatomical distribution of coronary artery lesions (proximal, mid or distal portions) and ras in a study by danesh,2 but two and 3-vessel coronary disease reported as an independent predictor of significant ras. although the therapeutic implications of incidentally detected ras has been remained controversial until now21 it may be valuable to be aware of this condition given the progressive nature of the disease, the precautions in prescribing angiotensin antagonists and possibly the need to revascularization in appropriately selected cases. given the considerable drawbacks of noninvasive imaging techniques1,2,13 and safety of renal angiography13,16,17 recognizing potential candidates for screening of ras based on readily available variables at the time of cardiac catheterization is important from a practical point of view. patients with incidental ras may deserve aggressive medical treatment and more close follow - up. simultaneous renal angiography at the time of coronary angiography might be justified in patients older than 62 years, with increased intra - arterial systolic pressure (150 mmhg) and pulse pressure (60) and rca disease in the absence of traditional clinical clues. | aims : we evaluated the relationship between distribution of lesions in coronary tree and atherosclerotic renal artery stenosis (ras).methods and results : data collected prospectively on 500 consecutive patients who underwent simultaneous renal angiography following coronary angiography. overall prevalence of ras was 26.2% (131 patients). significant (50% luminal diameter stenosis) ras was present in 70 patients (14%). in 346 individuals of the study population, significant cad was present (69.2%). significant ras was more common (18.4%) in this group. older age, higher intra - arterial systolic blood pressure (sbp) and pulse pressure (pp) at the time of catheterization, and 3-vessel coronary artery disease (cad) were associated with significant ras in univariate analysis. relationship between involved locations of coronary arteries [left anterior descending (lad), left circumflex (lcx), right coronary artery (rca), and their ostio - proximal portions ] and ras were significant except for left main (lm) disease. in multivariate model, age more than 62 years, sbp greater than 150 mmhg, pp in excess of 60 mmhg and rca involvement were independent predictors of significant ras.conclusion:simultaneous renal angiography following coronary angiography might be justified in patients with significant rca disease who are older with increased levels of intra - arterial sbp and pp. |
a 12-year - old female visited the hospital with the chief complaint of an abnormal finding on a simple chest x - ray. a discontinuity of the diaphragm was observed in the retrosternal portion by chest ct and mesenteric fat was herniated through this discontinuity (fig. the lack of an intestinal hernia was confirmed by barium esophagography, and there was no finding of incarceration. although the patient was 12 years old, her height was above average for her age at 164 cm. thus, it was determined to perform a laparoscopic procedure considering that the surgical space for the laparoscopic procedure would be large enough, given her height. a 10-mm 30 degree laparoscope was inserted through an upper umbilical incision. to insert additional surgical instruments, a 10-mm troca was inserted through the left flank, the right lower rib, and the right flank incision. the diaphragm injury was observed on the left side of the falciform ligament in the peritoneal cavity (fig. the great omentum and fat from the falciform ligament were put in the thoracic cavity. the great omentum, which was pushed into the thoracic cavity by the diaphragm injury, was recovered. the falciform ligament was separated from the margin of the thoracic wall to secure space for the operation. after resection of adipose tissue from the falciform ligament, the hernia sac was closed by tying up the mediastinal pleura and the right diaphragm as is done for plication. to avoid creating a dead space in the pleura cavity, an incision of about 1 cm was made in the mediastinal pleura before tie - up to make the dead space disappear by inflation of the lung. the right margin of the diaphragm defect was stretched in the lateral direction to adjust the edge of the diaphragm. a chest tube was inserted to treat a pneumothorax which occurred right after the operation. the patient was discharged on the third day after the surgery without special complications, and her progress was observed in the outpatient department (fig. morgagni hernias, which are the rarest type among the congenital diaphragmatic hernias, comprise 3~5% of all diaphragmatic hernias. however, other cases are found in adulthood and pregnancy, the latter of which makes abdominal pressure increase because it is usually asymptomatic. according to the research of comer. acute symptoms form in only 14% of cases ; however, surgical treatment should be considered because there is a risk of incarceration by an intestinal hernia. in addition, this approach has an advantage in case of adhesion between the pericardia and lungs. on the other hand, disadvantages of this approach are that it is difficult to repair bilateral hernias and to diagnose the obstruction of intestin. herniated organs can be returned to their original places easily with an approach through the abdomen. particularly, this approach should be used in case of bilateral and central hernias, and combined abnormalities in the abdomen. therefore, to prevent excessive pressure on the intestine, the diaphragm injury lesion should be anchored retrosternally. however, the argument for the minimally invasive surgical technique still stands because this procedure is difficult. to compliment this procedure, the method in which the diaphragm defect is sutured on the abdominal wall and the suture is fixed to the subcutaneous layer of the abdominal wall has been developed. in the present case, a horizontal procedure with laparoscopy to treat a morgagni hernia is not a difficult method. however, follow up for cases of pregnancy and trauma, which cause an increase in abdominal pressure, is essential. | a 12-year - old female presented with the abnormal findings on the chest pa. the chest ct revealed a retrosternal defect of the diaphragm and a fatty opacity in the pleural cavity, resulting in a diagnosis of morgagni hernia. it was decided to undergo a laparoscopic surgery. the retrosternal defect of the diaphragm measuring 3.5 cm in diameter was found, through which a portion of the greater omentum and the fatty tissue connected with the falciform ligament were herniated into the pleural cavity. the greater omentum was pushed back into the peritoneal cavity and the fatty tissue connected with falciform ligament was excised. the mediastinal pleura was plicated and the defect of the diaphragm was repaired primarily. immediately after the operation, the patient developed a right pneumothorax for which a chest tube was inserted. she was discharged at the post - operative third day without any further complications. |
the clinical and radiographic findings of traumatic dental injury reveal a loss of tooth structure with pulp exposure, which is commonly referred to in dental literature as complicated crown fracture. the majority of these injuries occur in recently erupted or young permanent teeth that have immature roots. the treatment planning is influenced by the extent of the exposure of the pulp to the oral environment, the degree of root development and the time interval between the traumatic dental injury and the examination. the treatment strategy of the crown fracture after pulp exposure is dictated by the concern for vitality of the dental pulp for continued root development of immature permanent teeth. pulp preservation by vital pulp therapy includes pulp capping and pulpotomy in complicated crown fractures of immature permanent incisors. pulp capping is recommended for small exposure that occurred not more than a few hours previously. as the exposure site was large or the elapsed time was long between the accident and examination, partial pulpotomy was considered as the treatment of choice. conversely, in mature teeth with closed apex, root canal therapy is recommended due to the extensive loss of tooth structure. cvek 's vital pulpotomy technique has been used until 1983 with a calcium hydroxide mixture in order to initiate reparative dentin formation by controlling infection and stimulating the wound healing process. in the past decade, mineral trioxide aggregate (mta) became popular for maintaining the healing of the pulp tissues as it provides good sealing ability, biocompatibility, and low cytotoxicity and also induces odontoblast to form a hard barrier. mta was first described in the dental scientific literature in 1993 and was given approval for endodontic use by the us food and drug administration in 1998. up to 2002, only one mta material consisting of gray - colored powder was available, and in that year, white mineral trioxide aggregate (wmta) was introduced as proroot mta (dentsply endodontics, tulsa, ok, usa) to address esthetic concerns. both formulae contain 75% portland cement, 20% bismuth oxide, and 5% gypsum by weight. however, few clinical studies exist in the dental literature, showing the success of vital pulpotomy by using mta with long follow - up. the aim of presenting these case reports is to describe vital pulpotomy treatment with mta in complicated crown fractures of permanent immature anterior teeth in two cases with long - term follow - up. a 9-year - old boy was referred to the pediatric dentistry clinic with a chief complaint of a trauma that caused fracture in maxillary central incisors by a bicycle accident. the intraoral examination revealed horizontal mid - crown fractures of his maxillary central incisors with pulp exposure. the teeth were not mobile and electronic pulp testing showed vital pulpal responses within normal limits. the radiographic examination revealed that both the teeth had immature apices without any root fracture or periapical radiolucent area [figure 1a ]. (a) preoperative radiograph of case 1 ; (b) postoperative radiograph of case 1 ; (c) 4.5 years follow - up radiograph showing apical root development a decision was made to perform partial pulpotomies instead of direct pulp capping due to the size of exposure. it was suggested that the teeth should be treated using wmta (proroot, dentsply, tulsa dental, ok, usa) as a partial pulpotomy therapy. under local anesthesia and rubber dam isolation, the coronal pulp tissues were gently removed to a depth of 2 mm by using a high - speed sterile round diamond bur (dentsply maillefer, tulsa, ok, usa) under water cooling. hemorrhage was controlled with sterile cotton pellets and sterile saline solution to avoid clot formation. when pulpal bleeding stopped within 3 min, mta powder was mixed with distilled water according to the recommended consistency and placed without any pressure to cover the exposed pulps. a moist cotton pellet was placed on the mta and the cavity was sealed temporarily with glass ionomer cement (fuji ix, gc corporation, tokyo, japan). after 3 days, upper fractured central incisors were reexamined. as the teeth were asymptomatic to percussion sensitivity and gave vital response to electronic pulp test ; permanent composite resin restorations (z250, 3m / espe, st. the patient was followed for 4.5 years with 3-month examination intervals. during this period, the clinical and radiographic evaluations of maxillary central incisors presented no spontaneous pain or discomfort to percussion with evidence of continuing apical root development [figure 1c ]. an 8-year - old boy was referred to pediatric dental clinic for the treatment of maxillary left central incisor tooth. the tooth was not mobile and gave a vital pulpal response on electronic pulp testing. radiographic examination revealed that the maxillary central incisors had open apices and the left maxillary central incisor was fractured. (a) preoperative radiograph of case 2 ; (b) postoperative radiograph of case 2 ; (c) 2 years follow - up radiograph showing apical root development with coronal dentin bridge formation due to the open apices of the maxillary incisors, it was decided to perform vital partial pulpotomy of maxillary left central incisor with wmta. the patient and his mother were informed about the advantages and possible complications of the treatment plan. upon approval of the patient and his parents, the tooth was treated as in careful follow - up examinations were done at 3-month intervals in order to observe the root development of left maxillary central incisor. at the 2-years follow - up examination, no problems were detected and periapical radiograph showed that the apex of the maxillary left central incisor tooth was closed without any sign of pathology, and a dentine bridge was apparent at the pulpotomy site [figure 2c ]. a 9-year - old boy was referred to the pediatric dentistry clinic with a chief complaint of a trauma that caused fracture in maxillary central incisors by a bicycle accident. the intraoral examination revealed horizontal mid - crown fractures of his maxillary central incisors with pulp exposure. the teeth were not mobile and electronic pulp testing showed vital pulpal responses within normal limits. the radiographic examination revealed that both the teeth had immature apices without any root fracture or periapical radiolucent area [figure 1a ]. (a) preoperative radiograph of case 1 ; (b) postoperative radiograph of case 1 ; (c) 4.5 years follow - up radiograph showing apical root development a decision was made to perform partial pulpotomies instead of direct pulp capping due to the size of exposure. it was suggested that the teeth should be treated using wmta (proroot, dentsply, tulsa dental, ok, usa) as a partial pulpotomy therapy. under local anesthesia and rubber dam isolation, the coronal pulp tissues were gently removed to a depth of 2 mm by using a high - speed sterile round diamond bur (dentsply maillefer, tulsa, ok, usa) under water cooling. hemorrhage was controlled with sterile cotton pellets and sterile saline solution to avoid clot formation. when pulpal bleeding stopped within 3 min, mta powder was mixed with distilled water according to the recommended consistency and placed without any pressure to cover the exposed pulps. a moist cotton pellet was placed on the mta and the cavity was sealed temporarily with glass ionomer cement (fuji ix, gc corporation, tokyo, japan). after 3 days, upper fractured central incisors were reexamined. as the teeth were asymptomatic to percussion sensitivity and gave vital response to electronic pulp test ; permanent composite resin restorations (z250, 3m / espe, st. the patient was followed for 4.5 years with 3-month examination intervals. during this period, the clinical and radiographic evaluations of maxillary central incisors presented no spontaneous pain or discomfort to percussion with evidence of continuing apical root development [figure 1c ]. an 8-year - old boy was referred to pediatric dental clinic for the treatment of maxillary left central incisor tooth. the tooth was not mobile and gave a vital pulpal response on electronic pulp testing. radiographic examination revealed that the maxillary central incisors had open apices and the left maxillary central incisor was fractured. (a) preoperative radiograph of case 2 ; (b) postoperative radiograph of case 2 ; (c) 2 years follow - up radiograph showing apical root development with coronal dentin bridge formation due to the open apices of the maxillary incisors, it was decided to perform vital partial pulpotomy of maxillary left central incisor with wmta. the patient and his mother were informed about the advantages and possible complications of the treatment plan. upon approval of the patient and his parents, the tooth was treated as in careful follow - up examinations were done at 3-month intervals in order to observe the root development of left maxillary central incisor. at the 2-years follow - up examination, no problems were detected and periapical radiograph showed that the apex of the maxillary left central incisor tooth was closed without any sign of pathology, and a dentine bridge was apparent at the pulpotomy site [figure 2c ]. in complicated crown fractures, preserving the vital dental pulp or part of it in a healthy state is the main goal in treating young permanent teeth. it was revealed in several studies that inflammation is confined to the surface 23 mm of the pulp when traumatically exposed and left untreated for up to 168 hours.[1618 ] therefore, it is universally accepted that vital techniques are recommended for immature teeth. for many years, calcium hydroxide has been used in vital pulpotomy to cause a coagulation necrosis, inducing a low - grade irritation that leads to differentiation of the undifferentiated pulp cells. these cells synthesize predentine which is subsequently mineralized, while the coagulated tissue is calcified. mta is one of the materials of choice which has been suggested for use in vital pulpotomy treatment, with a reparation mechanism similar to that of calcium hydroxide. when compared with calcium hydroxide, mta produces significantly more dentinal bridging in a shorter period of time with significantly less inflammation and also provides a hard - setting, non - resorbable surface without the presence of tunnels in the dentine barrier. therefore, in the cases presented here, wmta was used for the vital pulpotomy treatment. many in vivo and histological studies have reported the superior physical and biological properties of mta in a short follow - up period.[2125 ] in the cases presented here, after long - term follow - up periods, injured tooth treated with wmta pulpotomy showed successful clinical and radiographical outcomes. this result should be attributed to excellent sealing ability of wmta to prevent microleakage of bacteria and their by - products. long - term follow - up of these case reports have shown that vital pulpotomy with wmta would be a successful treatment choice, showing good long - term prognosis for immature teeth with complicated crown fracture. | this case report describes the partial pulpotomy treatment of complicated crown fractures of two cases by using white mineral trioxide aggregate (wmta) with long - term follow - up. in the cases presented here, to injured incisor teeth were open apices and the pulp exposure site was large, so it was decided to perform vital pulpotomy with wmta. long - term follow - up examinations revealed that the treatment preserved pulpal vitality with continued root development and apex formation. wmta may be considered as an alternative option for the treatment of traumatized immature permanent teeth. |
psoriasis is a chronic, recurrent, inflammatory disorder of the skin affecting approximately 2% of the world 's population. psoriasis vulgaris (psv) is the most common type of psoriasis, which manifests as well - demarcated, scaly patches on the skin. psoriasis is considered severe if more than 10% of a patient 's body surface is affected or if a patient scores more than 10 on the psoriasis area severity index (pasi). topical therapies are used for mild or localized disease while systemic therapies and phototherapy are used for patients with moderate to severe disease. extracts of the chinese herb tripterygium wilfordii hook f (twhf), also known as lei gong teng, are used as one of the most common systemic treatments for inflammatory and immune disorders including psoriasis, rheumatoid arthritis (ra), crohn 's disease, nephritis, and systemic lupus erythematosus. recently, extracts of twhf have been tested in western countries, with good efficacy. however, acitretin is a teratogen, causes dyslipidemia, the therapeutic effect is slow to onset, and it has other side effects that limit widespread use. although both therapies are effective, twhf has never been directly compared with systemic acitretin in a randomized clinical study. we compared the efficacy and safety of twhf and acitretin for the treatment of moderate to severe psv in a randomized, double - blind, double - dummy, parallel - group clinical trial. adults (age > 18 years and 18 years and < 75 years) with moderate to severe psv were eligible to enroll in the study. eligible patients had a pasi score greater than or equal to 10 or a psoriasis - affected body surface area 10% or higher. exclusion criteria included : participation in other clinical studies during the past 30 days ; patients with psoriatic erythroderma, psoriasis pustulosa, psoriasis arthritis, or guttate psoriasis ; patients taking systemic corticoid, immunosuppressive agents, or biologicals therapy during the past 4 weeks ; patients taking local corticoid therapy or phototherapy during the past 2 weeks ; patients that are pregnant, breastfeeding, planning to become pregnant within 2 years ; patients with acute or chronic systemic infections, hepatitis b virus, hepatitis c virus, hiv, a history of malignancy, severe systemic disease, or elevation of aspartate transaminase, alanine transaminase, blood - lipoids reaching 1.5 times the normal range ; and patients with allergies to twhf or acitretin. this randomized, double - blind, double - dummy, parallel - group clinical study was conducted at peking union medical college hospital in china. the first patient was enrolled on september 27, 2011, and the last patient completed the study on june 14, 2014. patients were randomly assigned in a 1:1 ratio to receive either a chloroform methanol extract of twhf 20 mg 3 times a day plus a placebo matching acitretin 30 mg once a day or acitretin 30 mg once a day plus a placebo matching a chloroform methanol extract of twhf 20 mg three times daily, both for 8 weeks. a permuted block randomization design using a block size of four the random sequence was generated by an independent randomization group, principal investigators enrolled the study participants, and a third - party vendor assigned the interventions using the web response system. patients were assessed at baseline and at weeks 2, 4, and 8. at the screening visit, the laboratory tests consisted of urinalysis, hematology, and blood biochemistry (urea nitrogen, creatinine, cholesterol, triglycerides, high - density lipoprotein, aspartate transaminase, alanine transaminase) at baseline, week 4, and week 8. differences from baseline to weeks 2, 4, and 8 were tested using paired samples t - test. differences in the primary outcomes between the two groups at weeks 2, 4 and 8 were performed by analysis of variance (anova) test. all analyses were performed using spss software program version 15.0 for windows (spss inc., safety was evaluated by all adverse events (aes), laboratory tests, and physical examinations. at each visit, patients were queried for any aes, and an assessment was made as to whether there were any possible, probable or definite relationships between study drug and aes. an ae was defined as any undesirable symptom or sign that occurred after the initiation of the treatment, regardless of its relation to the study drug. a serious ae was defined as any untoward medical occurrence that, at any dose, resulted in death, was life - threatening, required hospitalization, or resulted in persistent or significant disability, incapacity, or a congenital malformation, or required medical intervention. the endpoint of the study was the response of 75% improvement in pasi score (pasi 75) at weeks 2, 4, and 8. efficacy assessments (pasi) were performed by doctors who were blinded to the treatment group assignments. of the 115 patients enrolled, 58 were allocated to the twhf group and 57 were allocated to the acitretin group most patients were men (70.4%), and the mean age sd was 41.1 12.2 years. there was a higher attrition rate in the acitretin group (5.2% in the twhf group and 7.0% in the acitretin group by week 2, and 8.6% in the twhf group and 12.3% in the acitretin group by week 8). discontinuation occurred because of withdrawn consent (n = 1), loss to follow - up (n = 8), ae (n = 1), and disease progression (n = 2). reasons for discontinuing the study were similar between groups. baseline demographics and clinical characteristics of the patients sd : standard deviation ; twhf : tripterygium wilfordii hook f ; bmi : body mass index ; pasi : psoriasis area severity index. the pasi score decreased from a median of 23.8 (range 7.559.5) to 11.1 (range 0.346.9) in the twhf group (n = 58 ; p < 0.0001) and from 23.2 (range 7.256.7) to 13.0 (range 0.446.4) (n = 57 ; p < 0.0001) in the acitretin group within 8 weeks [table 2 ]. the median pasi score improved in the twhf group by 50.4 31.0% and in the acitretin group by 42.7 45.7%. there was no significant difference in median pasi improvement between the two groups (p = 0.317). there was also no statistically significant difference between two groups at week 8 for pasi 25 (70.7% vs. 64.9% in the twhf and acitretin groups, respectively ; adjusted p = 0.553) [figure 1a ], pasi 50 (51.7% vs. 43.9% ; nominal p = 0.457) [figure 1b ], pasi 75 (19.0% vs. 17.5% ; nominal p = 1) [figure 1c ], and pasi 90 (5.2% vs. 5.3% ; nominal p = 0.204) [figure 2, 3 ]. at week 2, there was also no significant difference in median pasi score improvement (18.7 41.1% vs. 18.3 16.5% in the twhf and acitretin groups, respectively ; adjusted p = 0.985) [figure 1d ], pasi 25 (50.0% vs. 31.6% ; nominal p = 0.058) [figure 1a ], pasi 50 (12.1% vs. 3.5% ; nominal p = 0.162) [figure 1b ], and pasi 75 (1.7% vs. 0% ; nominal p = 1) [figure 1c ]. similarly, at week 4, there was no significant difference in median pasi score improvement (34.4 34.9% vs. 31.6 24.7% in the twhf and acitretin groups, respectively ; adjusted p = 0.635) [figure 1d ], pasi 25 (55.2% vs. 59.6% ; nominal p = 0.707) [figure 1a ], pasi 50 (37.9% vs. 24.6% ; nominal p = 0.160) [figure 1b ], and pasi 75 (1.7% vs. 1.8% ; nominal p = 1) [figure 1c ]. treatment efficacy of twhf and acitretin sd : standard deviation ; twhf : tripterygium wilfordii hook f ; pasi : psoriasis area severity index. (a) pasi 25 response ; (b) pasi 50 response ; (c) pasi 75 response ; (d) percentage pasi improvement. clinical appearance of a patient from tripterygium wilfordii hook f group at baseline, weeks 2, 4, and 8. clinical appearance of a patient from acitretin group at baseline, weeks 2, 4, and 8. at week 2, the median pasi score improvement rate of the head, upper limbs, trunk, and lower limbs in the twhf group was 14.9 33.7%, 10.6 49.2%, 25.5 22.9%, and 9.7 61.0%, respectively. there was no significant difference in median pasi improvement between different parts of the body (p = 0.222) [tables 3 and 4 ]. there was also no significant difference in median pasi improvement between different parts of the body at weeks 4 (p = 0.462) and 8 (p = 0.882) in the twhf group. similarly, in the acitretin group, there was no significant difference in median pasi improvement between different parts of the body at weeks 2, 4, and 8 (p = 0.339, p = 0.144, and p = 0.764, respectively). treatment efficacy of different parts of the body (meansd) twhf : tripterygium wilfordii hook f ; pasi : psoriasis area severity index. median pasi improvement of different parts of the body (%, meansd) twhf : tripterygium wilfordii hook f ; pasi : psoriasis area severity index. there was no statistically significant difference between two groups at week 2 for median pasi improvement rate of the head (14.9 33.7% vs. 22.9 33.3% in the twhf and acitretin groups, respectively ; adjusted p = 0.221), upper limbs (10.6 49.2% vs. 7.2 78.9% ; nominal p = 0.787), trunk (25.5 22.9% vs. 18.0 21.4% ; nominal p = 0.084), and lower limbs (9.7 61.0% vs. 14.9 19.8% ; nominal p = 0.550). at week 8, there was also no statistically significant difference between the two groups for median pasi improvement rate of the head (49.9 40.3% vs. 49.6 45.0% in the twhf and acitretin groups, respectively ; adjusted p = 0.979), upper limbs (42.6 71.3% vs. 33.9 115.2% ; nominal p = 0.649), trunk (48.8 42.0% vs. 36.9 86.9% ; nominal p = 0.381), or lower limbs (45.5 39.7% vs. 38.9 38.2% ; nominal p = 0.394). at week 4, there was a statistically significant difference between two groups for median pasi improvement rate of the trunk (39.5 30.8% vs. 23.8 40.3% in the twhf and acitretin groups, respectively ; adjusted p = 0.026). however, there was no statistically significant difference between the two groups at week 4 for median pasi improvement rate of the head (35.1 41.4% vs. 39.2 37.9% in the twhf and acitretin groups, respectively ; adjusted p = 0.597), upper limbs (35.7 44.0% vs. 31.3 36.9% ; nominal p = 0.575), and lower limbs (27.1 45.3% vs. 27.1 28.0% ; nominal p = 0.991). more aes were reported in the acitretin group (78.1%) compared with the twhf group (43.6%). the most frequent complaints in twhf group were menstrual disorders in females, dry mouth, gastrointestinal complaints, and swelling of the lower limbs. the results of blood biochemistry (alanine transaminase, aspartate transaminase, urea nitrogen, creatinine, cholesterol, triglyceride, and high - density lipoprotein) are shown in table 5. compared with baseline, there was a significant increase in the level of aspartate transaminase and triglycerides at week 8 in the twhf group (p = 0.026 and p = 0.011, respectively). in the acitretin group, there was a significant increase in the level of alanine transaminase, cholesterol, and high - density lipoprotein at week 8 (p = 0.03, p < 0.01, and p < 0.01, respectively). laboratory tests of twhf and acitretin (meansd) twhf : tripterygium wilfordii hook f ; alt : alanine transaminase ; ast : aspartate transaminase ; hdl : high - density lipoprotein. of the 115 patients enrolled, 58 were allocated to the twhf group and 57 were allocated to the acitretin group most patients were men (70.4%), and the mean age sd was 41.1 12.2 years. there was a higher attrition rate in the acitretin group (5.2% in the twhf group and 7.0% in the acitretin group by week 2, and 8.6% in the twhf group and 12.3% in the acitretin group by week 8). discontinuation occurred because of withdrawn consent (n = 1), loss to follow - up (n = 8), ae (n = 1), and disease progression (n = 2). reasons for discontinuing the study were similar between groups. baseline demographics and clinical characteristics of the patients sd : standard deviation ; twhf : tripterygium wilfordii hook f ; bmi : body mass index ; pasi : psoriasis area severity index. the pasi score decreased from a median of 23.8 (range 7.559.5) to 11.1 (range 0.346.9) in the twhf group (n = 58 ; p < 0.0001) and from 23.2 (range 7.256.7) to 13.0 (range 0.446.4) (n = 57 ; p < 0.0001) in the acitretin group within 8 weeks [table 2 ]. the median pasi score improved in the twhf group by 50.4 31.0% and in the acitretin group by 42.7 45.7%. there was no significant difference in median pasi improvement between the two groups (p = 0.317). there was also no statistically significant difference between two groups at week 8 for pasi 25 (70.7% vs. 64.9% in the twhf and acitretin groups, respectively ; adjusted p = 0.553) [figure 1a ], pasi 50 (51.7% vs. 43.9% ; nominal p = 0.457) [figure 1b ], pasi 75 (19.0% vs. 17.5% ; nominal p = 1) [figure 1c ], and pasi 90 (5.2% vs. 5.3% ; nominal p = 0.204) [figure 2, 3 ]. at week 2, there was also no significant difference in median pasi score improvement (18.7 41.1% vs. 18.3 16.5% in the twhf and acitretin groups, respectively ; adjusted p = 0.985) [figure 1d ], pasi 25 (50.0% vs. 31.6% ; nominal p = 0.058) [figure 1a ], pasi 50 (12.1% vs. 3.5% ; nominal p = 0.162) [figure 1b ], and pasi 75 (1.7% vs. 0% ; nominal p = 1) [figure 1c ]. similarly, at week 4, there was no significant difference in median pasi score improvement (34.4 34.9% vs. 31.6 24.7% in the twhf and acitretin groups, respectively ; adjusted p = 0.635) [figure 1d ], pasi 25 (55.2% vs. 59.6% ; nominal p = 0.707) [figure 1a ], pasi 50 (37.9% vs. 24.6% ; nominal p = 0.160) [figure 1b ], and pasi 75 (1.7% vs. 1.8% ; nominal p = 1) [figure 1c ]. treatment efficacy of twhf and acitretin sd : standard deviation ; twhf : tripterygium wilfordii hook f ; pasi : psoriasis area severity index. (a) pasi 25 response ; (b) pasi 50 response ; (c) pasi 75 response ; (d) percentage pasi improvement. clinical appearance of a patient from tripterygium wilfordii hook f group at baseline, weeks 2, 4, and 8. clinical appearance of a patient from acitretin group at baseline, weeks 2, 4, and 8. at week 2, the median pasi score improvement rate of the head, upper limbs, trunk, and lower limbs in the twhf group was 14.9 33.7%, 10.6 49.2%, 25.5 22.9%, and 9.7 61.0%, respectively. there was no significant difference in median pasi improvement between different parts of the body (p = 0.222) [tables 3 and 4 ]. there was also no significant difference in median pasi improvement between different parts of the body at weeks 4 (p = 0.462) and 8 (p = 0.882) in the twhf group. similarly, in the acitretin group, there was no significant difference in median pasi improvement between different parts of the body at weeks 2, 4, and 8 (p = 0.339, p = 0.144, and p = 0.764, respectively). treatment efficacy of different parts of the body (meansd) twhf : tripterygium wilfordii hook f ; pasi : psoriasis area severity index. median pasi improvement of different parts of the body (%, meansd) twhf : tripterygium wilfordii hook f ; pasi : psoriasis area severity index. there was no statistically significant difference between two groups at week 2 for median pasi improvement rate of the head (14.9 33.7% vs. 22.9 33.3% in the twhf and acitretin groups, respectively ; adjusted p = 0.221), upper limbs (10.6 49.2% vs. 7.2 78.9% ; nominal p = 0.787), trunk (25.5 22.9% vs. 18.0 21.4% ; nominal p = 0.084), and lower limbs (9.7 61.0% vs. 14.9 19.8% ; nominal p = 0.550). at week 8, there was also no statistically significant difference between the two groups for median pasi improvement rate of the head (49.9 40.3% vs. 49.6 45.0% in the twhf and acitretin groups, respectively ; adjusted p = 0.979), upper limbs (42.6 71.3% vs. 33.9 115.2% ; nominal p = 0.649), trunk (48.8 42.0% vs. 36.9 86.9% ; nominal p = 0.381), or lower limbs (45.5 39.7% vs. 38.9 38.2% ; nominal p = 0.394). at week 4, there was a statistically significant difference between two groups for median pasi improvement rate of the trunk (39.5 30.8% vs. 23.8 40.3% in the twhf and acitretin groups, respectively ; adjusted p = 0.026). however, there was no statistically significant difference between the two groups at week 4 for median pasi improvement rate of the head (35.1 41.4% vs. 39.2 37.9% in the twhf and acitretin groups, respectively ; adjusted p = 0.597), upper limbs (35.7 44.0% vs. 31.3 36.9% ; nominal p = 0.575), and lower limbs (27.1 45.3% vs. 27.1 28.0% ; nominal p = 0.991). more aes were reported in the acitretin group (78.1%) compared with the twhf group (43.6%). the most frequent complaints in twhf group were menstrual disorders in females, dry mouth, gastrointestinal complaints, and swelling of the lower limbs. the results of blood biochemistry (alanine transaminase, aspartate transaminase, urea nitrogen, creatinine, cholesterol, triglyceride, and high - density lipoprotein) are shown in table 5. compared with baseline, there was a significant increase in the level of aspartate transaminase and triglycerides at week 8 in the twhf group (p = 0.026 and p = 0.011, respectively). in the acitretin group, there was a significant increase in the level of alanine transaminase, cholesterol, and high - density lipoprotein at week 8 (p = 0.03, p < 0.01, and p < 0.01, respectively). laboratory tests of twhf and acitretin (meansd) twhf : tripterygium wilfordii hook f ; alt : alanine transaminase ; ast : aspartate transaminase ; hdl : high - density lipoprotein. to our knowledge, this trial is the first randomized, double - blind, double - dummy, parallel - group clinical comparison of twhf with acitretin in moderate to severe psv. because twhf has several important advantages over acitretin (e.g., low risk for teratogenicity, dyslipidemia, or photosensitivity), it is important to analyze whether or not the two therapies have similar efficacy. the data from this trial clearly indicate that twhf and acitretin significantly improved psv within 8 weeks, indicating that both medicines are highly efficient for the treatment for moderate to severe psv. our results demonstrated no statistically significant difference in treatment efficacy between the two groups in patients with moderate to severe psv in 8 weeks. a decrease in safety signals psoriasis vulgaris is a common chronic relapsing inflammatory cutaneous disease. for patients with moderate - to - severe psv, currently, four conventional systemic agents : cyclosporin a, methotrexate, fumaric acid esters (germany only), and acitretin, and five biological agents : infliximab, etanercept, adalimumab, alefacept (united states and switzerland only), and ustekinumab are approved for chronic plaque psoriasis. twhf, is approved in china and often used to treat a variety of immune and inflammation - related diseases such as psoriasis and ra. acitretin is currently approved by the us food and drug administration for the treatment of severe psoriasis in adults. because it is teratogenic and should not be used in women who are pregnant, breast - feeding, or may become pregnant within 3 years of discontinuing acitretin, its use is substantially limited in female patients of childbearing potential. dyslipidemia may also occur and require dose reduction or treatment with lipid - lowering agents. the extracts of twhf contain more than 70 ingredients, but triptolide is the most potent bioactive substance. it has been shown to possess potent anti - inflammatory and immunosuppressive properties in vitro as well as in different animal models in numerous preclinical studies. inflammatory myeloid dendritic cells release interleukin-23 (il-23) and il-12 to activate il-17-producing t cells, th1 cells, and th22 cells to produce abundant psoriatic cytokines il-17, interferon-, tumor necrosis factor (tnf), and il-22. these inhibit the immunological response and maintain cutaneous immunological homeostasis, which prevents autoimmunity against autoantigens. triptolide can suppress the activation of t - lymphocytes, promote the differentiation of cd4 + t cells to foxp3 + tregs, and downregulate the level of tnf-. in addition, a recent study showed that the triptolide inhibits il-2 expression in t cells, and can inhibit the maturation and allogenicity of dendritic cells. these mechanisms might explain the pharmacological effects of twhf in psv treatment. in a literature search including the databases : medline, the cochrane library, china biomedical literature on disc, national science and technology library, vip, and all chinese medical journals, only ten randomized controlled trials (rcts) using twhf extracts to treat psoriasis patients were found. nine of these studies were performed using a group of patients who received a comparator drug, and only one was designed with a placebo group. the comparator drugs included erythromycin, azithromycin, glycyrrhizin, di yin pian, or ampepitidium et elementum, indigo naturalis (a dark blue plant used to treat psoriasis in traditional chinese medicine), and bimolane. all the rcts demonstrated a statistically significant improvement of the disease after treatment with twhf extracts. however, most of these claims were derived from uncontrolled clinical trials or retrospective reports. the design of the above studies does not meet the criteria required for rigorous clinical trials. in addition, significant improvement of pasi in patients with only mild forms of psoriasis may bias a favorable efficacy outcome. therefore, rcts in a defined patient population with moderate to severe psv are needed to gain further insight into the potential benefits and risks of the use of twhf extracts. the major side effects of twhf extracts documented in the literature are gastrointestinal complaints, skin rash and pigmentation, decreases in white and red blood cells and platelets, and dysfunction of the male and female reproductive system such as dysmenorrhea, irregular menstruation, or reversible sterility. an adverse effect on renal function such as a decrease in creatinine clearance in elderly patients was also documented. the side effect - related withdrawal rate for the chloroform methanol extract of twhf in a 3-month trial was only 2.9%. dry mouth, gastrointestinal complaints, and swelling of the lower limbs were also common complaints. no significant abnormalities in urinalysis or blood panels were seen during the 8 weeks. compared with baseline, there was a significant increase in the level of aspartate transaminase and triglycerides within 8 weeks in the twhf group. we prescribed oral lipid - lowering drugs and hepatinica to patients whose level of serum aspartate transaminase and triglycerides reached 1.5 times the normal value. with this intervention, the blood biochemistry values returned to normal or were maintained. in conclusion, tripterygium wilfordii hook f might be an effective and safe treatment in patients with moderate to severe psv. twhf could be considered a suitable treatment option for certain patient populations, specifically patients who have contraindications to other therapies. increases in aspartate transaminase and triglyceride levels can be treated symptomatically. because there is a lack of long - term trials or reports of open studies running for more than 1 year, it is difficult to judge the full potential of twhf for use in psoriasis. further multi - center, large - sample clinical trials are needed to evaluate the long - term effects of twhf, including efficacy, safety, and tolerability. | background : few clinical trials have evaluated the efficacy and safety of tripterygium wilfordii hook f (twhf) compared with acitretin in psoriasis. we aimed to compare the efficacy and safety of twhf compared with acitretin in the treatment of moderate to severe psoriasis vulgaris.methods:adults with psoriasis area severity index (pasi) score 10 and psoriasis - affected body surface area 10% were randomized into a twhf (20 mg, 3 times a day) or acitretin group (30 mg, once a day). the treatment course lasted for 8 weeks. patients were assessed at baseline and at 2, 4, and 8 weeks. laboratory tests were performed at baseline, week 4, and week 8. the data were analyzed using paired samples t - test or analysis of variance (anova).results : a total of 115 patients was enrolled (58 twhf ; 57 acitretin). the median pasi score improved in the twhf group by 50.4% and in the acitretin group by 42.7%. there was no significant difference in median pasi improvement between two groups at 2, 4, and 8 weeks. there was also no significant difference in pasi 25, pasi 50, pasi 75, and pasi 90 response between the two groups at 2, 4, and 8 weeks. there was a significant increase in the level of aspartate transaminase and triglycerides in the twhf group (p = 0.026 and p = 0.011, respectively). in the acitretin group, there was a significant increase in the level of alanine transaminase, cholesterol, and high - density lipoprotein (p = 0.030, p < 0.01, and p < 0.01, respectively).conclusions : there was no significant difference in treatment efficacy between the twhf and acitretin groups within 8 weeks, but there were fewer treatment - related adverse events in the twhf group. |
the liver is the most common, and often the only, site of distant metastases from colorectal cancer (crc).1 hepatic resection is the only effective therapy for a subset of patients with crc metastatic to the liver, and is associated with 5-year survival rates ranging from 25 to 40%.25 from 60 to 65% of patients will, however, develop recurrent tumors after hepatic resection, indicating that they had harbored unrecognized intra- or extrahepatic tumor foci at the time of liver resection.6 moreover, several studies report unresectable disease in 4070% of patients that undergo laparotomy for liver resection.2,7,8 these data indicate that better patient selection is needed to avoid unnecessary operations. there are several potential ways of improving patient selection, one of which is the administration of neoadjuvant therapy followed by reevaluation and better preoperative staging. positron emission tomography with the glucose analog 18-fluoro-2-deoxy - d - glucose (fdg - pet) is a sensitive diagnostic tool for the detection of colorectal metastases. approximately 25% of patients are discovered to have new intra- or extrahepatic tumors on fdg - pet performed after standard imaging.913 screening with fdg - pet before hepatic resection for crc significantly improves the survival rates of resected patients, probably by improving patient selection.14 the role of neoadjuvant chemotherapy to down - stage nonresectable liver metastases and to improve outcome following hepatic resection of resectable liver metastases is an evolving concept, but one that is not yet established. with the recent application of new chemotherapeutic agents, such as irinotecan, oxaliplatin, and bevacizumab, improved response rates can be achieved and the use of these agents in the neoadjuvant setting would appear to be especially relevant for patients with nonresectable disease or patients with high risk of recurrence.1518 the aim of our study was to examine the effect of neoadjuvant chemotherapy for hepatic colorectal metastases on ct and fdg - pet / ct findings and to define the role of these imaging techniques in this setting. to do so, we compared ct and fdg - pet / ct findings with histopathological reports. patients with colorectal liver metastases were assigned to receive either an immediate liver resection (group 1) or neoadjuvant chemotherapy (group 2). the criteria for neoadjuvant treatment were : nonresectable tumors due to size, location, and number and assessment of the surgical team that complete (r0) resection was not technically possible.high risk of recurrence according to the memorial sloan kettering cancer center (mskcc) clinical risk score to assess risk of recurrence.19 specifically, patients with two or more risk factors [number of metastases > 1, disease - free survival 200 ng / ml, metastases from the colonic tumor to regional lymph nodes, size of the largest metastases > 5 cm ] were assigned to neoadjuvant treatment.presence of extrahepatic disease.oncologists preference this applied to patients with mskcc > 2 that were referred from other hospitals for immediate surgery. the decision not to administer neoadjuvant therapy was not necessarily in agreement with our policy.patients preference patients who refused neoadjuvant therapy were assigned to immediate surgery when feasible. nonresectable tumors due to size, location, and number and assessment of the surgical team that complete (r0) resection was not technically possible. kettering cancer center (mskcc) clinical risk score to assess risk of recurrence.19 specifically, patients with two or more risk factors [number of metastases > 1, disease - free survival 200 ng / ml, metastases from the colonic tumor to regional lymph nodes, size of the largest metastases > 5 cm ] were assigned to neoadjuvant treatment. the decision not to administer neoadjuvant therapy was not necessarily in agreement with our policy. patient s preference patients who refused neoadjuvant therapy were assigned to immediate surgery when feasible. treatment consisted of a neoadjuvant chemotherapeutic combination of 5-fluorouracil, leucovorin, and either oxaliplatin (folfox) or irinotecan (folfiri). most of the group 2 patients were given neoadjuvant irinotecan unless they were enrolled on a multicenter study whose protocol consisted of the administration of neoadjuvant oxaliplatin. before undergoing neoadjuvant chemotherapy, all group 2 patients underwent a triphasic contrast - enhanced ct scan, and a fdg - pet / ct was performed in 30 (62.5%) of them. all 75 patients in group 1 and group 2 underwent fdg - pet / ct and abdominal ct before liver surgery. the time interval between the last course of chemotherapy and the fdg - pet / ct scan was at least 2 weeks, and surgical exploration took place within 1 month following the fdg - pet / ct scan in most of the cases. because we used an integrated pet / ct technique, precise anatomical localization could be achieved and confirmed with the standard triphasic abdominal ct findings. the patients were asked to fast for at least 4 h before undergoing pet / ct. earlier lab tests had shown that they all had glucose levels 1, disease - free survival 200 ng / ml, metastases from the colonic tumor to regional lymph nodes, size of the largest metastases > 5 cm ] were assigned to neoadjuvant treatment.presence of extrahepatic disease.oncologists preference this applied to patients with mskcc > 2 that were referred from other hospitals for immediate surgery. the decision not to administer neoadjuvant therapy was not necessarily in agreement with our policy.patients preference patients who refused neoadjuvant therapy were assigned to immediate surgery when feasible. nonresectable tumors due to size, location, and number and assessment of the surgical team that complete (r0) resection was not technically possible. kettering cancer center (mskcc) clinical risk score to assess risk of recurrence.19 specifically, patients with two or more risk factors [number of metastases > 1, disease - free survival 200 ng / ml, metastases from the colonic tumor to regional lymph nodes, size of the largest metastases > 5 cm ] were assigned to neoadjuvant treatment. the decision not to administer neoadjuvant therapy was not necessarily in agreement with our policy. patient s preference patients who refused neoadjuvant therapy were assigned to immediate surgery when feasible. treatment consisted of a neoadjuvant chemotherapeutic combination of 5-fluorouracil, leucovorin, and either oxaliplatin (folfox) or irinotecan (folfiri). most of the group 2 patients were given neoadjuvant irinotecan unless they were enrolled on a multicenter study whose protocol consisted of the administration of neoadjuvant oxaliplatin. before undergoing neoadjuvant chemotherapy, all group 2 patients underwent a triphasic contrast - enhanced ct scan, and a fdg - pet / ct was performed in 30 (62.5%) of them. all 75 patients in group 1 and group 2 underwent fdg - pet / ct and abdominal ct before liver surgery. the time interval between the last course of chemotherapy and the fdg - pet / ct scan was at least 2 weeks, and surgical exploration took place within 1 month following the fdg - pet / ct scan in most of the cases. because we used an integrated pet / ct technique, precise anatomical localization could be achieved and confirmed with the standard triphasic abdominal ct findings. the patients were asked to fast for at least 4 h before undergoing pet / ct. earlier lab tests had shown that they all had glucose levels b in colonic specimen)81.5%82%0.73no. of liver tumors (mean) (std deviation)1.19 (0.4)2.52 (1.9)0.0001max tumor diameter (largest) (std deviation)3.53 cm (2.84)3.9 cm (1.84)0.49extrahepatic disease (no. of patients)790.56prior liver resection461mean mskcc risk score (range)1.82 (04)2.48 (25)0.003group 1, immediate hepatic resection ; group 2, hepatic resection following neoadjuvant chemotherapyln = lymph nodetable 2operative proceduresoperative procedure (no. of patients ; lesions)group 1(n = 27)group 2 (n = 48)right hepatic lobectomy58left hepatic lobectomy44central hepatectomy03right trisegmentectomy10nonanatomic resections1529left lat segmentectomy22explorative laparotomy (no resection)02group 1, immediate hepatic resection ; group 2, hepatic resection following neoadjuvant chemotherapy study patients profiles group 1, immediate hepatic resection ; group 2, hepatic resection following neoadjuvant chemotherapy group 1, immediate hepatic resection ; group 2, hepatic resection following neoadjuvant chemotherapy the overall findings, the sensitivity, specificity, and accuracy of triphasic contrast - enhanced ct and fdg - pet / ct in the detection of viable liver metastases compared to the pathological results are presented in table 3. fdg - pet and ct had a statistically significant higher sensitivity in detecting liver metastases in patients who did not receive chemotherapy compared to patients who received chemotherapy (table 3). statistical analysis also revealed that triphasic contrast - enhanced ct had a higher sensitivity than pet / ct in detecting colorectal metastasis following neoadjuvant treatment (65.3 vs 49%, respectively, p 3 cmgroup 1 sensitivity (total no. of lesions)33% (n = 3)100% (n = 15)92% (n = 13)group 2 sensitivity (total no. of lesions)17% (n = 35)78% (n = 41)100% (n = 22)group 1, immediate hepatic resection ; group 2, hepatic resection following neoadjuvant chemotherapy sensitivity of fdg - pet : correlation with tumor size group 1, immediate hepatic resection ; group 2, hepatic resection following neoadjuvant chemotherapy we also compared the sensitivity of ct and fdg - pet for patients who received folfiri or folfox (n = 31) with patients who received the same regimen plus bevacizumab (n = 17). the results are outlined in table 5. we found that the sensitivity of fdg - pet, but not of ct, was lower in patients who received bevacizumab, although the difference did not reach statistical significance. table 5fdg - pet and ct in patients who received chemotherapy with or without bevacizumab : comparison with pathological results bevacizumab bevacizumab + p valuepet tp2919 true negative (complete response)173 fp22 fn2030 sensitivity59%39%0.068ct tp3331 true negative (complete response)135 fp60 fn1618 sensitivity67%63%0.9 fdg - pet and ct in patients who received chemotherapy with or without bevacizumab : comparison with pathological results in group 1, there were one fp result for extrahepatic disease (suspected recurrence in colonic anastomosis, abdominal wall), one true - positive (tp) result (recurrence in mesocolic lymph nodes), and one false negative (fn) result (in a patient with peritoneal metastases). in group 2, there was one fp result (for suspected peritoneal metastasis), three tp results (recurrence in paraaortic lymph nodes and solitary lung metastasis), and two fn results (for peritoneal metastases). between june 2002 and september 2005, 75 patients with 155 suspected metastatic lesions from a primary crc underwent hepatic resection in our department. group 1 included 27 patients with 33 lesions who underwent immediate liver resection and group 2 included 48 patients with 122 lesions who first received neoadjuvant chemotherapy before subsequently undergoing liver resection. the patient s profiles are outlined in table 1. table 2 lists the operative procedures that were performed in the two groups. table 1study patients profiles group 1 (n = 27)group 2 (n = 48)p valuesex ratio (f / m)0.500.920.22mean age, years (std deviation)66 (9.8)61.25 (10.9)0.06site colon9 (71%)32 (66%)0.74 rectum8 (29%)16 (33%)ln metastases (duke s > b in colonic specimen)81.5%82%0.73no. of liver tumors (mean) (std deviation)1.19 (0.4)2.52 (1.9)0.0001max tumor diameter (largest) (std deviation)3.53 cm (2.84)3.9 cm (1.84)0.49extrahepatic disease (no. of patients)790.56prior liver resection461mean mskcc risk score (range)1.82 (04)2.48 (25)0.003group 1, immediate hepatic resection ; group 2, hepatic resection following neoadjuvant chemotherapyln = lymph nodetable 2operative proceduresoperative procedure (no. of patients ; lesions)group 1(n = 27)group 2 (n = 48)right hepatic lobectomy58left hepatic lobectomy44central hepatectomy03right trisegmentectomy10nonanatomic resections1529left lat segmentectomy22explorative laparotomy (no resection)02group 1, immediate hepatic resection ; group 2, hepatic resection following neoadjuvant chemotherapy study patients profiles group 1, immediate hepatic resection ; group 2, hepatic resection following neoadjuvant chemotherapy group 1, immediate hepatic resection ; group 2, hepatic resection following neoadjuvant chemotherapy the overall findings, the sensitivity, specificity, and accuracy of triphasic contrast - enhanced ct and fdg - pet / ct in the detection of viable liver metastases compared to the pathological results are presented in table 3. fdg - pet and ct had a statistically significant higher sensitivity in detecting liver metastases in patients who did not receive chemotherapy compared to patients who received chemotherapy (table 3). statistical analysis also revealed that triphasic contrast - enhanced ct had a higher sensitivity than pet / ct in detecting colorectal metastasis following neoadjuvant treatment (65.3 vs 49%, respectively, p 3 cmgroup 1 sensitivity (total no. of lesions)33% (n = 3)100% (n = 15)92% (n = 13)group 2 sensitivity (total no. of lesions)17% (n = 35)78% (n = 41)100% (n = 22)group 1, immediate hepatic resection ; group 2, hepatic resection following neoadjuvant chemotherapy sensitivity of fdg - pet : correlation with tumor size group 1, immediate hepatic resection ; group 2, hepatic resection following neoadjuvant chemotherapy we also compared the sensitivity of ct and fdg - pet for patients who received folfiri or folfox (n = 31) with patients who received the same regimen plus bevacizumab (n = 17). we found that the sensitivity of fdg - pet, but not of ct, was lower in patients who received bevacizumab, although the difference did not reach statistical significance. table 5fdg - pet and ct in patients who received chemotherapy with or without bevacizumab : comparison with pathological results bevacizumab bevacizumab + p valuepet tp2919 true negative (complete response)173 fp22 fn2030 sensitivity59%39%0.068ct tp3331 true negative (complete response)135 fp60 fn1618 sensitivity67%63%0.9 fdg - pet and ct in patients who received chemotherapy with or without bevacizumab : comparison with pathological results in group 1, there were one fp result for extrahepatic disease (suspected recurrence in colonic anastomosis, abdominal wall), one true - positive (tp) result (recurrence in mesocolic lymph nodes), and one false negative (fn) result (in a patient with peritoneal metastases). in group 2, there was one fp result (for suspected peritoneal metastasis), three tp results (recurrence in paraaortic lymph nodes and solitary lung metastasis), and two fn results (for peritoneal metastases). the role of neoadjuvant chemotherapy followed by hepatectomy for colorectal liver metastases has not yet been clearly established. new chemotherapeutic agents, including irinotecan, oxaliplatin, and the biologic agent bevacizumab, have yielded improved response rates in the treatment of advanced crc. these agents may have a potential role in the neoadjuvant setting for down - staging both nonresectable disease to resectability15,16 and resectable disease, probably mostly for patients with high risk of recurrence.17 our policy is to administer neoadjuvant treatment to patients with nonresectable disease, those with extrahepatic disease, and those with resectable disease who have two or more risk factors according to the mskcc clinical risk score.19 one of the theoretical benefits of neoadjuvant treatment is that patients who develop additional extrahepatic or intrahepatic metastases during this time period are spared a futile major operative procedure. accurate staging before the beginning of neoadjuvant treatment and restaging following the treatment are crucial for optimal patient selection. the standard preoperative staging of patients with colorectal liver metastases includes combined abdominal ct and chest x - ray or chest ct. it was recently demonstrated that fdg - pet as a complementary staging method improves the therapeutic management of patients with colorectal liver metastases.20 preoperative screening with fdg - pet results in an increased survival rate of patients who undergo liver resection.14 this can be explained by the detection of occult intra- and extrahepatic metastatic disease, thus obviating futile explorations. in the current study, the sensitivity of fdg - pet / ct following neoadjuvant therapy was only 49% compared to a sensitivity of 93.3% in patients who did not receive neoadjuvant treatment (p < 0.0001). the influence of the chemotherapeutic drugs on the sensitivity of fdg - pet in detecting extrahepatic metastases is not known, but we could assume that it is influenced in a similar way. this may result in a higher - than - expected rate of nonresectable disease discovered at the time of laparotomy and more extrahepatic recurrences following resection. in our series, only three of the 48 patients (6.25%) who received neoadjuvant chemotherapy were found to have nonresectable disease (one had diffuse liver metastases and two had peritoneal spread) that was not discovered preoperatively by either abdominal ct or fdg - pet / ct. we believe that one of the reasons for the high operability rate is the fact that a significant number of patients underwent a baseline fdg - pet / ct before the administration of neoadjuvant chemotherapy. we therefore recommend performing a baseline fdg - pet scan for all candidates for liver resection before the administration of neoadjuvant treatment. a longer follow - up the decreased sensitivity of fdg - pet / ct in detecting liver metastases should also be a consideration when planning the extent of liver resection. we believe that the extent of resection should be guided by additional imaging modalities, including abdominal ct and ious, in patients who received neoadjuvant treatment. in our series, triphasic contrast - enhanced abdominal ct had a higher sensitivity than fdg - pet / ct in detecting colorectal metastasis in patients who received neoadjuvant chemotherapy (65.3 vs 49%, p < 0.0001). the higher sensitivity of ct alone compared to fdg - pet / ct in detecting small colorectal metastasis has been reported by ruers.,20 and this may be even greater in patients who received chemotherapy. an attractive solution is the integrated pet / ct scanner on which a diagnostic triphasic abdominal ct scan can be performed at the same setting as the pet scan. there are several possible explanations for the decreased sensitivity of fdg - pet / ct in the detection of colorectal metastases following neoadjuvant therapy : size of the lesion. the sensitivity of fdg - pet in detecting colorectal metastasis was reported as being directly related to the size of the lesions.20 we found similar results in our series (table 4). the average size of the metastases following neoadjuvant treatment was significantly smaller than that in patients who did not receive chemotherapy (33.9 mm in group 1 and 18.9 mm in group 2, p < 0.0001). two fn results in group 1 and 32 in group 2 involved tumors smaller than 1 cm. we can assume that one of the main reasons for the decreased sensitivity of fdg - pet following chemotherapy is the decrease in size of the metastases.chemotherapy and metabolic shutdown. it has been demonstrated that the sensitivity of fdg - pet is diminished in cancer patients who undergo the examination less than 2 weeks following the administration of chemotherapy,21 presumably due to a temporary metabolic shutdown. although the scans in our study were done with a minimal interval of 2 weeks from the last course of chemotherapy, partial response to therapy may have caused decreased fdg uptake in metastatic lesions, making them undetectable in comparison to the physiological background uptake of fdg in the liver. we found a lower sensitivity of fdg - pet (but not of ct) in detecting liver metastases following regimens including bevacizumab compared to regimens that did not include bevacizumab, although the difference did not reach statistical significance. this result may have significant clinical implications ; however, it needs to be verified in larger series.time interval between the fdg - pet and surgery. in our series, two patients with fn results (two hepatic lesions) underwent surgery more than 2 months after fdg - pet was performed. viable tumors were discovered at the site of the original metastases which had disappeared on fdg - pet following neoadjuvant treatment. although it is conceivable that the relatively long interval between fdg - pet and surgery may have contributed to the fn results, we believe that these tumors may have been fn due the small size of the lesions following partial pathological response to chemotherapy.nonavid tumors. pet avidity of the tumors can be assessed only in patients who undergo a baseline fdg - pet before neoadjuvant treatment. it has been reported that fdg - pet is less sensitive for mucinous adenocarcinoma.22 in our series, ten fn results (lesions) were in patients with nonavid mucinous adenocarcinoma (sensitivity 37.5%). the sensitivity of fdg - pet in detecting colorectal metastasis was reported as being directly related to the size of the lesions.20 we found similar results in our series (table 4). the average size of the metastases following neoadjuvant treatment was significantly smaller than that in patients who did not receive chemotherapy (33.9 mm in group 1 and 18.9 mm in group 2, p < 0.0001). two fn results in group 1 and 32 in group 2 involved tumors smaller than 1 cm. we can assume that one of the main reasons for the decreased sensitivity of fdg - pet following chemotherapy is the decrease in size of the metastases. it has been demonstrated that the sensitivity of fdg - pet is diminished in cancer patients who undergo the examination less than 2 weeks following the administration of chemotherapy,21 presumably due to a temporary metabolic shutdown. although the scans in our study were done with a minimal interval of 2 weeks from the last course of chemotherapy, partial response to therapy may have caused decreased fdg uptake in metastatic lesions, making them undetectable in comparison to the physiological background uptake of fdg in the liver. we found a lower sensitivity of fdg - pet (but not of ct) in detecting liver metastases following regimens including bevacizumab compared to regimens that did not include bevacizumab, although the difference did not reach statistical significance. this result may have significant clinical implications ; however, it needs to be verified in larger series. two patients with fn results (two hepatic lesions) underwent surgery more than 2 months after fdg - pet was performed. viable tumors were discovered at the site of the original metastases which had disappeared on fdg - pet following neoadjuvant treatment. although it is conceivable that the relatively long interval between fdg - pet and surgery may have contributed to the fn results, we believe that these tumors may have been fn due the small size of the lesions following partial pathological response to chemotherapy. pet avidity of the tumors can be assessed only in patients who undergo a baseline fdg - pet before neoadjuvant treatment. it has been reported that fdg - pet is less sensitive for mucinous adenocarcinoma.22 in our series, ten fn results (lesions) were in patients with nonavid mucinous adenocarcinoma (sensitivity 37.5%). there were two fp results in group 1 and four fp results in group 2. four of the six fp results were in patients who had undergone a previous hepatic resection, for which follow - up fdg - pet detected uptake in the same location of the resected metastasis. the pathological examination revealed only foreign body reaction without any tumor cells. in our current study, fdg uptake in the tumor bed following previous resection had a positive predictive value of 33% (2/6). we believe that fdg uptake in the tumor bed following a previous liver resection is not specific for tumor recurrence, especially if the cea levels are normal. nguyen. demonstrated that fdg uptake may be high in various granulomatous lesions,23 possibly explaining the fp results along resection margins. the sensitivity of fdg - pet in detecting colorectal hepatic metastases decreases significantly following neoadjuvant chemotherapy. this may result in a higher - than - expected rate of nonresectable disease discovered at the time of laparotomy and in more extrahepatic recurrences following resection. we recommend staging patients with a baseline contrast - enhanced fdg - pet / ct both before and after the administration of neoadjuvant therapy. the extent of hepatic resection should be guided by systematic integration of data from all additional imaging modalities (abdominal ct, ious), as well as by the original imaging findings (before the neoadjuvant treatment). we recommend resection of all metastases that achieved complete radiological response, whenever technically possible. longer follow - up and further studies are required to justify neoadjuvant treatment and screening with fdg - pet / ct in patients with colorectal metastases to the liver who are at high risk of recurrence. | backgroundrecent data confirmed the importance of 18-fluoro-2-deoxy - d - glucose positron emission tomography (fdg - pet) in the selection of patients with colorectal hepatic metastases for surgery. neoadjuvant chemotherapy before hepatic resection in selected cases may improve outcome. the influence of chemotherapy on the sensitivity of fdg - pet and ct in detecting liver metastases is not known.methodspatients were assigned to either neoadjuvant treatment or immediate hepatic resection according to resectability, risk of recurrence, extrahepatic disease, and patient preference. two - thirds of them underwent fdg - pet / ct before chemotherapy ; all underwent preoperative contrast - enhanced ct and fdg - pet / ct. those without extensive extrahepatic disease underwent open exploration and resection of all the metastases according to original imaging findings. operative and pathological findings were compared to imaging results.resultstwenty-seven patients (33 lesions) underwent immediate hepatic resection (group 1), and 48 patients (122 lesions) received preoperative neoadjuvant chemotherapy (group 2). sensitivity of fdg - pet and ct in detecting colorectal (cr) metastases was significantly higher in group 1 than in group 2 (fdg - pet : 93.3 vs 49%, p < 0.0001 ; ct : 87.5 vs 65.3, p = 0.038). ct had a higher sensitivity than fdg - pet in detecting cr metastases following neoadjuvant therapy (65.3 vs 49%, p < 0.0001). sensitivity of fdg - pet, but not of ct, was lower in group 2 patients whose chemotherapy included bevacizumab compared to patients who did not receive bevacizumab (39 vs 59%, p = 0.068).conclusionsfdg - pet / ct sensitivity is lowered by neoadjuvant chemotherapy. ct is more sensitive than fdg - pet in detecting cr metastases following neoadjuvant therapy. surgical decision - making requires information from multiple imaging modalities and pretreatment findings. baseline fdg - pet and ct before neoadjuvant therapy are mandatory. |
... the aim of oxygen therapy should be to increase the delivery of oxygen rather than to reach any arbitrary concentration in the arterial blood. is administration of oxygen, the most widely prescribed drug in the formulary, free of risks to nonhypoxemic patients with regional ischemia ? hyperoxia marginally increases the arterial blood oxygen content (cao2), theoretically increasing tissue oxygen delivery (do2) assuming no reduction in tissue blood flow. however, oxygen causes constriction of the coronary, cerebral, renal and other key vasculatures - and if regional perfusion decreases concomitantly with blood hyperoxygenation, one would have a seemingly paradoxical situation in which the administration of oxygen may place tissues at increased risk of hypoxic stress. any tissue damage in the course of oxygen administration would plausibly be attributed to the underlying disease process. ascribing hypoxic damage to oxygen administration is counter - intuitive and is difficult to accept without a receptive mindset. considering the ubiquity of oxygen therapy, the continued low threshold for its administration, and the widespread belief that its use is justified and safe, we believe it is important to revisit the arguments made to justify the status quo. owing to the vasoconstrictor effects on the coronary, cerebral, renal and other key vasculatures, there are many scenarios in which administration of oxygen decreases the perfusion to vital organs to a greater extent than the small increase in cao2, thereby actually reducing do2. the calculated cao2 increases with normobaric hyperoxia (assuming all hemoglobin is already saturated) by only 0.03 ml / l per mmhg. with increases in alveolar pao2 from 100 to 600 mmhg, cao2 increases by 15 ml / l, or about ~7.5% assuming a hemoglobin concentration of 150 g / l. in healthy adults, administration of high oxygen concentrations is therefore likely to decrease brain do2. despite this known effect of hyperoxia on cerebral blood flow, and the published recommendations, patients with stroke - even those with satisfactory arterial saturations - although singhal and colleagues reported transient improvement in patients with ischemic strokes, survival at 7 months for patients with mild or moderate strokes is significantly greater in those administered air than in those given 100% oxygen for the first 24 hours after the event. normobaric hyperoxia reduces coronary blood flow by 8 to 29% in normal subjects and in patients with coronary artery disease or chronic heart failure. the reduction in coronary artery flow as early as 1950 russek and colleagues reported that supplemental oxygen failed to reduce electrocardiographic signs of ischemia or reduce anginal pain in patients with myocardial infarction. in 1969 bourassa and colleagues proposed that hyperoxia - induced decreases in coronary blood flow provoke myocardial ischemia in patients with severe coronary artery disease. then in 1976, in a double - blind randomized controlled trial, rawles and kenmure reported greater serum aspartate aminotransferase levels, indicating increased infarct size, in patients with acute myocardial infarction receiving high - flow oxygen compared with room air. they also observed a nonsignificant tripling of the death rate in those patients. given these concerns, the emergency oxygen guideline group of the british thoracic society called for ' large randomised trials of oxygen therapy for non - hypoxaemic patients with acute cardiac and cerebral ischaemia '. conti, in a recent editorial, reminded readers that that there is only level c evidence for the administration of supplemental oxygen to patients with uncomplicated st elevation in myocardial infarction during the first 6 hours. based on currently available evidence, the uk national institute for health and clinical excellence guidelines have recently emphasized that ' supplementary oxygen should not be routinely administered to patients with acute chest pain of suspected cardiac origin, but that oxygen saturation levels should be monitored and used to guide its administration '. similar cautions have been expressed about the use of oxygen for the treatment of traumatic brain injury. the mechanisms by which hyperoxia causes systemic vasoconstriction remain uncertain. recent work focuses on the inhibition of vasodilators (prostaglandins, nitric oxide) by reactive oxygen species generated as a result of the hyperoxia [19 - 23 ]. other work suggests that reactive oxygen species activate brainstem respiratory neurons, but this suggestion needs to be established as occurring under normobaric conditions. the role of hyperoxia - induced hypocapnia (that is, the reverse haldane effect) remains contentious. regardless of the underlying mechanism(s), the importance of considering the effects of both pao2 and paco2 on vascular tone is evident in a study in which both hyperoxia and hypocapnia independently increased cerebrovascular resistance and reduced cerebral blood flow. indeed, in some situations, the vasoconstrictive effects of hyperoxia may be predominantly due to the concomitant hypocapnia. positron emission tomography provides similar results : the reduction of cerebral blood flow and the increase in oxygen extraction during inhalation of 100% oxygen is completely reversed when subjects breathe carbogen (5% carbon dioxide, 95% oxygen). these observations emphasize the importance of independent control of arterial pco2 and po2 - possibly using dynamic forcing of alveolar gases (for example) or sequential gas delivery (for example) - when studying the independent effects of po2 and pco2 on regional perfusions. these observations also suggest that adding carbon dioxide to oxygen may offset the vasoconstriction due to hyperoxia or hypoxia - induced hypocapnia. there are other clinical situations in which the routine administration of high - concentration oxygen may lead to worse outcomes, although primarily through mechanisms other than changes in regional perfusion. austin and colleagues recently reported in a randomized controlled trial that patients with acute exacerbations of chronic obstructive pulmonary disease have a twofold to fourfold increased mortality when treated with high - flow oxygen compared with oxygen titrated to result in an arterial oxygen saturation between 88 and 92%. although several mechanisms may account for these findings, worsening respiratory failure is probably the predominant mechanism. of the patients whose arterial blood gases were measured within 30 minutes of presentation to hospital, those who received high - concentration oxygen were more likely to have hypercapnia (mean difference paco2 34 mmhg) or respiratory acidosis (mean difference ph 0.12). adverse outcomes with hyperoxia have also been reported in critically ill patients admitted to the intensive care unit ; a high pao2 in the first 24 hours after admission is independently associated with in - hospital mortality. in this study a u - shaped curve of mortality with pao2 was observed, illustrating the risks of both hypoxia and hyperoxia. kilgannon and colleagues recently reported that patients administered high - concentration oxygen resulting in hyperoxia (pao2 > 300 mmhg) following cardiac arrest have increased in - hospital mortality, a finding they attributed to increased oxidative stress associated with hyperoxia. however, because a subsequent study was unable to replicate these findings, randomized controlled trials will be required to resolve the clinical uncertainty. neonatal resuscitation is the clinical situation in which administration of 100% oxygen has most clearly been demonstrated to increase the risk of death. this has resulted in a radical change in practice whereby room air rather than oxygen is now the recommended resuscitation regime. considering the ubiquity of the administration of supplemental oxygen, there are surprisingly few randomized clinical trials that demonstrate its beneficial role when hypoxemia is absent. this may reflect the fact that its usage is so embedded in clinical practice that it is accepted as safe. nevertheless, there are some situations in which supplemental oxygen administration is useful : treatment of cluster headache, reducing the oxidative stress associated with colon surgery, and the prevention of desaturation during endoscopy. supplemental oxygen administration can, however, have the unintended side effect of delaying recognition by oximetry of hypoventilation. until recently many studies had indicated that supplementary oxygen reduced postoperative nausea and vomiting, but the current status is ambiguous (for example [43 - 46 ]). similarly, oxygen was thought to reduce postsurgical infections - but more recent studies (see for a partial summary) have cast doubt on the original findings. moreover, ventilation with high inspired oxygen concentrations during surgery leads to subsequent impairment of pulmonary gas exchange [48 - 50 ] that may be of clinical significance. traumatic injury and compartment syndrome may appear to be obvious applications for supplementary oxygen - an increased po2 would help overcome the reductions in perfusion - but hyperbaric rather than normobaric oxygen is the treatment of choice [51 - 53 ]. oxygen is used for the treatment of carbon monoxide poisoning, but this is probably less effective than it should be if the accompanying hypocapnia is not prevented. in the case of breathlessness, which has long been treated with supplementary oxygen, a recent randomized double - blind controlled trial established that nasal oxygen was no better than air in relieving breathlessness and improving quality of life in palliative care patients with refractory breathlessness. in conclusion, nasa managers demanded in 1986 that their counterparts at martin - thiokol prove that it was not safe to launch the space shuttle challenger despite concerns expressed by engineers about the integrity at low temperatures of the o - rings joining the segments of the solid rocket boosters. in the case of supplementary oxygen, failure to ask the right question reinforces complacency about its use in patients who may have regional hypoxia or ischemia but are not hypoxemic. cao2 : arterial blood oxygen content ; do2 : oxygen delivery ; paco2 : arterial partial pressure of carbon dioxide ; pao2 : arterial partial pressure of oxygen ; pco2 : partial pressure of carbon dioxide ; po2 : partial pressure of oxygen. si and jaf have participated in the development of devices suitable for increasing the efficiency of oxygen delivery. the protection of the related intellectual property and distribution of income from sales (if any) follow the guidelines set by the university health network. | supplementary oxygen is routinely administered to patients, even those with adequate oxygen saturations, in the belief that it increases oxygen delivery. but oxygen delivery depends not just on arterial oxygen content but also on perfusion. it is not widely recognized that hyperoxia causes vasoconstriction, either directly or through hyperoxia - induced hypocapnia. if perfusion decreases more than arterial oxygen content increases during hyperoxia, then regional oxygen delivery decreases. this mechanism, and not (just) that attributed to reactive oxygen species, is likely to contribute to the worse outcomes in patients given high - concentration oxygen in the treatment of myocardial infarction, in postcardiac arrest, in stroke, in neonatal resuscitation and in the critically ill. the mechanism may also contribute to the increased risk of mortality in acute exacerbations of chronic obstructive pulmonary disease, in which worsening respiratory failure plays a predominant role. to avoid these effects, hyperoxia and hypocapnia should be avoided, with oxygen administered only to patients with evidence of hypoxemia and at a dose that relieves hypoxemia without causing hyperoxia. |
rotavirus gastroenteritis (rvge) is the most common cause of severe diarrhea among infants and young children aged 90% of episodes of rvge in europe and north america, with regional and seasonal variations in the most prevalent types. data from a large european study conducted in 20045 indicate that serotypes g1, g2, g3, g4, and g9 accounted for > 98% of cases of rvge. these data highlight the importance of rotavirus vaccines that mimic natural rotavirus infection and protect against the most common serotypes of rotavirus, as reflected in international guidelines advocating universal vaccination of infants and children against rotavirus.[4,1720 ] despite these guidelines, which recommend either of the orally administered rotavirus vaccines currently available (a two - dose series of the monovalent vaccine rix4414 [rotarix ] or a three - dose series of the pentavalent rotavirus vaccine [rotateq ]), vaccination of infants and children against rotavirus is a much - debated topic often entangled in issues of cost effectiveness and health economics. this article focuses on the rotavirus vaccine rix4414, which is composed of a monovalent, live, attenuated, human rotavirus strain of g1p type.[2123 ] data on the protective efficacy of rotavirus vaccine rix4414 against rvge in developed countries are available primarily from a large, randomized, double - blind, phase iii trial conducted in six european countries (czech republic, finland, france, germany, italy, and spain), although supporting data from other relevant studies are also available. the large european study evaluated the efficacy of the vaccine in terms of its effects on the incidence of rvge (including severe rvge) and on healthcare resource use, such as hospitalization due to rvge, among infants during their first 2 years of life. a total of 3994 healthy infants aged 614 weeks were randomized to receive two oral doses of rotavirus vaccine rix4414 (n = 2646) or placebo (n = 1348), which were administered at the same time as the first two doses of other, routine childhood vaccinations. the primary endpoint was vaccine efficacy against rvge of any severity during a follow - up period from 2 weeks after administration of the second dose to the end of the first rotavirus season (20045), and all efficacy analyses were conducted in the per - protocol population. vaccine efficacy was calculated using the following formula : 1 incidence of rvge in the vaccine group / incidence of rvge in the placebo group. for the primary endpoint, the protective efficacy of rotavirus vaccine rix4414 against rvge of any severity was 87.1% (95% ci 79.6, 92.1 ; p < 0.0001) during the first follow - up period. similar results were also demonstrated in the second follow - up period (20056) and when both follow - up periods were combined. for all follow - up periods, vaccine efficacy was also significant (p < 0.0001) against severe rvge (defined as a score of 11 on the 20-point vesikari scale), rvge requiring hospitalization, and rvge requiring medical attention. in addition, vaccine efficacy against any and severe rvge caused by each of the rotavirus g types identified (g1, g2, g3, g4, and g9) was significant (p 0.02) in the combined efficacy follow - up period. various naturalistic studies conducted in developed countries have demonstrated the real - world effectiveness of rotavirus vaccination after introduction of the vaccine for routine use in the community setting. typically, these studies compared various outcomes, such as the numbers of rvge cases, rvge - related hospitalizations, and/or emergency department visits, that occurred during the pre - vaccination period with those that occurred during a specific period after widespread or universal introduction of a rotavirus vaccination program. studies conducted in the australian state of queensland and in european countries[2830 ] involved rotavirus vaccination programs with either the monovalent or pentavalent rotavirus vaccine, whereas studies conducted in the us generally focused only on the pentavalent vaccine (reviewed elsewhere). rotavirus vaccine rix4414 was generally well tolerated in clinical trials, with an overall tolerability profile similar to that of placebo. there was no increased risk of intussusception with rotavirus vaccine rix4414 in a large (n = 63 225), placebo - controlled, pre - licensure safety study conducted in latin america and finland. however, interim results from a postmarketing active surveillance study conducted in mexico, along with worldwide passive surveillance data, suggest that there may be an increased risk of intussusception during the first 7 days after administration. both the us prescribing information and the eu summary of product characteristics state that rotavirus vaccine rix4414 should not be administered to infants with a previous history of intussusception or to those with uncorrected congenital malformation of the gastrointestinal tract (e.g. meckel s diverticulum) that would predispose them to intussusception. most of the published cost - effectiveness analyses of rotavirus vaccine rix4414 were conducted in european countries and used decision - tree or markov models that incorporated data from various sources, including protective efficacy results from large phase iii trials with rotavirus vaccines and local cost data, to evaluate the cost effectiveness of introducing a universal rotavirus vaccination program compared with no universal vaccination program against rotavirus. in several analyses, both the healthcare payer and societal perspectives were used,[3340 ] whereas other studies were conducted from either a societal or a healthcare payer perspective. limited societal perspective, which excluded indirect costs but included out - of - pocket medical expenses along with other direct medical costs. some studies focused only on rix4414,[36,37,4244 ] while others also included indirect comparisons with the pentavalent rotavirus vaccine or, in some cases, the universal rotavirus vaccination program being evaluated allowed for the use of either rix4414 or the pentavalent rotavirus vaccine. a wide range of results was reported across the cost - effectiveness analyses, which appears to be related, at least in part, to the substantial heterogeneity among the models used in the studies. the analyses typically showed that the cost of a universal rotavirus vaccination program was partly offset by reductions in rvge - related healthcare resource use and that the program was associated with quality - adjusted life - year (qaly) gains. however, the universal rotavirus vaccination program was deemed to be cost effective from the perspective of the healthcare payer only in some studies, but not in others,[3335,3840,43 ] when applying commonly reported cost - effectiveness thresholds, such as 20 00050 000, $ us50 000, or 20 00030 000 per qaly gained.[4649 ] a consistent finding across studies that were conducted from both a healthcare payer and a societal (or limited societal) perspective was that incremental cost - effectiveness ratios (icers) were more favorable from a societal perspective,[3340,43 ] as might be expected because additional costs associated with rvge (e.g. out - of - pocket medical expenses and/or lost productivity of parents of children who develop rvge) were included. another consistent finding of the studies was that, compared with no universal vaccination program, icer values for a two - dose oral series of rotavirus vaccine rix4414 were more favorable than those for a three - dose oral series of pentavalent rotavirus vaccine when cost effectiveness of the two vaccines was evaluated separately in the same study. however, modelled analyses directly comparing the two vaccines would require head - to - head clinical trial data, which are currently lacking. in addition, there are inherent uncertainties in comparing icer values of the available rotavirus vaccines because of the tender process that would be used to establish the vaccine price in a universal program. although results of the cost - effectiveness analyses were sensitive to a number of parameters, which often varied between studies, there were also some common findings in the sensitivity analyses. variations in the cost of vaccine, qaly losses for a caregiver, and the number of caregivers affected, as well as annual discount rates (particularly for outcomes), were among the most frequently reported parameters having the greatest impact on icer values. economic models such as those used in the cost - effectiveness analyses with rotavirus vaccine rix4414 have, out of necessity, the inherent limitations of using data from a variety of sources and extrapolating shorter - term clinical trial data to project longer - term costs and outcomes. moreover, data or assumptions used to populate the models (e.g. waning of vaccine protection, rate of vaccine uptake, protective efficacy of partial vaccination, time period over which infections could be acquired, incidence of rvge, probability of rvge hospitalization) often varied between studies, which, together with results of sensitivity analyses, highlights some of the uncertainties in results from these modelled analyses. along with differences in the selection of data sources used in the analyses, other factors contributing to the wide variability in results include differences in the study perspective, year of costing, and discount rates, as well as country- or region - specific differences in estimates of healthcare resource use and associated costs. the type of model used in vaccine cost - effectiveness analyses can also affect results ; for example, whether the main features of the model change over time (dynamic model) or not (static model).[5054 ] the effects of herd immunity, whereby vaccination of part of a population confers partial indirect protection for the remainder, are not captured in static models (e.g. decision - tree, markov), which results in an underestimation of the cost effectiveness of a vaccination program. two analyses of rotavirus vaccine rix4414 included the effects of herd immunity, using data from dynamic transmission models in the sensitivity analyses, and in both cases the inclusion of herd immunity effects markedly improved icer values. | the most common cause of severe diarrhea in infants and young children is rotavirus gastroenteritis (rvge), which is associated with significant morbidity, healthcare resource use, and direct and indirect costs in industrialized nations. the monovalent rotavirus vaccine rix4414 (rotarix) is administered as a two - dose oral series in infants and has demonstrated protective efficacy against rvge in clinical trials conducted in developed countries. in addition, various naturalistic studies have demonstrated real - world effectiveness after the introduction of widespread rotavirus vaccination programs in the community setting.numerous cost - effectiveness analyses have been conducted in developed countries in which a universal rotavirus vaccination program using rix4414 was compared with no universal rotavirus vaccination program. there was a high degree of variability in base - case results across studies even when the studies were conducted in the same country, often reflecting differences in the selection of data sources or assumptions used to populate the models. in addition, results were sensitive to plausible changes in a number of key input parameters. as such, it is not possible to definitively state whether a universal rotavirus vaccination program with rix4414 is cost effective in developed countries, although results of some analyses in some countries suggest this is the case. in addition, international guidelines advocate universal vaccination of infants and children against rotavirus. it is also difficult to draw conclusions regarding the cost effectiveness of rotavirus vaccine rix4414 relative to that of the pentavalent rotavirus vaccine, which is administered as a three - dose oral series. although indirect comparisons in cost - effectiveness analyses indicate that rix4414 provided more favorable incremental cost - effectiveness ratios when each vaccine was compared with no universal rotavirus vaccination program, results were generally sensitive to vaccine costs. actual tender prices of a full vaccination course for each vaccine were not known at the time of the analyses and therefore had to be estimated. |
life - time prevalence of acute pain, in particular headache, is assumed to be nearly 100%. this means that, i.e. almost every individual will suffer from some kind of pain (e.g. headache) during their lifetime. life - time headache prevalence alone is 66% worldwide, in adults in europe it is 80% for tension - type headache, and 15% for migraine. in most cases analgesics pain - relieving therapy is generally symptom - prompted and common types of acute pain, such as episodic tension - type headache or migraine with or without aura, often does not require intervention by a doctor. this explains why analgesics are among the drugs most frequently used in self - medication. however, adverse effects of drugs intended for self - medication can not be completely ruled out. since most adverse drug effects are dose - related, consumption of drugs for self - medication in particular analgesics is often in the focus of public interest. reliable long - term data on the use of analgesics in different countries are only available for the period from 1970 to 1986. the purpose of the present examination was to close the gap over the past 20 years and to provide a database for further analyses such as epidemiological studies or studies on the usage pattern of otc analgesics. data on pharmacies purchases including all sales of the distributors to pharmacies and, in addition, direct purchases of pharmacies from pharmaceutical companies were obtained for a sample of 1,000 pharmacies (statistics by the institute of medical statistics, ims, frankfurt). these consumption data represent the quantities available for sale in the pharmacies. correction for unused tablets due to expiry is not possible since no valid data are available. figures from 1986 onwards were available for the following countries : germany, switzerland, austria, belgium, canada, australia, france, sweden, and the usa. except for the first reading point in 1986, the analysis was performed in 5-year intervals. we began by comparing the different countries that were compared over the period from 1986 to 2005 and followed this with an exemplary analysis of the situation in germany since 1980 by comparing germany (west) with germany (all) and prescription with non - prescription analgesics. long - term trends could only be shown for the former western part of germany as no long - term data are available for the former eastern part. only analgesics of atc code (anatomical therapeutic chemical classification system as provided by the who) n2b (general analgesics) were included ; narcotics (n2a), migraine remedies (n2c), and spasmolytics (a3d) were not considered for this analysis. their compositions were identified by using international drug directories (e.g. rote liste for germany, physician s desk reference for the usa). in germany, the substances of non - prescription n2b analgesics include acetylsalicylic acid (asa), paracetamol or ibuprofen, either as single agents or in combination. the n2b prescription drugs in germany include substances such as metamizole, tilidine plus naloxone, codeine, or tramadol as single agents or in combination. sales figures were converted into standardised units (su) in order to control for the different package sizes and formulations (see appendix 1). the conversion formulas varied depending on the different formulations. for many products one tablet equaled 1 su. per - capita consumption was calculated by dividing the defined su by the official population figures in the respective countries and years (data obtained from statistisches bundesamt, wiesbaden, germany). for further analysis the following categories were created according to the number of agents : (a1) single analgesics (contain 1 agent) ; (a2) combination analgesics (contain two agents at least, e.g. paracetamol, acetylsalicylic acid, codeine, caffeine, or phenacetin). in another analysis, category a2 was subdivided into : (a21) caffeine - containing multiple combination analgesics with two analgesic agents at least plus caffeine, and (a22) other combination analgesics. low - dose asa products (300 mg for platelet aggregation inhibition / prevention of vascular events) were not counted among the analgesics and remained unconsidered. the international comparison for 2005 shows that the highest per - capita consumption among the countries included was observed in sweden (147 su per inhabitant, fig. 1), followed by france with 141 su, australia with 106, canada with 81, belgium with 66, and the usa with 61 su. germany (all) with 51 su, switzerland with 46, and austria with 42 su showed the lowest per - capita consumption in international comparison. per - capita consumption in austria, switzerland and germany is only one - third to one half that in other countries. the development from 1986 to 2005 reveals two groups : one group with germany, austria, switzerland and belgium, whose per - capita consumption was between 40 and 65 su per year and remained constant or showing a minor increase only (switzerland, belgium) during this period (fig. 2). in the other group, comprising canada, australia, france, and sweden, per - capita consumption in 1986 was distinctly higher than in the first group, ranging between 57 su (aus) to 105 su (s).fig. 2trend of analgesic consumption comparing all analgesics with single analgesics in the years 1986, 1990, 1995, 2000, 2005 (data for 1995 in usa not available) international comparison of analgesics consumption in the year 2005 trend of analgesic consumption comparing all analgesics with single analgesics in the years 1986, 1990, 1995, 2000, 2005 (data for 1995 in usa not available) the up to threefold higher consumption in sweden and france can not be explained by use of products with basically different compositions, e.g. asa products. comparisons between an unweighted presentation and a presentation weighted with mg asa reveals no significant differences in the distribution of the su values of asa single products (table 1). the weighting takes into account the different asa doses per su by multiplying the respective consumption figures (su figures) per product by the corresponding weighting factor (equivalent asa dose in mg) and dividing by the sum of the weighting factors.table 1relative proportions of standardised medication units (su) among different countries comparing weighted and unweighted proportionsrelative proportions of asa consumptionunweighted (%) weighted by mg asa (%) usa48.358.2germany (west)19.716.0canada10.712.2france16.79.7sweden1.31.1switzerland0.80.7austria1.81.7belgium0.60.3australia0.10.1total 9 countries100100100% = overall consumption of acetylic salicylic acid (asa) among 9 countries relative proportions of standardised medication units (su) among different countries comparing weighted and unweighted proportions 100% = overall consumption of acetylic salicylic acid (asa) among 9 countries it stands out that in countries with high analgesic use, such as sweden and france, consumption increased again considerably between 1986 and 2005 to 141 (f) and 147 (s) (fig. 2). in both absolute figures and percentages, this increase is clearly higher than in the first group. in the first group annual consumption increased in germany (west) from 45 to 52 su (+ 16%) and in switzerland from 35 to 46 su (+ 31%). in the second group, in contrast, consumption increased in australia from 57 to 106 su (+ 86%), in france from 74 to 141 (+ 91%), and in sweden from 105 to 147 su (+ 40%). the high per - capita consumption in sweden and france is based on a high increase in single products in the past 15 to 20 years (fig. 2) ; this is also true of australia (third highest analgesic use). in germany and switzerland the use of single analgesics has also increased over the past 20 years associated with a (minor) increase in overall analgesic consumption over the last 5 years. in the past a short - term increase in single analgesics in austria caused a higher overall consumption in 1995, which subsided thereafter, accompanied by a decrease in single analgesics. today austria has the lowest rate of single products in international comparison and the lowest total consumption of all countries studied. in the usa a reduction in the use of single products caffeine - containing multiple combinations (at least two analgesic agents plus caffeine) are of minor relevance in international comparison and do not explain the increasing overall use in some countries (fig. 3).fig. 3trend of analgesic consumption comparing all analgesics with caffeine - containing multiple analgesic combinations in the years 1986, 1990, 1995, 2000, 2005 (data for 1995 in usa not available) trend of analgesic consumption comparing all analgesics with caffeine - containing multiple analgesic combinations in the years 1986, 1990, 1995, 2000, 2005 (data for 1995 in usa not available) the correlation analysis across different countries and time points confirms the association between the use of single analgesics and overall analgesic use (fig. 4). we showed that a strong relationship (r = 81.8%) exists between the consumption of single analgesics and total analgesic consumption.fig. 4ecological analysis between consumption of all analgesics with single analgesics in the years 1986, 1990, 1995, 2000, 2005 considering the countries switzerland, germany (west), france, usa, canada, belgium, austria, sweden and und australia (data for 1995 in usa not available) ecological analysis between consumption of all analgesics with single analgesics in the years 1986, 1990, 1995, 2000, 2005 considering the countries switzerland, germany (west), france, usa, canada, belgium, austria, sweden and und australia (data for 1995 in usa not available) in contrast, only a very weak correlation and a negative one can be observed between caffeine - containing multiple combinations and overall consumption (r = 19.7% ; fig. 5). this supports the statement that this product group of multiple combinations does not contribute to the increased use on a population level, but rather even counteracts higher overall consumption.fig. 5ecological analysis between consumption of all analgesics with caffeine - containing multiple analgesic combinations in the years 1986, 1990, 1995, 2000, 2005 considering the countries switzerland, germany (west), france, usa, canada, belgium, austria, sweden and und australia (data for 1995 in usa not available) ecological analysis between consumption of all analgesics with caffeine - containing multiple analgesic combinations in the years 1986, 1990, 1995, 2000, 2005 considering the countries switzerland, germany (west), france, usa, canada, belgium, austria, sweden and und australia (data for 1995 in usa not available) per - capita consumption of analgesics in germany (west) has been very stable over the past 25 years and, at 52 su per capita per year, is currently slightly higher than the long - term average (increase since 2000 : approx. there is no difference between germany (west) and germany (all) (fig. 6).fig. 6trend of analgesic consumption in germany (west) from 1980 until 2005 ; comparison between germany (west) and germany (all) in the year 2005 trend of analgesic consumption in germany (west) from 1980 until 2005 ; comparison between germany (west) and germany (all) in the year 2005 most of the analgesics of the n2b market considered here (market for general analgesics) can be bought without prescription over the counter in pharmacies (fig. 7). between 1986 and 1995 the consumption of prescription - free analgesics increased markedly from 25.0 to 42.7 su per capita per year and in 1995 comprised approx. 88% of all analgesics used, while the percentage of prescription analgesics decreased by approx. this picture has been changing since 1995 with prescription analgesics tripling to approx. 18 su per capita per year, while the otc analgesics decreased by approx. the increase in prescription drug consumption since 1995 is primarily responsible for the slight increase in overall analgesics and is now back at the level of 1986. no consumption of any phenacetin - containing analgesics has been observed in germany since 1986.fig. 7trend of analgesic consumption in germany (west) from 1980 until 2005 comparing prescription - free (otc) with prescription (rx) analgesics trend of analgesic consumption in germany (west) from 1980 until 2005 comparing prescription - free (otc) with prescription (rx) analgesics in 2005 in the prescription (rx) group of n2b analgesics, combination products accounted for approx. the most important combinations are codeine plus paracetamol (18% of all su in the rx group), followed by tilidine plus naloxone (12%). the predominating single products in the rx group are metamizole (28%) and tramadol (23%). growth in the rx group can be seen for both the combinations named and the single products. the international comparison for 2005 shows that the highest per - capita consumption among the countries included was observed in sweden (147 su per inhabitant, fig. 1), followed by france with 141 su, australia with 106, canada with 81, belgium with 66, and the usa with 61 su. germany (all) with 51 su, switzerland with 46, and austria with 42 su showed the lowest per - capita consumption in international comparison. per - capita consumption in austria, switzerland and germany is only one - third to one half that in other countries. the development from 1986 to 2005 reveals two groups : one group with germany, austria, switzerland and belgium, whose per - capita consumption was between 40 and 65 su per year and remained constant or showing a minor increase only (switzerland, belgium) during this period (fig. 2). in the other group, comprising canada, australia, france, and sweden, per - capita consumption in 1986 was distinctly higher than in the first group, ranging between 57 su (aus) to 105 su (s).fig. 2trend of analgesic consumption comparing all analgesics with single analgesics in the years 1986, 1990, 1995, 2000, 2005 (data for 1995 in usa not available) international comparison of analgesics consumption in the year 2005 trend of analgesic consumption comparing all analgesics with single analgesics in the years 1986, 1990, 1995, 2000, 2005 (data for 1995 in usa not available) the up to threefold higher consumption in sweden and france can not be explained by use of products with basically different compositions, e.g. asa products. comparisons between an unweighted presentation and a presentation weighted with mg asa reveals no significant differences in the distribution of the su values of asa single products (table 1). the weighting takes into account the different asa doses per su by multiplying the respective consumption figures (su figures) per product by the corresponding weighting factor (equivalent asa dose in mg) and dividing by the sum of the weighting factors.table 1relative proportions of standardised medication units (su) among different countries comparing weighted and unweighted proportionsrelative proportions of asa consumptionunweighted (%) weighted by mg asa (%) usa48.358.2germany (west)19.716.0canada10.712.2france16.79.7sweden1.31.1switzerland0.80.7austria1.81.7belgium0.60.3australia0.10.1total 9 countries100100100% = overall consumption of acetylic salicylic acid (asa) among 9 countries relative proportions of standardised medication units (su) among different countries comparing weighted and unweighted proportions 100% = overall consumption of acetylic salicylic acid (asa) among 9 countries it stands out that in countries with high analgesic use, such as sweden and france, consumption increased again considerably between 1986 and 2005 to 141 (f) and 147 (s) (fig. 2). in both absolute figures and percentages, this increase is clearly higher than in the first group. in the first group annual consumption increased in germany (west) from 45 to 52 su (+ 16%) and in switzerland from 35 to 46 su (+ 31%). in the second group, in contrast, consumption increased in australia from 57 to 106 su (+ 86%), in france from 74 to 141 (+ 91%), and in sweden from 105 to 147 su (+ 40%). the high per - capita consumption in sweden and france is based on a high increase in single products in the past 15 to 20 years (fig. 2) ; this is also true of australia (third highest analgesic use). in germany and switzerland the use of single analgesics has also increased over the past 20 years associated with a (minor) increase in overall analgesic consumption over the last 5 years. in the past a short - term increase in single analgesics in austria caused a higher overall consumption in 1995, which subsided thereafter, accompanied by a decrease in single analgesics. today austria has the lowest rate of single products in international comparison and the lowest total consumption of all countries studied. in the usa a reduction in the use of single products caffeine - containing multiple combinations (at least two analgesic agents plus caffeine) are of minor relevance in international comparison and do not explain the increasing overall use in some countries (fig. 3).fig. 3trend of analgesic consumption comparing all analgesics with caffeine - containing multiple analgesic combinations in the years 1986, 1990, 1995, 2000, 2005 (data for 1995 in usa not available) trend of analgesic consumption comparing all analgesics with caffeine - containing multiple analgesic combinations in the years 1986, 1990, 1995, 2000, 2005 (data for 1995 in usa not available) the correlation analysis across different countries and time points confirms the association between the use of single analgesics and overall analgesic use (fig. 4). we showed that a strong relationship (r = 81.8%) exists between the consumption of single analgesics and total analgesic consumption.fig. 4ecological analysis between consumption of all analgesics with single analgesics in the years 1986, 1990, 1995, 2000, 2005 considering the countries switzerland, germany (west), france, usa, canada, belgium, austria, sweden and und australia (data for 1995 in usa not available) ecological analysis between consumption of all analgesics with single analgesics in the years 1986, 1990, 1995, 2000, 2005 considering the countries switzerland, germany (west), france, usa, canada, belgium, austria, sweden and und australia (data for 1995 in usa not available) in contrast, only a very weak correlation and a negative one can be observed between caffeine - containing multiple combinations and overall consumption (r = 19.7% ; fig. 5). this supports the statement that this product group of multiple combinations does not contribute to the increased use on a population level, but rather even counteracts higher overall consumption.fig. 5ecological analysis between consumption of all analgesics with caffeine - containing multiple analgesic combinations in the years 1986, 1990, 1995, 2000, 2005 considering the countries switzerland, germany (west), france, usa, canada, belgium, austria, sweden and und australia (data for 1995 in usa not available) ecological analysis between consumption of all analgesics with caffeine - containing multiple analgesic combinations in the years 1986, 1990, 1995, 2000, 2005 considering the countries switzerland, germany (west), france, usa, canada, belgium, austria, sweden and und australia (data for 1995 in usa not available) per - capita consumption of analgesics in germany (west) has been very stable over the past 25 years and, at 52 su per capita per year, is currently slightly higher than the long - term average (increase since 2000 : approx. there is no difference between germany (west) and germany (all) (fig. 6trend of analgesic consumption in germany (west) from 1980 until 2005 ; comparison between germany (west) and germany (all) in the year 2005 trend of analgesic consumption in germany (west) from 1980 until 2005 ; comparison between germany (west) and germany (all) in the year 2005 most of the analgesics of the n2b market considered here (market for general analgesics) can be bought without prescription over the counter in pharmacies (fig. the consumption of prescription - free analgesics increased markedly from 25.0 to 42.7 su per capita per year and in 1995 comprised approx. 88% of all analgesics used, while the percentage of prescription analgesics decreased by approx. this picture has been changing since 1995 with prescription analgesics tripling to approx. 18 su per capita per year, while the otc analgesics decreased by approx. the increase in prescription drug consumption since 1995 is primarily responsible for the slight increase in overall analgesics and is now back at the level of 1986. no consumption of any phenacetin - containing analgesics has been observed in germany since 1986.fig. 7trend of analgesic consumption in germany (west) from 1980 until 2005 comparing prescription - free (otc) with prescription (rx) analgesics trend of analgesic consumption in germany (west) from 1980 until 2005 comparing prescription - free (otc) with prescription (rx) analgesics in 2005 in the prescription (rx) group of n2b analgesics, combination products accounted for approx. the most important combinations are codeine plus paracetamol (18% of all su in the rx group), followed by tilidine plus naloxone (12%). the predominating single products in the rx group are metamizole (28%) and tramadol (23%). growth in the rx group can be seen for both the combinations named and the single products. this is the first investigation providing reliable, internationally comparable analyses of analgesic use in nine countries over a period of 20 years. the data from the database of the institute of medical statistics provide a sound and valid basis. the consumption data collected represent the supplies available for sale in the pharmacies. because of the nature of per - capita consumption data it is theoretically possible that the amounts of unused (expired) medications might be different in the various countries. however, no data on this are available. even if we assume that such differences exist, it is hardly conceivable that the differences in unused medication between countries would be high enough to explain the considerable differences in per - capita consumption alone. on international comparison, austria, switzerland and germany show the lowest per - capita consumption of analgesics. in sweden and france consumption is three times as high, which is attributed to the high increase in single products over the past 1520 years. it is difficult to assess to what extent these differences can be explained by different prevalences of painful disorders, such as shown for headache disorders at least for various world regions, since the required country - specific data are not available. austria today has the lowest proportion of single products in international comparison. in the usa a decrease in the use of single products is accompanied by a decrease in overall analgesic consumption. the correlation analysis over the different countries and time points confirms the positive correlation between use of single analgesics and overall analgesics consumption. caffeine - containing multiple combinations containing two analgesic agents plus caffeine are of minor relevance in international comparison and thus can not be causally related to the sharply rising overall consumption in some countries. the correlation analysis also shows that this product group does not contribute to the high analgesic consumption on a population level on the contrary it rather counteracts this phenomenon. it is currently 52 su per capita per year and thus slightly higher than the long - term average. these consumption figures, which are very stable in international comparison, were already seen from 1970 to 1986. they varied between 47 and 54 su per capita per year, with a continuous decrease after 1979, the year with the highest consumption. two - thirds of the current per - capita consumption in germany comprises otc analgesics. the increase in the use of prescription drugs since 1995 has led to a slight increase in overall consumption, which is now back at the level of 1986. this could be explained by an increasing number (possibly age - related) pain patients or a certain previous undersupply with analgesics of the latter. phenacetin - containing analgesics, which in the 1970s still held a market share of up to 40%, were taken from the market in 1986.. 1 su per year) since 1980. for all otc analgesics (except the 400 mg ibuprofen products) the epidemiological headache study conducted by the german migraine and headache society between 2003 and 2005 (first results were presented at the german pain congress 2006) showed that the headache prevalence over 1 year is approx. 60% and that about one - third of the german adult population suffer from headaches more than once per month. if we assume that these patients suffer from their migraine or headaches for 23 days per month and that they treat their condition with half the recommended maximum daily dose, i.e. 3 1 tablet per day, we receive a calculated per - capita consumption of 40 su per year for the total population (estimated 45% affected). the appropriate use of analgesics for the self - treatment of these indications alone can explain the per - capita consumption of approx. some of the affected people will undoubtedly successfully treat their headaches without medication or with an even lower daily dose. in addition to headache treatment, there is the treatment of pain of other etiologies (e.g. toothache, menstruation - induced pain, cold - induced pain, low back pain) and almost all analgesic use prescribed by doctors. consequently the consumption figures in germany and other countries do not support the claim of an inappropriately high use of analgesics on a population level. the prevalence of the conditions in which analgesic use is indicated, as the most relevant criterion for analgesic consumption, is frequently underestimated. although we definitely do not wish to trivialise the risks of analgesic abuse in general, or of medication overuse headache in particular, it is important to realise that estimates from analgesic consumption studies on population level as this are not adequate to provide scientifically founded data on analgesic consumption by specific patient groups. in conclusion, it has been shown that great differences exist in the per - capita consumption of analgesics between different countries. these differences seem to be due to the impact of single analgesics : countries with increasing amounts of single analgesics likewise show an increase in their total supplies of analgesics. in countries such as austria, where consumption with single analgesics is decreasing, the total consumption of analgesics also decreases and actually shows the lowest per - capita consumption among the considered countries. on the other hand, there is no correlation between the consumption of caffeine - containing analgesics and total analgesic consumption. per - capita taking into account the prevalence of the conditions prompting analgesics consumption, we can conclude appropriate use of analgesics on total population level. table 2table 2example for an su calculationcountry a(a)(b)(c)(d)(a) (b) (a) (c / factor)(e)(f)(d)/(e)packs per year (000)tablets per packml liquid per packsuinhabitants (000)per - capita consumptionyear 2000 asa8002016,000 6003018,000 paracetamol 1,0001010,000 4003012,000 50025012,500 total68,5001,50046year 2005 asa7002014,000 9003027,000 paracetamol800108,000 3503010,500 4502009,000 total 68,5001,40049 example for an su calculation | there are no reliable data at present on use of analgesics in various countries. we compared per - capita consumption in nine different countries during the period 19862005. the per - capita consumption was calculated on the basis of the sales figures of distributors to pharmacies and direct purchases by pharmaceutical companies in a sample of 1,000 pharmacies. the countries studied were : australia, austria, belgium, canada, france, germany, sweden, switzerland, and the usa. in international comparison austria, switzerland, and germany showed the lowest per - capita consumption of analgesics (approx. 4050 standard units (su) per capita per year), while in sweden and france consumption was three times as high. the correlation analysis over the various countries and time points confirmed a significant correlation between use of single analgesics and overall use of analgesics. in germany, where an allegedly particularly high and constantly rising analgesic use has been discussed controversially (meiner, pharm ind 49:12471251, 1987), per - capita consumption of analgesics from 1980 to 2005 remained practically unchanged at approx. 50 su per capita per year. the prevalence of conditions inducing analgesic use shows appropriate analgesics use on an overall population level. |
it is effective in attaining various pharmacological actions like deepana, pachana, shamana, shodhana, tarpana etc., when prepared according to classical guidelines, used in appropriate concentrations and clinical conditions. manjishtadi kashayam (mk), rasna erandadi kashayam (rek), sahacharadhi kashayam (sk), maharasnadi (also known as rasna dwiguna bhagam) kashayam (mrk), are quoted in the classical text sahasra yogam and dhanwantharam kashayam (dk) is quoted in the classical text ashtanga hrudayam. these five formulations are extensively used in the treatment of vata and vata - rakta diseases. in the management of vata vikaras, it is clearly indicated that in conditions where the vata alone is vitiated (no association with other doshas : pitta and kapha), one should initiate the treatment with snehana karma. but if other doshas are involved in pathogenesis along with vata, then snehana karma is contraindicated for treatment of the diseases. the formulations taken for the study are described in the texts of ayurveda in the kashayam form. it is observed in clinical practice that in the management of vatavikaras with the association of other doshas especially kapha, the kashaya formulations are very effective and they do the function of amahara also. an attempt is made to correlate the clinical indications of these kashayams with the antioxidant property. diseases of connective tissues, bones, joints and nervous system are broadly included in vatavyadhis and vatarakta vikaras. ayurveda as a system of medicine contributes profoundly to the wellness, curative and preventive aspects of these conditions. in vatavyadhis, there is persistent inflammation in synovial membranes of joints, with migration of activated phagocytes and other leukocytes into synovial and periarticular tissue. free radicals are generated in the process of phagocytosis, and these cytotoxic reactive oxygen species cause oxidative damage to the cells like destruction of membrane lipids, proteins, deoxyribonucleic acid and cartilage. the enzymatic or free radical damage to proteins like igg or collagen is also considered as a possibility that causes chronic inflammations in joints, local destruction of cartilage, bone and the systemic manifestations in rheumatoid arthritis. a molecule capable of inhibiting the oxidation of other molecules is known as an antioxidant ; it either prevents the reactive oxygen species from being formed or removes them before it damages vital components of the cell. oxidative stress occurs when there is an imbalance between oxidants and antioxidants in favour of oxidants which causes damage. oxidative stress has been implicated in a wide variety of chronic inflammatory diseases like rheumatoid arthritis, lupus erythematosus and psoriatic arthritis. the free radical scavenging role of the phenolic compounds is attributed to their unique structure. the phenolic compounds in the water extracts thereby take the role of antioxidants and play a protective role in vatavyadhis where the damage of bones and cartilage takes place. all the five formulations included in this study were manufactured by the arya vaidya pharmacy (coimbatore) limited. appropriately diluted standard and samples were made up to 3.5 ml with distilled water in a series of test tubes. these tubes were then treated with 0.5 ml 2 n folin - ciocalteus reagent and incubated for three minutes at room temperature. the reaction mixture was then incubated at room temperature for ninety minutes after which the absorbance was read at 760 nm (shimadzu uv vis spectrophotometer, 1800). the results were expressed as gallic acid equivalent in milligram per gram of sample, using a standard curve generated with gallic acid. the antioxidant activities of the ayurvedic formulations were estimated by dpph (22-diphenyl-1-picryl hydrazine) radical scavenging activity. briefly, to various concentrations of the sample, methanolic solution containing dpph radicals (0.1 mm) was added and shaken vigorously. the reaction mixture was then left to stand for thirty minutes in dark. after the incubation period the percentage inhibition of dpph free radical was calculated using the formula, % inhibition=(control - sample)/control)100. the sample concentration providing 50% inhibition (ec50) was calculated from the graph of rsa (radical scavenging activity) percentage against sample concentration. all the five formulations included in this study were manufactured by the arya vaidya pharmacy (coimbatore) limited. appropriately diluted standard and samples were made up to 3.5 ml with distilled water in a series of test tubes. these tubes were then treated with 0.5 ml 2 n folin - ciocalteus reagent and incubated for three minutes at room temperature. the reaction mixture was then incubated at room temperature for ninety minutes after which the absorbance was read at 760 nm (shimadzu uv vis spectrophotometer, 1800). the results were expressed as gallic acid equivalent in milligram per gram of sample, using a standard curve generated with gallic acid. the antioxidant activities of the ayurvedic formulations were estimated by dpph (22-diphenyl-1-picryl hydrazine) radical scavenging activity. briefly, to various concentrations of the sample, methanolic solution containing dpph radicals (0.1 mm) was added and shaken vigorously. the reaction mixture was then left to stand for thirty minutes in dark. after the incubation period the percentage inhibition of dpph free radical was calculated using the formula, % inhibition=(control - sample)/control)100. the sample concentration providing 50% inhibition (ec50) was calculated from the graph of rsa (radical scavenging activity) percentage against sample concentration. the results from total phenolic content determination and dpph radical scavenging activity show that the ayurvedic formulation, mk exhibited higher activity when compared to other kashayams [tables 1, 2 and figure 1 ]. dk with phenolic content 12.056 0.004 mg / g and 22 mg / ml effective concentration for 50% inhibition comes next in the present study. the other three kashayams namely sk, mrk and rek exhibits almost similar activity with total phenolic content 10.61 0.002 mg / g, 10.63 0.006 mg / g, 10.31 0.004 mg / g and percentage inhibition 36 g / ml, 26 g / ml and 40.48 g / ml respectively. total phenolic content of kashayams in vitro antioxidant property of kashayams dpph radical scavenging activity of various kashayams. mk which shows the maximum phenol content and most antioxidant property is a combination of forty five herbs as shown in the table 1. the combination has a broad spectrum of activity ranging from astadasa kusta (a wide spectrum of skin diseases), vatarakta (rheumatoid arthritis), arditha (bells palsy) upadamsa (veneral diseases), sleepada (filariasis), prasupthi (numbness), pakshaghat (hemi plegia), medodosha (diseases related to fatty depositions) netra roga (eye disease) etc. in clinical practice it is observed that in conditions like rheumatoid arthritis, skin disorders and degenerative disorders like hemiplegia, bell 's palsy and eye diseases, where oxidative stress causes or worsens the disease process, mk is very effective. the combination of mk has many potent vatahara herbs which are also antioxidants like rubia cordifolia, cedrus deodar, zingiber officinale, tinospora cordifolia, sida rhombifolia, triphala, hemidesmus indicus etc. the antioxidant effect of the combination possibly makes it a useful combination in the treatment of vatavyadhis. it is a versatile product with forty ingredients. even though it is indicated in the context of garbha vyapad (anomalies of pregnancy), it is effective in vatarogas (diseases affecting many connective tissues like bone, joints, ligaments, muscles and nervous system). it is also effective in traumatic conditions of bone and vital points. it is effectively used in bala rogas (pediatric diseases) and soothika vikaras (post partum care). it is also indicated in jwara (pyrexia), gulma (flatulence), graham unmada (mental disorders) moothraghata (urinary obstructions) and andra vriddhi (hernia). it is clinically seen that in conditions subsequent to trauma and also in arthritis or degenerative conditions subsequent to trauma, this formulation is exceptionally advantageous. in traumatic cartilage and joint injury there is an increased production of reactive oxidant species and reduced antioxidant defence. as a result of this imbalance, cell death and degradation of extracellular matrix there is significant improvement in chondrocyte viability and protection against extracellular matrix damage, following joint injury when there is brief exposure to free radical scavengers. os (oxygen species) play a role in multiple physiological processes from oocyte maturation to fertilization and embryo development. oxidative stress occurs due to excessive production of free radicals and/or impaired antioxidant defence mechanism. it is combination of twenty five herbs indicated in the context of vatavyadhis in sahasrayoga. it is effective in vatavyadhis, sarvangavata, pakshagata, apabhahugam, gridrasi, filariasis, andravridhi, sukla roga, linga roga, yoni roga, infertility. it is anabolic to body and helps in the management of infertility. in clinical practice it is observed that the brihmana (nourishing) nature of this formulation helps in the correction and redeeming painful conditions of vatavyadhis and also in correcting infertility. in both these conditions antooxidants play a vital role. the ingredients asparagus racemosus, withania somnifera, kaempferia galangal, are all nourishing herbs which are vatahara and also anti oxidant. it is indicated in vatavyadhis prakarana of sahasrayoga and is a combination of three herbs as shown in the table. rek shows the least antioxidant property of the five kashayams indicated in vatarakta chapter of sahasrayoga. it is also indicated in ekanga vatam (hemiplegia) swasa kasa (respiratory disorders) kanda rogas (disease of throat) soothika roga (diseases in post partum stage), jihwa sthambam (paralysis of lingual muscles), (filariasis). the phenol content of the formulations mrk, sk and rek is comparable. from the observations, it is seen that the antioxidant property of the products substantiate the clinical indications of the products, where oxidative stress affects the system and the products provide corrective or protective functions in the conditions. the comparison of the antioxidant property is done to provide an idea on the sequence of the formulations which could be effectively used in an oxidative stress induced condition associated with the vata and vatarakta disorders. the sequence of the kashayams in the descending order of the antioxidant property is as follows : mk, dk, mrk, sk and rek. the antioxidant properties of the kashayams justify the protective and corrective effects produced by the products. | background : the five kashayams (kwaths - decoctions) manjishtadi kashayam (mk), rasna erandadi kashayam (rek), sahacharadhi kashayam (sk), maharasnadi (or rasna dwiguna bhagam) kashayam (mrk) and dhanwantharam kashayam (dk) are widely used in the management of diseases manifested due to vitiation of vata and vatarakta (mostly diseases of connective tissues, bones, joints and nervous system). free radicals are generated subsequent to the inflammatory changes in such conditions, and these cytotoxic reactive oxygen species cause oxidative damage to the cells. phenolic compounds are the most common water soluble antioxidant compounds in plants.objective:the present study aims at evaluating the phenolic content and antioxidant properties of these five kashayams and their probable protective role in the management of vata vyadhis.materials and method : the total phenolic contents of these five ayurvedic decoctions were determined using folin - ciocalteu method and the antioxidant properties were estimated by dpph (22-diphenyl-1-picryl hydrazine) radical scavenging activity.result:mk exhibited higher property (total phenolic content-15.61 0.006 mg / g wt, ec50 - 7.2g / ml) when compared to other kashayams. dk with phenolic content 12.056 0.004 mg / g wt and 22 g / ml effective concentration for 50% inhibition comes next in the present study. rek, sk and mrk show almost similar phenolic content and antioxidant property.conclusion:from the observations, it is seen that the total phenolic content and the antioxidant property of the products justify the protective and corrective effects produced by the products in vata and vatarakta disorders. |
a 15-year - old boy was admitted to the emergency room with anterior chest pain, nonproductive cough, progressive dyspnea, and subcutaneous emphysema of the neck. all symptoms had occurred suddenly, although the night before and the morning of the acute respiratory distress he had experienced heavy breathing, for which he had arbitrarily taken oral corticosteroids. he denied accidental trauma of the chest, surgical maneuvers, or acute infections prior to the respiratory event., he was eupnoic at rest, respiratory rate was 18/min, oxygen saturation was 98% on room air, pulse rate was regular, blood pressure was 110/60 mm hg, and body temperature was 36.5c. physical examination revealed extensive subcutaneous crepitations along the neck region and the superior part of the rib cage. laboratory studies revealed hb 15.7 gr / dl, and wbc counts11.2 10/l (neutrophils 92.7%, lymphocytes 6.7%, and eosinophils 0.1%). the patient was referred for posteroanterior and lateral chest x - ray (figures 1(a) and 1(b)), which demonstrated linear streaks of air in the mediastinum extending into the upper parts of the lung, more evident in the lateral projection. the radiological signs of pneumomediastinum are multiple and include radiolucent linear streaks of air in the mediastinum, often extending into the neck, air surrounding the mediastinal structures ; the presence of subcutaneous emphysema of soft tissues is often described. the lateral view increases the sensitivity in detecting signs of pneumomediastinum, in that, it may reveal radiolucent bands in the retrosternal areas, such as in our patient. for further evaluation, he underwent thoracic high - resolution computed tomography (hrct), where air was demonstrated around the esophagus, trachea, ascending aorta, peribronchial, and perivascular connective tissue ; partial pneumothorax on both sides was detected ; this was more prominent at the level of the left apex of the lung. finally, diffuse subcutaneous emphysema was present. no other abnormalities of the bronchial tree, such as fistulae, were detected. the patient was treated with systemic and inhaled corticosteroids plus inhaled long - acting bronchodilators ; analgesic treatment was administered when necessary. a follow - up ct scan performed one week after the admission showed almost complete resolution of the radiological alterations. the patient was discharged with no medication and was strongly advised to maintain a resting lifestyle. he was invited to return to the outpatient clinic for follow - up investigations. at the follow - up visits, he was asymptomatic, and physical and radiological examinations were normal. however, his mother reported an episode of acute dyspnea when he was 4 years old. in the year before the event, he had suffered from frequent episodes of rhinorrea, sneezing, nasal blockage, and sometimes chest tightness, especially during exposure to pets and/or windy weather. skin prick testing was performed, revealing sensitivities to dermatophagoides pteronyssinus and farinae, grass pollen, and dog dander. total ige were 146 iu / ml ; specific ige for dermatophagoides pteronyssinus and farinae was 0.470 ku / l and 13.9 ku / l, respectively ; ige values for dog dander were 11.3 ku / l, and for grass pollen 4.9 ku / l (normal range for specific ige < 0.10 ku / l). spirometry documented significant improvement in lung function after short - acting bronchodilator (15% from baseline fev1), which allowed for the diagnosis of asthma to be made. the diagnosis of asthma is rarely a challenge for physicians ; usually, a suspicion arising from the clinical manifestations of the disease is supported by the results of the functional pulmonary tests showing signs of reversible bronchial obstruction, which render the diagnosis an uncomplicated task. in some circumstances, however, the diagnostic process may be delayed or influenced by unusual presentation of asthma. this occurs especially in elderly patients, in whom multiple respiratory symptoms may be confusing or in subjects with noncharacteristic symptoms, in whom the single respiratory symptom may be misleading. in our case, pneumomediastinum and pneumotorax represented the first presentation of allergic asthma. atypical presentations of asthma have been associated with cough as the only respiratory symptom (cough - variant asthma) ; urticaria or anaphylactic shock is known as first appearance of an underlying asthma. pneumomediastinum has been reported as an unusual, rare complication of bronchial asthma [4, 5 ]. reports on this topic are scarce and all refer to spontaneous pneumomediastinum and subcutaneous emphysema following an acute attack of asthma. to our knowledge, this is the first clinical report of pneumomediastinum as initial sign of undiagnosed asthma. on the basis of the documented sensitization to grass pollen in our patient, and given that the episode occurred in september when the pollen concentration is still elevated in southern italy, the role of the allergic component as the precipitating factor can not be excluded. the air leakage is usually the result of alveolar wall rupture secondary to high intra - alveolar pressures. histological studies have shown that air dissects into the connective tissue, resulting in interstitial emphysema. because of a pressure gradient between the periphery of the lung and the hilus, air tracks along the vascular sheath into the hilum, thus reaching the mediastinum and moving to the neck and the subcutaneous space. sometimes, air may also decompress into the pericardium or retroperitoneal tissues. causes of increased alveolar pressure include barotrauma in patients receiving mechanical ventilation, deep inspiratory maneuvers such as during strenuous exercise or diabetic ketoacidosis, extreme respiratory efforts such as violent cough or prolonged valsalva manoeuver, and obstructed expiratory flow with overinflation. the clinical description of our patient leaves no doubt on the obstructive origin of the pneumomediastinum. asthma has been described in different studies as a predisposing factor for the development of spontaneous pneumomediastinum in up to 50% of cases [710 ]. in the retrospective study of macia 9 out of 41 patients (22%) had a recent of remote history of asthma, and a severe attack precipitating the pneumomediastinum was demonstrated in only one out of four asthmatics. nonrespiratory causes of pneumomediastinum are represented by disruption of the esophagus, penetrating thoracic injuries, or mediastinal infection with gas - forming bacteria. the clinical history and the absence of symptoms suggestive of the above - described conditions in our patient allowed ruling out nonrespiratory causes of the pneumomediastinum. the incidence of spontaneous pneumomediastinum is difficult to establish due to the paucity of studies [11, 12 ]. the clinical diagnosis is based on the symptom triad of dyspnea, nonspecific chest pain, and subcutaneous emphysema, which was evident in our case. however, symptoms are nonspecific, and sometimes the patient may be asymptomatic. the only patognomonic sign (hamman 's sign), characterized by a crunching or bubbling sound that is synchronous with the heart beat, is rarely noticed by the patient or the physician. in a revision of 41 cases with pneumomediastinum, hamman 's sign was detected in only 12% of patients. our patient only referred progressive dyspnea and chest pain ; no other respiratory and nonrespiratory symptoms were present ; hamman 's sign was not detected at the time of presentation. the computed tomography (ct) scanning is considered the gold standard of imaging tests because it is able to detect even small amounts of mediastinal air. the ct scan has the advantage over the chest radiographs of offering superior contrast resolution and lack of superimposition ; therefore, the sensitivity of the methodology is greater, enabling the false negative cases to be reduced. in our case, the ct scans allowed to detect the initial condition and to follow the clinical and radiological evolutions. in addition to the evaluation and treatment of the underlying condition, the typical management of pneumomediastinum consists of rest, oxygen therapy, and analgesia. our patient was treated with bed rest and analgesics ; oxygen and cough sedatives were not required. after the discharge, he was strongly invited to follow a rigid resting lifestyle, and asthma was treated according to the most recent guidelines. in conclusion, pneumomediastinum with pneumothorax and subcutaneous emphysema may represent a very unusual presentation of underlying allergic asthma. pneumomediastinum is a benign condition, which occurs spontaneously or as a complication of an underlying respiratory disease, such as during an asthma attack. this is, to our knowledge, the first report of pneumomediastinum as asthma symptom, rather than asthma complication. in the diagnostic evaluation of the causes of pneumomediastinum in young subjects, the presence of underlying asthma | we describe the case of an adolescent who was admitted to the hospital because of sudden occurrence of chest pain, dyspnea and subcutaneous emphysema. on admission, physical examination revealed subcutaneous crepitations in the superior part of the rib cage, and auscultation of the chest showed widespread wheezing. the radiological assessment confirmed the diagnosis of pneumomediastinum and pneumothorax. a follow - up ct scan performed one week after the admission showed almost complete resolution of the radiological alterations. at the following visits, the patient was asymptomatic, but reported to have suffered from frequent episodes of rhinorrea, sneezing, nasal blockage, and sometimes, chest tightness, especially during exposure to pets and/or windy weather. skin prick testing showed sensitivities to dermatophagoides pteronyssinus and farinae, grass pollen and dog dander. spirometry documented significant improvement in lung function after short - acting bronchodilator, allowing for the diagnosis of asthma to be made. although pneumomediastinum may be a complication of various respiratory diseases, including asthma, it has never been reported as the first presentation of underlying bronchial asthma. herein, the physiopathological mechanisms, the diagnostic procedures and treatment of pneumomediastinum in asthma are discussed. we suggest that the diagnosis of asthma should be considered in the differential diagnosis of pneumomediastinum in adolescence. |
research in the invertebrate model drosophila melanogaster has driven the discovery of innate immunity, and has facilitated the understanding of various aspects of blood cell development. drosophila hematopoiesis can be divided into the lineage of embryonic / larval hemocytes, which originate in the embryo and expand in the larva, and the lineage of lymph gland hemocytes. here, we present a protocol that focuses on the lineage of embryonic / larval hemocytes, which in the drosophila larva mainly comprises plasmatocytes (macrophages) and few crystal cells. in the larva, hemocytes of the embryo persist and colonize segmentally repeated and terminal hematopoietic pockets (hps) located between the epidermis and muscle layers of the larval body wall. based on their nature as self - renewing macrophages, their predominant residence in local tissue microenvironments, and their lineage from the earliest blood cells emerging during development, this blood cell population is considered similar to vertebrate self - renewing tissue macrophages, an independent myeloid lineage recently identified in a variety of species. however, in drosophila, some or all of these resident cells also show plasticity to give rise to other blood cell types such as crystal cells. larval hemocytes are predominantly resident (sessile), but are in a dynamic steady - state between various hps. they are progressively released into circulation, in particular as the 3 instar larva approaches pupariation. immune challenges, injury or mechanical disturbance lead to a premature, in the latter case reversible, mobilization of resident hemocytes into the hemolymph. previous studies have suggested that resident and circulating larval hemocytes are of the same lineage, but differ in their adhesive or homing properties. selective isolation of circulating versus resident hemocytes revealed elevated levels of proliferation in the resident hemocyte population, suggesting their exposure to inductive cues from the hps. drosophila larval hps are lined by epidermis and muscle layers and further harbor sensory neuron clusters of the peripheral nervous system (pns) and liver function resembling oenocytes. functionally, mutant and genetic cell ablation experiments have demonstrated that sensory neurons present in the hps support the trophic survival and localization of larval hemocytes. here we describe a method for the specific isolation and quantification of resident and circulating hemocytes from single drosophila larvae, and a protocol for mechanical hemocyte mobilization. the methods can be used for the ex vivo study of hemocytes and can further be adapted to other drosophila developmental stages such as the pupa and adult, and other invertebrate systems. since previous studies did not distinguish between resident and circulating hemocytes, this protocol provides a common standard for the study of resident blood cells and will help to increase the consistency of invertebrate blood cell research. first, the hemocyte bleed / scrape assay describes the differential isolation and automated quantification of fluorescent protein - marked resident and circulating hemocyte populations from single drosophila larvae ; the protocol provides two options for regular and tile scan - equipped microscopes (figure 1). as a result, the percentage of circulating hemocytes and the total number of hemocytes per larva are obtained. the method relies on transgenic drosophila larvae that express fluorescent protein among their blood cell population. the choice of hemocyte driver or reporter determines the outcome, i.e., which population of blood cells is visualized and quantified. to label mainly macrophages (plasmatocytes), which comprise the vast majority of the resident and circulating hemocyte population of the drosophila larva, suitable transgenes include hml-dsred, hml-gal4, pxn - gal4, crq - gal4 (by h. agaisse), or eater - gal4 ; for labeling the relatively small population of crystal cells, suitable lines are bcf6-cfp and -gfp, or lz - gal4 (by j. pollock) ; for labeling lamellocytes, a specialized cell type mainly induced by immune challenges and injury, e.g., msnf9mo - mcherry may be used. some transgenic drivers are expressed in a range of differentiated blood cells and progenitors, such as he - gal4, which labels about 80% of all larval blood cells. please note that in all cases where gal4 drivers are used, combination with uas - gfp or another fluorescent protein uas - transgene is required. in the results section, this method is used to monitor blood cell number and circulation behavior over the course of larval development. second, the hemocyte disturbance assay describes a preceding step designed to detach resident hemocytes by external manipulation, which subsequently allows the evaluation of the ability of hemocytes to re - adhere and home to hps within a limited time frame (30 - 60 min). typically this assay is followed by the bleed / scrape assay to determine the percentage of circulating hemocytes per larva. we present a simplified protocol for this assay (figure 1d), which uses disturbance by vortexing with glass beads, rather than manipulation of single larva with a paint brush as described previously. in the results section, this assay is used to demonstrate that transiently detached hemocytes float in the hemolymph and can be recovered in the fraction of circulating hemocytes. the assay is also useful to quantify differences of hemocytes in their homing / adhesion to resident sites, comparing e.g., various genetic backgrounds or stimulation conditions. please note that this mechanical manipulation reflects a reversible process and is distinct from infection- or injury - induced resident hemocyte mobilization, which typically are not reversible in a short time frame. preparation of slides : option 1 for microscopes without tile scanning function : for each larva to be analyzed, prepare one glass slide with about 5 pap - pen wells of 2 mm squares each, corresponding to the field viewing area of the microscope ; add approximately 5 - 10 l of s2 media to each (figure 1a). keep slides in moist chamber to prevent wells from drying out.option 2 for microscopes with tile scanning function : for each larva to be analyzed, prepare one glass slide with 3 to 4 pap - pen wells of ~3 - 4 mm squares each ; add approximately 15 - 20 l of s2 media to each well (figure 1b). keep slides in moist chamber to prevent wells from drying out. note : the above recommended number of wells is sufficient for totals of up to 3,000 hemocytes per larva (late 2 instar larvae, ~2.5 - 3 mm length, transgene labeling the majority of larval blood cells). when assessing larger blood cell numbers, more wells might be needed to avoid over - crowding. option 1 for microscopes without tile scanning function : for each larva to be analyzed, prepare one glass slide with about 5 pap - pen wells of 2 mm squares each, corresponding to the field viewing area of the microscope ; add approximately 5 - 10 l of s2 media to each (figure 1a). option 2 for microscopes with tile scanning function : for each larva to be analyzed, prepare one glass slide with 3 to 4 pap - pen wells of ~3 - 4 mm squares each ; add approximately 15 - 20 l of s2 media to each well (figure 1b). keep slides in moist chamber to prevent wells from drying out. note : the above recommended number of wells is sufficient for totals of up to 3,000 hemocytes per larva (late 2 instar larvae, ~2.5 - 3 mm length, transgene labeling the majority of larval blood cells). when assessing larger blood cell numbers, more wells might be needed to avoid over - crowding. collection of larvae : squirt water into a fly vial containing larvae and flush larvae into a petri dish, or scoop some food that contains larvae into a petri dish and dilute with water using a squirt bottle.gently pick larvae out of the petri dish using a paintbrush and place them in water in a cavity dish or on a slide on a cold block. note : larvae can be kept for a limited time in water or on a cold block ; use specimens within 45 min or less to avoid larval death or unwanted effects on hemocytes. squirt water into a fly vial containing larvae and flush larvae into a petri dish, or scoop some food that contains larvae into a petri dish and dilute with water using a squirt bottle. gently pick larvae out of the petri dish using a paintbrush and place them in water in a cavity dish or on a slide on a cold block. note : larvae can be kept for a limited time in water or on a cold block ; use specimens within 45 min or less to avoid larval death or unwanted effects on hemocytes. dissection : select larvae under a fluorescence microscope on a cold metal block. measure sizes and image larvae if desired.isolation of circulating hemocytes (bleed) : once larvae are selected, place one larva in the first pap - pen well (figure 1c, 2a).use 2 clean needles or dissecting scissors and forceps to make an incision at both the posterior and anterior ends of the larva. to avoid disturbing resident hemocytes, it is best to make these incisions on the ventral side of the larva. for consistent results, make the incisions in the same locations for every larva. for 1 instar larvae, 1 incision (in the ventral anterior) is sufficient.allow larva to bleed for a few seconds without any pressure or physical agitation (figure 2a). note : if working on multiple larvae it is better to make these incisions for each one before proceeding to the next step to avoid keeping larvae on ice too long which could affect the samples integrity.gently lift the larva with the needles or forceps and dip it into the second well to rinse any remaining circulating hemocytes. isolation of resident hemocytes (scrape) : gently transfer the larva to the next well (figure 2c).identify the lymph gland of the larva, which typically is located approximately 1/3 from the anterior end of the larva, and which may fluoresce dorsally through the larval body wall. avoid the lymph gland while releasing resident hemocytes by pinning down the larva with a needle as near as possible to the lymph gland to avoid puncturing (figure 2c). note : during normal development the maturation of lymph gland hemocytes is delayed compared to larval hemocytes, and fluorescent reporters of differentiated hemocytes may not show a signal in the lymph gland of young larvae. in these instances, less attention needs to be paid to the lymph gland as no contamination of differentiated fluorescently - labeled larval hemocytes by lymph gland hemocytes is expected.release resident blood cells in a dissection process of scraping and/or jabbing. use one needlepoint to effectively pin down the larva near the lymph gland (see above) or other body areas as needed. use another needle to jab at the clusters of hemocytes that are visible through the larval body wall (figure 2c, e), aiming to separate the hemocytes. hemocytes can also be released in a scraping motion. however, tearing the epidermis early may release big clusters of blood cells, which could make automated counting more challenging. note : depending on the age and genotype of the larva, the number of total hemocytes will vary. distribute the release process described above over several wells to avoid overcrowding of some wells with blood cells, which could make single cell image analysis more difficult.if few hemocytes remain in the final carcass, count these hemocytes by observation through the microscope and use of a manual tally counter (figure 2e). to facilitate counting, place the carcass on a clean area of the same slide and spread it as thinly as possible to reduce the number of optical planes.once the dissection is complete, wait between 5 - 10 min for the cells to settle (but not necessarily adhere) before imaging the wells. incubate the slide in a moist chamber to avoid drying, and avoid rough handling of the slides, which could disturb the settled hemocytes. note : when determining hemocyte counts, released cells are not fixed and the cells must be imaged shortly after dissection, preferably within 30 min after release from the larva. depending on the volume of medium and cell properties, the vast majority of cells will have settled within 5 - 10 min, which should be confirmed by focusing through the optical planes of the medium in the well. however, only a fraction of blood cells will have adhered to the slide surface by this time, a fact that needs to be considered if modifying this protocol for cell fixation - based approaches. select larvae under a fluorescence microscope on a cold metal block. isolation of circulating hemocytes (bleed) : once larvae are selected, place one larva in the first pap - pen well (figure 1c, 2a).use 2 clean needles or dissecting scissors and forceps to make an incision at both the posterior and anterior ends of the larva. to avoid disturbing resident hemocytes, it is best to make these incisions on the ventral side of the larva. for consistent results, make the incisions in the same locations for every larva. for 1 instar larvae, 1 incision (in the ventral anterior) is sufficient.allow larva to bleed for a few seconds without any pressure or physical agitation (figure 2a). note : if working on multiple larvae it is better to make these incisions for each one before proceeding to the next step to avoid keeping larvae on ice too long which could affect the samples integrity.gently lift the larva with the needles or forceps and dip it into the second well to rinse any remaining circulating hemocytes. once larvae are selected, place one larva in the first pap - pen well (figure 1c, 2a). use 2 clean needles or dissecting scissors and forceps to make an incision at both the posterior and anterior ends of the larva. to avoid disturbing resident hemocytes, it is best to make these incisions on the ventral side of the larva. for consistent results, make the incisions in the same locations for every larva. for 1 instar larvae, 1 incision (in the ventral anterior) allow larva to bleed for a few seconds without any pressure or physical agitation (figure 2a). note : if working on multiple larvae it is better to make these incisions for each one before proceeding to the next step to avoid keeping larvae on ice too long which could affect the samples integrity. gently lift the larva with the needles or forceps and dip it into the second well to rinse any remaining circulating hemocytes. isolation of resident hemocytes (scrape) : gently transfer the larva to the next well (figure 2c).identify the lymph gland of the larva, which typically is located approximately 1/3 from the anterior end of the larva, and which may fluoresce dorsally through the larval body wall. avoid the lymph gland while releasing resident hemocytes by pinning down the larva with a needle as near as possible to the lymph gland to avoid puncturing (figure 2c). note : during normal development the maturation of lymph gland hemocytes is delayed compared to larval hemocytes, and fluorescent reporters of differentiated hemocytes may not show a signal in the lymph gland of young larvae. in these instances, less attention needs to be paid to the lymph gland as no contamination of differentiated fluorescently - labeled larval hemocytes by lymph gland hemocytes is expected.release resident blood cells in a dissection process of scraping and/or jabbing. use one needlepoint to effectively pin down the larva near the lymph gland (see above) or other body areas as needed. use another needle to jab at the clusters of hemocytes that are visible through the larval body wall (figure 2c, e), aiming to separate the hemocytes. hemocytes can also be released in a scraping motion. however, tearing the epidermis early may release big clusters of blood cells, which could make automated counting more challenging. note : depending on the age and genotype of the larva, the number of total hemocytes will vary. distribute the release process described above over several wells to avoid overcrowding of some wells with blood cells, which could make single cell image analysis more difficult.if few hemocytes remain in the final carcass, count these hemocytes by observation through the microscope and use of a manual tally counter (figure 2e). to facilitate counting, place the carcass on a clean area of the same slide and spread it as thinly as possible to reduce the number of optical planes.once the dissection is complete, wait between 5 - 10 min for the cells to settle (but not necessarily adhere) before imaging the wells. incubate the slide in a moist chamber to avoid drying, and avoid rough handling of the slides, which could disturb the settled hemocytes. note : when determining hemocyte counts, released cells are not fixed and the cells must be imaged shortly after dissection, preferably within 30 min after release from the larva. depending on the volume of medium and cell properties, the vast majority of cells will have settled within 5 - 10 min, which should be confirmed by focusing through the optical planes of the medium in the well. however, only a fraction of blood cells will have adhered to the slide surface by this time, a fact that needs to be considered if modifying this protocol for cell fixation - based approaches. identify the lymph gland of the larva, which typically is located approximately 1/3 from the anterior end of the larva, and which may fluoresce dorsally through the larval body wall. avoid the lymph gland while releasing resident hemocytes by pinning down the larva with a needle as near as possible to the lymph gland to avoid puncturing (figure 2c). note : during normal development the maturation of lymph gland hemocytes is delayed compared to larval hemocytes, and fluorescent reporters of differentiated hemocytes may not show a signal in the lymph gland of young larvae. in these instances, less attention needs to be paid to the lymph gland as no contamination of differentiated fluorescently - labeled larval hemocytes by lymph gland hemocytes is expected. release resident blood cells in a dissection process of scraping and/or jabbing. use one needlepoint to effectively pin down the larva near the lymph gland (see above) or other body areas as needed. use another needle to jab at the clusters of hemocytes that are visible through the larval body wall (figure 2c, e), aiming to separate the hemocytes. hemocytes can also be released in a scraping motion. however, tearing the epidermis early may release big clusters of blood cells, which could make automated counting more challenging. note : depending on the age and genotype of the larva, the number of total hemocytes will vary. distribute the release process described above over several wells to avoid overcrowding of some wells with blood cells, which could make single cell image analysis more difficult. if few hemocytes remain in the final carcass, count these hemocytes by observation through the microscope and use of a manual tally counter (figure 2e). to facilitate counting, place the carcass on a clean area of the same slide and spread it as thinly as possible to reduce the number of optical planes. once the dissection is complete, wait between 5 - 10 min for the cells to settle (but not necessarily adhere) before imaging the wells. incubate the slide in a moist chamber to avoid drying, and avoid rough handling of the slides, which could disturb the settled hemocytes. note : when determining hemocyte counts, released cells are not fixed and the cells must be imaged shortly after dissection, preferably within 30 min after release from the larva. depending on the volume of medium and cell properties, the vast majority of cells will have settled within 5 - 10 min, which should be confirmed by focusing through the optical planes of the medium in the well. however, only a fraction of blood cells will have adhered to the slide surface by this time, a fact that needs to be considered if modifying this protocol for cell fixation - based approaches. quantification : take images of the settled hemocytes under a fluorescent microscope (figure 2b, d, f). follow with quantification of hemocytes using imagej software.prepare image for imagej cell counting algorithm : open image of well using imagej : file open (locate file and select).ensure that the image(s) is 8-bit or 16-bit. adjust the threshold for the image by selecting image then click adjust and select threshold. red and increase the lower threshold level (see black arrow) until each cell in the image is marked with a red dot (cells that are not being covered will be seen in grayscale ; figure 3b). as the lower threshold note : occasionally clusters of cells can not be resolved and would be counted as one by the particle counter. in such cases, the number of cells in a cluster can be estimated by examining the image (zoom in if needed) and manual counting using a tally counter. alternatively, the lower threshold can be increased to resolve clusters of cells ; any unmarked cells resulting from this manipulation can then be counted using a tally counter. analyze cell number using imagej : launch the particle analyzer to count the cells (figure 3c). select analyze and click on analyze particle. optionally select overlay outlines to see the particles the algorithm count (figure 3d). alternatively, set a limit to the size or pixel area of a unit (e.g., cell, clump of cells, etc.) for the algorithm to count.click ok. take images of the settled hemocytes under a fluorescent microscope (figure 2b, d, f). prepare image for imagej cell counting algorithm : open image of well using imagej : file open (locate file and select).ensure that the image(s) is 8-bit or 16-bit. adjust the threshold for the image by selecting image then click adjust and select threshold. red and increase the lower threshold level (see black arrow) until each cell in the image is marked with a red dot (cells that are not being covered will be seen in grayscale ; figure 3b). note : occasionally clusters of cells can not be resolved and would be counted as one by the particle counter. in such cases, the number of cells in a cluster can be estimated by examining the image (zoom in if needed) and manual counting using a tally counter. alternatively, the lower threshold can be increased to resolve clusters of cells ; any unmarked cells resulting from this manipulation can then be counted using a tally counter. open image of well using imagej : file open (locate file and select). adjust the threshold for the image by selecting image then click adjust and select threshold. observe the threshold window red and increase the lower threshold level (see black arrow) until each cell in the image is marked with a red dot (cells that are not being covered will be seen in grayscale ; figure 3b). note : occasionally clusters of cells can not be resolved and would be counted as one by the particle counter. in such cases, the number of cells in a cluster can be estimated by examining the image (zoom in if needed) and manual counting using a tally counter. alternatively, the lower threshold can be increased to resolve clusters of cells ; any unmarked cells resulting from this manipulation can then be counted using a tally counter. analyze cell number using imagej : launch the particle analyzer to count the cells (figure 3c). select analyze and click on analyze particle. optionally select overlay outlines to see the particles the algorithm count (figure 3d). alternatively, set a limit to the size or pixel area of a unit (e.g., cell, clump of cells, etc.) for the algorithm to count.click ok. optionally select overlay outlines to see the particles the algorithm count (figure 3d). alternatively, set a limit to the size or pixel area of a unit (e.g., cell, clump of cells, etc.) for the algorithm to count. click ok. observe a summary window with the count (figure 3e). to disturb hemocytes, select larvae and place them in a 2 ml microcentrifuge tube with approximately 0.5 g of glass beads (212 - 600 m) and add 0.5 ml water. retrieve the larvae from the glass beads by spilling the contents of the microcentrifuge tube into a petri dish and picking out the larvae with a paintbrush. for the recovery phase, place larvae in previously prepared petri dishes with small amounts of fly food. allow the larvae to re - establish their hemocyte pattern for a period of 45 min or as desired. note : discard any larvae that have stopped moving, as they have died in the process. however, we typically see little damage after 1 min of vortexing (see below and supplemental figure 1). after the recovery period, preparation of slides : option 1 for microscopes without tile scanning function : for each larva to be analyzed, prepare one glass slide with about 5 pap - pen wells of 2 mm squares each, corresponding to the field viewing area of the microscope ; add approximately 5 - 10 l of s2 media to each (figure 1a). keep slides in moist chamber to prevent wells from drying out.option 2 for microscopes with tile scanning function : for each larva to be analyzed, prepare one glass slide with 3 to 4 pap - pen wells of ~3 - 4 mm squares each ; add approximately 15 - 20 l of s2 media to each well (figure 1b). keep slides in moist chamber to prevent wells from drying out. note : the above recommended number of wells is sufficient for totals of up to 3,000 hemocytes per larva (late 2 instar larvae, ~2.5 - 3 mm length, transgene labeling the majority of larval blood cells). when assessing larger blood cell numbers, more wells might be needed to avoid over - crowding. option 1 for microscopes without tile scanning function : for each larva to be analyzed, prepare one glass slide with about 5 pap - pen wells of 2 mm squares each, corresponding to the field viewing area of the microscope ; add approximately 5 - 10 l of s2 media to each (figure 1a). option 2 for microscopes with tile scanning function : for each larva to be analyzed, prepare one glass slide with 3 to 4 pap - pen wells of ~3 - 4 mm squares each ; add approximately 15 - 20 l of s2 media to each well (figure 1b). keep slides in moist chamber to prevent wells from drying out. note : the above recommended number of wells is sufficient for totals of up to 3,000 hemocytes per larva (late 2 instar larvae, ~2.5 - 3 mm length, transgene labeling the majority of larval blood cells). when assessing larger blood cell numbers, more wells might be needed to avoid over - crowding. collection of larvae : squirt water into a fly vial containing larvae and flush larvae into a petri dish, or scoop some food that contains larvae into a petri dish and dilute with water using a squirt bottle.gently pick larvae out of the petri dish using a paintbrush and place them in water in a cavity dish or on a slide on a cold block. note : larvae can be kept for a limited time in water or on a cold block ; use specimens within 45 min or less to avoid larval death or unwanted effects on hemocytes. squirt water into a fly vial containing larvae and flush larvae into a petri dish, or scoop some food that contains larvae into a petri dish and dilute with water using a squirt bottle. gently pick larvae out of the petri dish using a paintbrush and place them in water in a cavity dish or on a slide on a cold block. note : larvae can be kept for a limited time in water or on a cold block ; use specimens within 45 min or less to avoid larval death or unwanted effects on hemocytes. dissection : select larvae under a fluorescence microscope on a cold metal block. measure sizes and image larvae if desired.isolation of circulating hemocytes (bleed) : once larvae are selected, place one larva in the first pap - pen well (figure 1c, 2a).use 2 clean needles or dissecting scissors and forceps to make an incision at both the posterior and anterior ends of the larva. to avoid disturbing resident hemocytes, it is best to make these incisions on the ventral side of the larva. for consistent results, make the incisions in the same locations for every larva. for 1 instar larvae, 1 incision (in the ventral anterior) is sufficient.allow larva to bleed for a few seconds without any pressure or physical agitation (figure 2a). note : if working on multiple larvae it is better to make these incisions for each one before proceeding to the next step to avoid keeping larvae on ice too long which could affect the samples integrity.gently lift the larva with the needles or forceps and dip it into the second well to rinse any remaining circulating hemocytes. isolation of resident hemocytes (scrape) : gently transfer the larva to the next well (figure 2c).identify the lymph gland of the larva, which typically is located approximately 1/3 from the anterior end of the larva, and which may fluoresce dorsally through the larval body wall. avoid the lymph gland while releasing resident hemocytes by pinning down the larva with a needle as near as possible to the lymph gland to avoid puncturing (figure 2c). note : during normal development the maturation of lymph gland hemocytes is delayed compared to larval hemocytes, and fluorescent reporters of differentiated hemocytes may not show a signal in the lymph gland of young larvae. in these instances, less attention needs to be paid to the lymph gland as no contamination of differentiated fluorescently - labeled larval hemocytes by lymph gland hemocytes is expected.release resident blood cells in a dissection process of scraping and/or jabbing. use one needlepoint to effectively pin down the larva near the lymph gland (see above) or other body areas as needed. use another needle to jab at the clusters of hemocytes that are visible through the larval body wall (figure 2c, e), aiming to separate the hemocytes. hemocytes can also be released in a scraping motion. however, tearing the epidermis early may release big clusters of blood cells, which could make automated counting more challenging. note : depending on the age and genotype of the larva, the number of total hemocytes will vary. distribute the release process described above over several wells to avoid overcrowding of some wells with blood cells, which could make single cell image analysis more difficult.if few hemocytes remain in the final carcass, count these hemocytes by observation through the microscope and use of a manual tally counter (figure 2e). to facilitate counting, place the carcass on a clean area of the same slide and spread it as thinly as possible to reduce the number of optical planes.once the dissection is complete, wait between 5 - 10 min for the cells to settle (but not necessarily adhere) before imaging the wells. incubate the slide in a moist chamber to avoid drying, and avoid rough handling of the slides, which could disturb the settled hemocytes. note : when determining hemocyte counts, released cells are not fixed and the cells must be imaged shortly after dissection, preferably within 30 min after release from the larva. depending on the volume of medium and cell properties, the vast majority of cells will have settled within 5 - 10 min, which should be confirmed by focusing through the optical planes of the medium in the well. however, only a fraction of blood cells will have adhered to the slide surface by this time, a fact that needs to be considered if modifying this protocol for cell fixation - based approaches. select larvae under a fluorescence microscope on a cold metal block. isolation of circulating hemocytes (bleed) : once larvae are selected, place one larva in the first pap - pen well (figure 1c, 2a).use 2 clean needles or dissecting scissors and forceps to make an incision at both the posterior and anterior ends of the larva. to avoid disturbing resident hemocytes, it is best to make these incisions on the ventral side of the larva. for consistent results, make the incisions in the same locations for every larva. for 1 instar larvae, 1 incision (in the ventral anterior) is sufficient.allow larva to bleed for a few seconds without any pressure or physical agitation (figure 2a). note : if working on multiple larvae it is better to make these incisions for each one before proceeding to the next step to avoid keeping larvae on ice too long which could affect the samples integrity.gently lift the larva with the needles or forceps and dip it into the second well to rinse any remaining circulating hemocytes. once larvae are selected, place one larva in the first pap - pen well (figure 1c, 2a). use 2 clean needles or dissecting scissors and forceps to make an incision at both the posterior and anterior ends of the larva. to avoid disturbing resident hemocytes, it is best to make these incisions on the ventral side of the larva. for consistent results, make the incisions in the same locations for every larva. for 1 instar larvae, 1 incision (in the ventral anterior) allow larva to bleed for a few seconds without any pressure or physical agitation (figure 2a). note : if working on multiple larvae it is better to make these incisions for each one before proceeding to the next step to avoid keeping larvae on ice too long which could affect the samples integrity. gently lift the larva with the needles or forceps and dip it into the second well to rinse any remaining circulating hemocytes. isolation of resident hemocytes (scrape) : gently transfer the larva to the next well (figure 2c).identify the lymph gland of the larva, which typically is located approximately 1/3 from the anterior end of the larva, and which may fluoresce dorsally through the larval body wall. avoid the lymph gland while releasing resident hemocytes by pinning down the larva with a needle as near as possible to the lymph gland to avoid puncturing (figure 2c). note : during normal development the maturation of lymph gland hemocytes is delayed compared to larval hemocytes, and fluorescent reporters of differentiated hemocytes may not show a signal in the lymph gland of young larvae. in these instances, less attention needs to be paid to the lymph gland as no contamination of differentiated fluorescently - labeled larval hemocytes by lymph gland hemocytes is expected.release resident blood cells in a dissection process of scraping and/or jabbing. use one needlepoint to effectively pin down the larva near the lymph gland (see above) or other body areas as needed. use another needle to jab at the clusters of hemocytes that are visible through the larval body wall (figure 2c, e), aiming to separate the hemocytes. hemocytes can also be released in a scraping motion. however, tearing the epidermis early may release big clusters of blood cells, which could make automated counting more challenging. note : depending on the age and genotype of the larva, the number of total hemocytes will vary. distribute the release process described above over several wells to avoid overcrowding of some wells with blood cells, which could make single cell image analysis more difficult.if few hemocytes remain in the final carcass, count these hemocytes by observation through the microscope and use of a manual tally counter (figure 2e). to facilitate counting, place the carcass on a clean area of the same slide and spread it as thinly as possible to reduce the number of optical planes.once the dissection is complete, wait between 5 - 10 min for the cells to settle (but not necessarily adhere) before imaging the wells. incubate the slide in a moist chamber to avoid drying, and avoid rough handling of the slides, which could disturb the settled hemocytes. note : when determining hemocyte counts, released cells are not fixed and the cells must be imaged shortly after dissection, preferably within 30 min after release from the larva. depending on the volume of medium and cell properties, the vast majority of cells will have settled within 5 - 10 min, which should be confirmed by focusing through the optical planes of the medium in the well. however, only a fraction of blood cells will have adhered to the slide surface by this time, a fact that needs to be considered if modifying this protocol for cell fixation - based approaches. identify the lymph gland of the larva, which typically is located approximately 1/3 from the anterior end of the larva, and which may fluoresce dorsally through the larval body wall. avoid the lymph gland while releasing resident hemocytes by pinning down the larva with a needle as near as possible to the lymph gland to avoid puncturing (figure 2c). note : during normal development the maturation of lymph gland hemocytes is delayed compared to larval hemocytes, and fluorescent reporters of differentiated hemocytes may not show a signal in the lymph gland of young larvae. in these instances, less attention needs to be paid to the lymph gland as no contamination of differentiated fluorescently - labeled larval hemocytes by lymph gland hemocytes is expected. release resident blood cells in a dissection process of scraping and/or jabbing. use one needlepoint to effectively pin down the larva near the lymph gland (see above) or other body areas as needed. use another needle to jab at the clusters of hemocytes that are visible through the larval body wall (figure 2c, e), aiming to separate the hemocytes. hemocytes can also be released in a scraping motion. however, tearing the epidermis early may release big clusters of blood cells, which could make automated counting more challenging. note : depending on the age and genotype of the larva, the number of total hemocytes will vary. distribute the release process described above over several wells to avoid overcrowding of some wells with blood cells, which could make single cell image analysis more difficult. if few hemocytes remain in the final carcass, count these hemocytes by observation through the microscope and use of a manual tally counter (figure 2e). to facilitate counting, place the carcass on a clean area of the same slide and spread it as thinly as possible to reduce the number of optical planes. once the dissection is complete, wait between 5 - 10 min for the cells to settle (but not necessarily adhere) before imaging the wells. incubate the slide in a moist chamber to avoid drying, and avoid rough handling of the slides, which could disturb the settled hemocytes. note : when determining hemocyte counts, released cells are not fixed and the cells must be imaged shortly after dissection, preferably within 30 min after release from the larva. depending on the volume of medium and cell properties, the vast majority of cells will have settled within 5 - 10 min, which should be confirmed by focusing through the optical planes of the medium in the well. however, only a fraction of blood cells will have adhered to the slide surface by this time, a fact that needs to be considered if modifying this protocol for cell fixation - based approaches. quantification : take images of the settled hemocytes under a fluorescent microscope (figure 2b, d, f). follow with quantification of hemocytes using imagej software.prepare image for imagej cell counting algorithm : open image of well using imagej : file open (locate file and select).ensure that the image(s) is 8-bit or 16-bit. adjust the threshold for the image by selecting image then click adjust and select threshold. red and increase the lower threshold level (see black arrow) until each cell in the image is marked with a red dot (cells that are not being covered will be seen in grayscale ; figure 3b). as the lower threshold note : occasionally clusters of cells can not be resolved and would be counted as one by the particle counter. in such cases, the number of cells in a cluster can be estimated by examining the image (zoom in if needed) and manual counting using a tally counter. alternatively, the lower threshold can be increased to resolve clusters of cells ; any unmarked cells resulting from this manipulation can then be counted using a tally counter. analyze cell number using imagej : launch the particle analyzer to count the cells (figure 3c). select analyze and click on analyze particle. optionally select overlay outlines to see the particles the algorithm count (figure 3d). alternatively, set a limit to the size or pixel area of a unit (e.g., cell, clump of cells, etc.) for the algorithm to count.click ok. take images of the settled hemocytes under a fluorescent microscope (figure 2b, d, f). prepare image for imagej cell counting algorithm : open image of well using imagej : file open (locate file and select).ensure that the image(s) is 8-bit or 16-bit. adjust the threshold for the image by selecting image then click adjust and select threshold. red and increase the lower threshold level (see black arrow) until each cell in the image is marked with a red dot (cells that are not being covered will be seen in grayscale ; figure 3b). note : occasionally clusters of cells can not be resolved and would be counted as one by the particle counter. in such cases, the number of cells in a cluster can be estimated by examining the image (zoom in if needed) and manual counting using a tally counter. alternatively, the lower threshold can be increased to resolve clusters of cells ; any unmarked cells resulting from this manipulation can then be counted using a tally counter. open image of well using imagej : file open (locate file and select). adjust the threshold for the image by selecting image then click adjust and select threshold. observe the threshold window red and increase the lower threshold level (see black arrow) until each cell in the image is marked with a red dot (cells that are not being covered will be seen in grayscale ; figure 3b). note : occasionally clusters of cells can not be resolved and would be counted as one by the particle counter. in such cases, the number of cells in a cluster can be estimated by examining the image (zoom in if needed) and manual counting using a tally counter. alternatively, the lower threshold can be increased to resolve clusters of cells ; any unmarked cells resulting from this manipulation can then be counted using a tally counter. analyze cell number using imagej : launch the particle analyzer to count the cells (figure 3c). select analyze and click on analyze particle. optionally select overlay outlines to see the particles the algorithm count (figure 3d). alternatively, set a limit to the size or pixel area of a unit (e.g., cell, clump of cells, etc.) for the algorithm to count.click ok. optionally select overlay outlines to see the particles the algorithm count (figure 3d). alternatively, set a limit to the size or pixel area of a unit (e.g., cell, clump of cells, etc.) for the algorithm to count. to disturb hemocytes, select larvae and place them in a 2 ml microcentrifuge tube with approximately 0.5 g of glass beads (212 - 600 m) and add 0.5 ml water. retrieve the larvae from the glass beads by spilling the contents of the microcentrifuge tube into a petri dish and picking out the larvae with a paintbrush. for the recovery phase, place larvae in previously prepared petri dishes with small amounts of fly food. allow the larvae to re - establish their hemocyte pattern for a period of 45 min or as desired. note : discard any larvae that have stopped moving, as they have died in the process. however, we typically see little damage after 1 min of vortexing (see below and supplemental figure 1). after the recovery period, to illustrate typical outcomes of the described methods, we first used the hemocyte bleed / scrape assay to outline the progression of larval hemocyte numbers and their residence over the course of larval development (figure 4). resident and circulating larval hemocyte populations were isolated from single larvae (hml-gal4, uas - gfp ; he - gal4 to labelthe vast majority of larval hemocytes) and quantified using imagej. cohorts of larvae sized 1.2 mm (~48 hr ael or 1 instar), 2.5 mm (~80 hr ael or late 2 instar), and 3.5 mm (~96 hr ael or 3 instar) were examined (figure 4). hemocyte numbers expanded over the course of larval development, correlating with and exceeding previous estimates based on light microscopy of dye stained larvae and live counting of fluorescent protein labeled hemocytes through the larval cuticle. in 1 instar larvae almost all hemocytes were resident, while the fraction of circulating hemocytes progressively increased over the course of larval development (figure 4b, c), consistent with previous publications. next we examined whether the method faithfully monitors the transition of hemocytes between the resident and circulating populations. taking advantage of the phenomenon that resident hemocytes can be transiently detached by mechanical disturbance and they re - adhere to their resident sites spontaneously, we dispersed resident hemocytes by vortexing with glass beads as described in the hemocyte disturbance assay. indeed, mechanical disturbance of larvae led to a dramatic increase in the population of circulating hemocytes at the expense of resident hemocytes (figure 5). after a recovery period of 45 min, hemocytes had largely returned to their adherent state, both by visual inspection and by the assessed percentage of circulating cells (figure 5d, e). as expected, total hemocyte numbers remained stable over time, despite the shift of hemocytes between the circulating and resident populations. several additional considerations were taken into account. to confirm that vortexing did not cause major tissue damage, vortexing with glass beads was performed in the presence of trypan blue (sigma) for various time periods (1, 5, 20 min). both 1 and 5 min vortexing did not cause any obvious tissue disruption, while 20 min vortexing resulted in small areas of damage, resembling damage caused by needle stitches used as positive control (supplemental figure 1). while internal damage of epidermis or other tissues without cuticle damage can not be excluded, this scenario seems rather unlikely as hemocytes of 1 min and 5 min - treated larvae re - adhered in the expected pattern and time frame, suggesting larval integrity was not compromised (supplemental figure 1). in contrast, larvae vortexed for 20 min suffered from a lack of re - adhesion, and did not even show attachment of circulating hemocytes to epidermal wound sites, as has been described previously. lastly, to demonstrate reproducibility of the method, we compared biological replicates of 2.5 mm larvae from the above two experiments, which were conducted by distinct experimenters. as illustrated in supplemental figure 2, both cohorts showed comparable total numbers of hemocytes per larva, and the percentage of circulating hemocytes. student s t testing showed no statistically significant differences, suggesting that the method is reproducible and broadly applicable. hemocyte bleed / scrape and disturbance assay setup and schematic. (a) single image slide setup : five 2 mm squares for imaging with a 5x objective. (b) tile scan slide setup : four 3 mm squares for imaging bleed / scrapes of 2.5 mm larvae with a tile scan microscope. recommended objectives for imaging are 5x or 10x. (d) in the disturbance assay, the hemocyte pattern is mechanically disrupted by vortexing larvae with glass beads. larvae are allowed to recover over a period of 45 min during which hemocytes re - adhere to the hematopoietic pockets. the adhesive properties of hemocytes can be assessed by this method, quantifying the percentage of hemocytes in circulation after disturbance (a) to bleed a larva, ventral incisions at the posterior and anterior ends of the larva are made (scissors symbol). (b) hemocytes in circulation will flow out of the incisions and settle on the surface of the slide. (c) the lymph gland (lg) is located and pinned down, without puncturing it. (e, f) the scrape process is repeated until all resident hemocytes are released. the larval carcass containing the intact lymph gland (a, b) after opening a hemocyte image file in imagej, the lower threshold level is adjusted to account for all the cells in the image. (c, d) analyze particles requires setting the cell pixel size, circularity, and the result readout format (e.g., overlay outlines). (a) overview of the larval stages used ; 1 instar (48 hr ael ; ~1.2 mm length) ; 2 instar (80 hr ael ; 2.5 mm length) ; 3 instar (96 hr ael ; ~3.5 mm length). (b) bar diagram of circulating and resident hemocyte numbers at the respective larval stages. note that the fraction of circulating hemocytes increases disproportionally over the course of larval development. (d) total hemocytes, resulting from the sum of circulating and resident hemocytes per larva. hemocytes were quantified using the bleed / scrape method ; n 6 larvae / condition, error bars show standard deviation, findings confirmed in 3 independent replicate experiments. (a - c) example of a larva before and after vortexing with glass beads, followed by 45 min recovery. (a) no disturbance control ; hemocytes are localized in hematopoietic pockets. (b) disrupted hemocyte pattern at 0 min after vortexing larvae in a suspension of glass beads and water. (c) hemocyte pattern at 45 min of recovery post - disturbance ; many hemocytes have relocated to the hematopoietic pockets ; note enlarged dorsal - vessel associated clusters and dorsal stripes which are predominant sites of early post - disturbance accumulation (arrows). (e) total hemocytes, resulting from the sum of circulating and resident hemocytes per larva. n 4 larvae / condition, error bars show standard deviation, findings confirmed in 3 independent replicate experiments. student s t - test to confirm significance, ns (not significant), (p 0.05), (p 0.01). here, we describe the first method to quantitatively recover resident and circulating blood cells from single drosophila larvae, and quantify these two hemocyte populations. the protocol comprises the sequential release of circulating and resident blood cells, followed by imaging and automated cell counting. larval resident hemocytes can be transiently mobilized into circulation by mechanical disturbance, a process that is known to be largely reversed within a 30 - 60 min recovery period. accordingly, this protocol was tested in two ways, (1) by assessing the total hemocyte number per larva and fraction of circulating hemocytes over the course of larval development, and (2) by experimentally dislodging resident hemocytes using an automated method, which confirmed the tight correlation of hemocyte localization and hemocyte number in the resident and circulating populations. in addition, the reproducibility of the method was demonstrated by comparing two datasets of biological replicates. in the past this protocol establishes a common standard to retrieve and quantify resident and circulating blood cell populations from drosophila larvae, providing an easily adaptable platform. the method described is critical for studies that focus on the role of resident hemocytes and their microenvironment, the hematopoietic pockets, and is suitable to study fluorescent protein transgene - carrying drosophila strains in wild type and genetically modified backgrounds. the protocol is also relevant for studies that focus on hemocyte mobilization after immune challenge or injury, and genetically or environmentally induced signaling that triggers mobilization of resident hemocytes or changes in total hemocyte number (reviewed in). it should be noted that, in cases of premature differentiation and release of hemocytes from the lymph gland, distinguishing embryonic / larval versus lymph gland lineages may be limited by the expression pattern of the fluorescent hemocyte reporter used. the protocol presented here relies on imaging live, fluorescently - labeled hemocytes. in the future, it may be modified to permit the detection of released cells after fixation, e.g., using immunocytochemistry. in this case, the protocol may need to be adapted to ensure complete adhesion of the blood cells, for example by increasing adhesion incubation times, and adding adhesive slide coating, such as concanavalin a. since the method allows retrieval of hemocytes and their manipulation ex vivo, it will benefit a wide range of developmental, cell biological and biochemical studies. resident and circulating blood cells are found during all postembryonic developmental stages of drosophila and other invertebrates, suggesting that adaptation of this method will benefit a wide range of studies beyond the drosophila larval hematopoietic system. | in vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). likewise, in the invertebrate model organism drosophila melanogaster, inductive microenvironments known as larval hematopoietic pockets (hps) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self - renewing macrophage lineage. hps are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. in the larva, resident (sessile) hemocytes are exposed to anti - apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the hps, such as the lining muscle and epithelial layers. during normal development, gradual release of resident hemocytes from the hps fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. the switch of larval hemocytes between resident locations and circulation raises the need for a common standard / procedure to selectively isolate and quantify these two populations of blood cells from single drosophila larvae. accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. the method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of drosophila and other invertebrate organisms. |
chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (clippers) is an inflammatory disorder in the central nervous system (cns) with distinct clinical, radiological, and pathological features. although the radiological lesions were reported to be located predominantly in the pons, brachium pontis, and cerebellum, other adjacent structures such as the white matter and spinal cord were very recently reported as involved regions in clippers. in this study, we report a case of clippers presenting with intracranial epstein barr virus (ebv) infection and diffuse white matter involvement. a 37-year - old male was diagnosed with mediastinal hodgkin 's lymphoma (lymphocyte predominance type) at the age of 26, and then obtained complete remission after treatment and remained free of relapse for 11 years. he was admitted with 7 months history of mental disorder, and 20 days history of gait and limb ataxia, dysphagia, and cough. the diagnosis of clippers was established based on the findings of punctate and nodular enhancing lesions in the bilateral pons, the basal ganglia, the mid - brain, the pontine brachium, and diffuse white matter in magnetic resonance imaging (mri), together with cd3 t - lymphocytic inflammatory infiltration in perivascular and parenchymal area revealed by bilateral parietal lobe brain biopsy. also, our patient exhibited a good response to steroid therapy and remained free of relapse for 5 months. clippers might be an autoimmune disorder, and intracranial ebv - infection raises the possibility that ebv - associated autoimmunity is associated with clippers pathogenesis. chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (clippers) is a recently defined immune - mediated and treatable central nervous system (cns) inflammatory disease with distinct clinical, radiological, and pathological characteristics. clippers patients present with a favorable but dependent response to steroids therapy and show a punctate and nodular pattern of gadolinium enhancement peppering in the pons, brainstem, white matter, and other adjacent structures. several studies suggested that clippers is an autoimmune disorder, but the related evidence still remains deficient. now, we report a case of clippers patient confirmed by clinical, radiological, and pathological characteristics. interestingly, a significantly elevated load of epstein barr virus (ebv) dna was detected in his cerebrospinal fluid (csf). this presentation raises the possibility that ebv - associated immunity dysfunction may be related to the pathogenesis of clippers. as far as we perceive, this is the first case of clippers with cns ebv infection. a 37-year - old male was diagnosed with mediastinal hodgkin 's lymphoma (lymphocyte predominance type) at the age of 26. after treated with abvd (adriamycin, bleomycin, vinblastine, dacarbazine), he obtained complete remission and remained free of relapse for 11 years. seven months prior to admission, he described sadness when feeling tough in his career. meanwhile, with the irritability, aypnia, and reduction of speech, he found the unwillingness of communication with others. after treated with escitalopram oxalate, risperidone, and sertraline, he gradually emerged symptoms of mania, which is characterized by increased grandiloquent speech and less need for sleep. diagnosed with bipolar disorder at the local hospital, he was given vpa - mg, buspirone and clonazepam, and then obtained an improvement. one month ago, he subacutely developed gait and limb ataxia featured by unstable walking and clumsy hand movements. he also had a generalized seizure (tonic) 2 weeks before admission, and then developed headache, shortness of breath, and somnolence. on admission, the patient 's temperature was normal. neurological examination revealed the right abducens nerve palsy and limited left eyeball movement to any direction. appearance of hoarseness, dysphagia, and cough had been noticed, with apparent pharyngeal reflex decreased. muscle strength scores of bilateral upper and lower limbs were 4 on the medical research council scale. the leukocytoclastic vasculitis was excluded because no palpable purpura, urticarial plaques, vesicles, bullae, or pustule was noted, nor were signs of joints injury or gastrointestinal injury. fever, malaise, weight loss, arthralgias, and myalgias were not detected in this case. hematuria and proteinuria were absent in this case. nor was any suspicious pulmonary hemorrhage noted. laboratory investigations revealed that the patient had normal complete and differential blood count, sedimentation rate, c - reactive protein, thyroid hormone, renal and liver function. serum immunological study, antinuclear antibodies, double - stranded dna antibodies, and antibodies against extractable nuclear antigens were all negative. paraneoplastic autoantibodies panels including anti - hu, anti - cv2, anti - ma2, anti - ri and anti - voltage - gated k channels were negative. nmda igg, ampa1 igg, ampa2 igg, lgl1 igg, caspr2 igg, and gaba b receptor igg were all negative. serum igg and/or igm of human immunodeficiency virus, hepatitis b virus, syphilis, and herpes simplex virus were all negative. barr nuclear antigens (ebnas) antibody and epstein barr viral capsid antigen igg (ebvca - igg > 22 ru / ml) but negative for epstein barr viral capsid antigen igm (ebvca - igm > 22 ru / ml), suggesting a possible history of ebv infection. the level of ebv dna in the blood (< 5000 copies) was also specified to rule out chronic active ebv disease (caebv) in this young patient. csf examination revealed mildly elevated protein levels (1.3 g / l, normal 0.150.40 notably, an elevated load of ebv - dna was detected in csf by pcr as 2.01 10 copies (normal < 5000 copies). brain magnetic resonance imaging (mri) showed hyperintense signal on t2-weighted images, associated with patchy gadolinium enhancement on t1-weighed images in the bilateral pons, the basal ganglia, the midbrain, the pontine brachium, and diffuse white matter (fig. the clinical and mri findings could suggest other disorders, such as neurosarcoidosis, cns vasculitis, autoimmune encephalitis, histiocytosis and paraneoplastic disease. bilateral parietal lobe brain biopsy revealed lymphocytic inflammatory infiltration in perivascular and parenchymal area (fig. 1c), consisting primarily of cd3 t lymphocytes (fig. 1d) and few detected cd1a cells, cd30 cells, cd10 cells and cd20 cells. neuroradiological images on admission and 3 weeks after steroid therapy and pathological findings from brain biopsy. representative t1-weighted imaging with gadolinium enhancement (top row) and corresponding t2-weighted imaging (bottom row) on cervical mri demonstrate changes of mri features after steroid therapy. the images performed on admission show punctate and nodular enhancing lesions in the bilateral pons, the basal ganglia, the midbrain, the pontine brachium, and extensively in cerebral white matter, with a perivascular enhancement pattern (a1, a2). a decrease in the number and extent of pathology is observed on a cervical mri scan after steroid treatment (b1, b2). in pathology findings from the parietal lobe specimen, hematoxylin and eosin staining showed lymphocytic inflammatory infiltration with perivascular and parenchymal cells (c ; 100). the lymphocytic infiltrates were mainly composed of cd3 t lymphocytes (d ; 100). proliferation rate, detected by ki67 antigen immunohistochemistry, was about 10% (e ; 100). (15 mg daily) was initiated, the patient responded well with gradually improved symptoms within a week from starting treatment, despite mild persistent mental abnormality and dysphagia. csf analysis results also showed a decrease of the cytology (white blood cells : 47/mm, 99% lymphocytes). the pontine lesions in mri decreased in numbers and extent 3 weeks from starting treatment (fig. four weeks later, the dexamethasone was tapered by 5 mg every 5 days until discontinuance. oral prednisolone (30 mg daily) was therefore initiated, ultimately maintained at 5 mg daily after sequential tapering. during the 5 months of steroids therapy, the patient was admitted with 7 months history of mental disorder, 20 days history of gait and limb ataxia, dysphagia, and cough. the mri images and histopathological features, along with the favorable clinical and radiological responses to corticosteroids led us to consider the diagnosis of clippers. however, the specialty in our patient should be noted : (i) this patient has a history of mediastinal hodgkin 's lymphoma (lymphocyte predominance type), and after treated with abvd (adriamycin, bleomycin, vinblastine, dacarbazine), he obtained complete remission and remained free of relapse for 11 years ; (ii) this is the first study reporting a clippers patient with an elevated load of ebv - dna in the csf ; (iii) this patient 's mri showed lesions of broad areas of cerebral white matter. although recurrence of hodgkin 's lymphoma in the brain is rarely reported, we did consider the possibility of newly emerged cns lymphoma occurring after treatment for hodgkin 's lymphoma because of the immunosuppression. however, findings in brain biopsy showed no evidence of cns lymphoma since the ki67 labeling index, which is strictly associated with cell proliferation, was 10%. in addition, the patient did not suffer from suspicious fever or body weight loss. nor was lymphadenopathy found. thus, we intended to diagnose the patient with clippers for now. still, it remains to be elucidated whether clippers is associated with cns lymphoma. some of the reported patients initially diagnosed with clippers finally progressed to cns lymphoma despite receiving timely treatment in literatures. these patients responded well to steroids therapy at first and gained symptoms improvement with decreased lesions. however, long - term follow - up revealed worsened symptoms and re - emerged lesions in mri, consequently leading to a second brain biopsy which gave the confirmed diagnosis of cns lymphoma. therefore, a long - term follow - up is necessary, aiming at monitoring the possible changes of symptoms or mri features to confirm whether there is coexisting or subsequent development of cns lymphoma. at the same time, a second biopsy is recommended if it is necessary, especially when worsening of clinical or radiological presentation appears in spite of proper immunosuppressive treatment, or when the immunosuppressive therapy is refractory. previous investigators proposed that clippers might be an autoimmune disorder due to the clinical response to immunosuppressive therapies, and the inflammatory infiltration around perivascular regions, where the possible targeted autoantigen is located. however, this hypothesis is incomplete because the specific mechanism is still unclear. in this clippers patient, a great attention should be paid to the early elevated load of ebv - dna in csf as well as the possible history of ebv infection. several autoimmune diseases, including multiple sclerosis, systemic lupus erythematosus, and sjgren 's syndrome, are associated with ebv infection. ebv causes autoimmune reaction through molecular mimicry, epitope spreading, and suppression of tlrs. growing evidence suggests that some of the ebv - encoded latent proteins, such as the ebnas, share linear and functional sequences identity with host proteins such as neuronal protein -synuclein. during acute or persistent reactivating infection, ebv can produce pro - inflammatory signals which promote anti - viral responses and cause tolerance abandon of immune system with mimicry to -synuclein, leading to the oligomerization of -synuclein. besides the main expression in neurons, -synuclein is also expressed in cerebral blood vessels, around where is exactly the place of inflammatory infiltration in clippers. thus, we propose a possible mechanism that the autoimmune response induced by ebv infection might be one of the possible pathological causes of clippers, contributing to the autoimmune hypothesis. the high load of ebv dna in the csf but not in the blood suggests that the affected cells are resident to the cns rather than recruited from the periphery. it is reported that the main target cells of ebv are epithelial cells and b cells. thus, the elevated ebv dna in csf may be due to the cns ebv reactivation or the activated resident ebv - positive cells in the cns by the local inflammation. the manifestation of bipolar disorder may be attributable to lesions of broad areas of cerebral white matter, which may also cause seizure - like activity at the early stage of the disease. although the reported lesions were located predominantly in the pons, brachium pontis, and cerebellum, a recent study showed that diffuse white matter might be involved in clippers as well. close attention should be paid to the clinical manifestations of lesions in other cerebral regions. this is the first study reporting a clippers case with an elevated load of ebv - dna in the csf. our findings indicate that intracranial ebv infection may be associated with the pathogenesis of clippers through autoimmunity. in addition, as it remains undetermined whether clippers is an independent disease entity or syndrome or a presage of other diseases, long - term follow - up of clippers patients, especially those who have a history of hodgkin 's lymphoma, is highly recommended. | abstractbackground : chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (clippers) is an inflammatory disorder in the central nervous system (cns) with distinct clinical, radiological, and pathological features. the pathophysiology of clippers still remains unclear and the reports are quite few. although the radiological lesions were reported to be located predominantly in the pons, brachium pontis, and cerebellum, other adjacent structures such as the white matter and spinal cord were very recently reported as involved regions in clippers. in this study, we report a case of clippers presenting with intracranial epstein barr virus (ebv) infection and diffuse white matter involvement.case summary : a 37-year - old male was diagnosed with mediastinal hodgkin 's lymphoma (lymphocyte predominance type) at the age of 26, and then obtained complete remission after treatment and remained free of relapse for 11 years. he was admitted with 7 months history of mental disorder, and 20 days history of gait and limb ataxia, dysphagia, and cough. the diagnosis of clippers was established based on the findings of punctate and nodular enhancing lesions in the bilateral pons, the basal ganglia, the mid - brain, the pontine brachium, and diffuse white matter in magnetic resonance imaging (mri), together with cd3 + t - lymphocytic inflammatory infiltration in perivascular and parenchymal area revealed by bilateral parietal lobe brain biopsy. also, our patient exhibited a good response to steroid therapy and remained free of relapse for 5 months. importantly, we found intracranial epstein barr virus infection in this patient.conclusion:clippers might be an autoimmune disorder, and intracranial ebv - infection raises the possibility that ebv - associated autoimmunity is associated with clippers pathogenesis. |
clozapine has been known to cause haematological side effects like agranulocytosis, neutropenia, leucocytosis, eosinophilia and thrombocytopenia. although some data is available with regard to leucopenia, data for thrombocytopenia is limited. in this report we present the case of a patient who developed thrombocytopenia with clozapine and review the existing literature. a woman aged 22, suffering from paranoid schizophrenia (as diagnosed by dsm - iv) for the past three years, presented with an acute episode. she had received adequate trials of olanzapine, risperidone and quetiapine along with electroconvulsive therapy. taking this into account she was started on clozapine. prior to starting of clozapine her hemogram including platelet count did not reveal any abnormality. for the management of acute symptoms in view of the suicidal risk, she was treated with electroconvulsive therapy, lorazepam up to 4 mg / day and was started on clozapine and the dose was gradually increased to 187.5 mg / day over the period of four weeks because of side effects of hypersalivation and constipation. while clozapine was increased, leucocyte count and platelet counts were monitored regularly on weekly basis and serial monitoring did not reveal any abnormality. with this she achieved symptomatic remission following which lorazepam was stopped. while on clozapine 187.5 mg / day, low platelet count (101,000 and 98,000/l on two occasions) was noted for the first time after 17 weeks of continuation of clozapine at the above said dose without reduction in the leucocyte count or haemoglobin levels. following this monitoring was increased and platelet count kept on fluctuating between 1,20,000/l to 1,35,000/l. over the period, platelet count started dropping. by another 24 weeks of clozapine therapy, the platelet count dropped to 60,000/l without any reduction in the leucocyte count and the haemoglobin levels. the drop occurred despite reduction in the dose of clozapine upto 125 mg / day. clozapine was stopped completely as a result. throughout this period she never had any fever, skin rash, purpura, excessive menstrual bleeding or bleeding from any other source, arthritis, arthralgia, muscle pain and muscle weakness. her investigations in the form of serum electrolytes, liver function test, renal function test, prothrombin time, bleeding time, clotting time and ultrasound abdomen and pelvis did not reveal any abnormality. she was also evaluated by physician and the opinion was that the low platelet count may be related to clozapine. within a week of stopping clozapine platelet counts started improving and by six weeks her platelet count was 1,80,000/l. in the meanwhile, with stoppage of clozapine, she had relapse of symptoms and required inpatient care, was treated with electroconvulsive therapy, lorazepam and chlorpromazine, with which she achieved remission. during this period her platelet count remained within the normal range. thrombocytopenia is defined as platelet count of less than 100 10/l. only two studies and a small case series have reported incidence of thrombocytopenia associated with clozapine and only few case reports have focused on this association.[410 ] the findings are summarized in table 1. literature reporting clozapine associated thrombocytopenia although in most of the literature thrombocytopenia associated with clozapine has been reported to be transitory to at best last for 13 weeks, in a case report thrombocytopenia persisted for 40 months after stopping clozapine. there are also few case reports of clozapine associated thrombocytosis. in one of this case reports thrombocytosis was seen in a patient treated with granulocyte colony stimulating factor for clozapine associated agranulocytosis. as with thrombocytopenia, an immunological mechanism thrombocytopenia was noted for the first time after 17 weeks of stable dose of clozapine, which kept on worsening with continuation of clozapine, even on the lower dose and subsided only after stoppage of clozapine and normalization of platelet count required six weeks. however, in contrast to some of the reports, in the index case, platelet count kept on falling consistently with continuation of clozapine and improved only after stoppage of clozapine, suggesting that in certain cases the side effects may be transitory and can lead to fatal outcome. from the above literature it can be concluded that there is a wide variation in the incidence of thrombocytopenia with clozapine, which possibly is influenced by the sample size studied. there is lack of consensus with regard to resolution, with some reports suggesting that resolution requires stoppage of clozapine, as was seen in the index case too ; whereas others suggest that thrombocytopenia resolves on its own. most of the available literature is silent about the possible mechanism, except for one report which demonstrated underlying immunological mechanism in a case of persistent thrombocytopenia even after stopping clozapine. considering the fact that the thrombocytopenia may be transitory as suggested in some of the case reports, an informed decision about rechallenge must be made after weighing the risk and benefits. from the above it hence, besides monitoring the leucocyte count, platelet count of patients receiving clozapine should also be monitored. | although agranulocytosis as a side effect of clozapine is well known, there is scarcity of data with regard to thrombocytopenia associated with clozapine. in this report we describe a case of clozapine induced thrombocytopenia and review the existing literature. a 22 year old female patient developed thrombocytopenia while on clozapine 187.5 mg / day for 17 weeks. thrombocytopenia persisted for 24 weeks even after reduction in the dose of clozapine and ultimately clozapine had to be stopped, which led to resolution of thrombocytopenia. clozapine - induced thrombocytopenia is a less well - known, but potentially serious, adverse effect that should be screened for in practice. the case highlights the fact that besides monitoring the leucocyte count, platelet count of patients receiving clozapine should also be monitored. |
alzheimer s disease (ad) is the leading cause of dementia worldwide and represents one of the most serious health issues for the elderly. an estimated 5.4 million americans have ad, and this number is expected to triple in 2050 due to lack of medical breakthroughs to stop, prevent, or slow the disease.1 over the years, research efforts have focused extensively on delineating the mechanisms and identifying targets involved in ad pathogenesis ; however, to date, no disease - modifying therapy has been clinically approved for ad. the 2 major neuropathological hallmarks of ad are extracellular deposition of amyloid beta (a)containing senile plaques and intracellular tau - containing neurofibrillary tangles (nfts) in the brain.2 a plaque formation is considered relatively specific to ad pathology ; however, nfts are found associated with other disorders as well.3 a plaque formation involves a cascade of events beginning with the sequential cleavage of the amyloid precursor protein (app), a large transmembrane protein, by proteolytic enzymes -secretase and -secretase. cleavage by -secretase releases a, peptides which are usually composed of 40 to 42 amino acid residues, into the extracellular space where they aggregate into oligomers, -sheet structured fibrils, followed by clumping into a plaques.4 some studies suggest that a pathology more closely reflects the preclinical (asymptomatic) stage of ad, whereas accumulation of tau tangles more closely reflects the onset of clinical symptoms in ad.5 among the numerous targets explored to treat ad, anti - a approaches directed toward removal of a and/or decreasing its production (eg, inhibition of -secretase, active immunotherapy, and anti - a antibodies) have shown promising results in animal models of ad68 and have been at the forefront of ad research. most of the studies investigating the effects of anti - a approaches have had limited success in mid- to late - stage clinical development and have failed to reach primary clinical end points.9 a recent study investigated the effect of a selective tau aggregation inhibitor in patients with mild - to - moderate ad. this anti - tau approach, however, failed to reach primary outcome measures.10 more recent research has pivoted focus to cytokine - mediated neuroinflammation as a major contributor to the development of ad, and evidence suggests that inflammation promotes pathological processes that lead to ad.1113 among the cytokines involved in neuroinflammation, tumor necrosis factor (tnf-) is the most studied and plays an essential role in the cytokine cascade during an inflammatory response. although the levels of tnf- in the periphery and central nervous system (cns) of healthy adults are maintained at very low levels, the levels of this cytokine are significantly elevated in blood14 and cns12 of patients with ad, and many clinical and animal studies have demonstrated a link between excess tnf- levels in the brain and ad.15 here, we focus on the involvement of tnf- in ad, challenges associated with the development of existing biologic tnf- inhibitors (tnfis) for ad, and potential therapeutic strategies for targeting tnf- for ad therapy. tnf-, first recognized for its antitumor activity,16 is one of the main inflammatory cytokines involved in initiating and propagating an inflammatory response. tnf- is a monomeric (17 kda) nonglycosylated type 2 transmembrane protein that belongs to a superfamily of ligand / receptor proteins called the tnf / tnf receptor (tnf / tnfr) superfamily proteins. tnf- is inserted into the membrane as a homotrimer which is then cleaved into a 51-kda soluble trimer via proteolytic cleavage by the tnf-converting enzyme.17 in the central nervous system, tnf- can be synthesized in the brain by microglia,18 neurons,19 and astrocytes.20 tnf- binds to 2 receptor subtypes, tnfr1 (p55-r) and tnfr2 (p75-r), each having distinct signaling pathways differentiated by the presence of an intracellular death domain (dd).21 tnfr1, which contains the dd, is ubiquitously expressed in various cell types and is activated through either soluble tnf- (stnf-) or membrane tnf- (mtnf-).22 tnfr1, once activated, is involved in divergent effects including cell proliferation, activation, antiviral activity, and primarily in tnf-mediated apoptosis and cytotoxicity. tnfr2 has a higher affinity for tnf-, is activated preferentially by mtnf-, and is primarily expressed at low levels by cells of the immune system and endothelial cells.22 tnfr2 lacks the dd and can trigger signaling cascades that activate pro - inflammatory and pro - survival pathways through the activation of the cellular inhibitor of apoptosis proteins 1 and 2, nuclear factor b, and phosphatidylinositol 3-kinase dependent signaling pathways.23 some studies also report an indirect role of tnfr2 in tnf-mediated cytotoxicity via the enhancement of tnfr1-mediated cytotoxicity, but no induction of cytotoxicity by tnfr2 alone.24 overall, the general consensus is that tnfr1 exerts pro - apoptotic functions, whereas tnfr2 typically promotes cell survival and proliferation.23 the role of tnf- in the pathophysiology of ad has been examined both in clinical and animal studies. one of the earliest evidence suggesting the involvement of tnf- in ad pathophysiology was its presence around the a plaque in postmortem human ad brains.25 following studies in human ad brains showed that tnfr1 signaling is required for a-induced neuronal death, and although tnfr1 protein levels are significantly greater in ad brains compared with nondemented brains, tnfr2 protein levels are lower.26 furthermore, an increase in the binding affinity of tnf- to tnfr1, but a decrease in binding affinity to tnfr2, was observed.26 genetic association studies showed that chromosome 12p region encoding for the tnfr1 gene and the chromosome 1p region encoding for the tnfr2 gene are associated with late - onset ad.27 the role of tnf- in ad pathology was further suggested by studies in which significant elevation of tnf- levels in the cerebrospinal fluid (csf)28 and serum29,30 of patients with ad correlated with disease progression.30 the reported clinical findings regarding the role of tnf- in ad pathology are consistent with observations made in mouse models of ad. elevated tnf- levels were observed in the brain tissues of ad transgenic mice.31 furthermore, elevated tnf- levels were associated with intraneuronal a immunoreactivity in the entorhinal cortex, and these elevated tnf- levels correlated with cognitive deficits in ad mice.32 the role of tnf- signaling in abnormal app processing, a plaque accumulation, tau - related pathology, and cell death has also been reported. deletion of tnfr1 in ad transgenic mice lowered a formation, a plaque burden, -secretase 1 (bace1) expression, and cognitive deficits.33 however, ablation of both tnfr1 and tnfr2 exacerbated a and tau pathology due to aggravation of tnfr1-mediated ad pathology resulting from silencing of tnfr2.34 furthermore, genetic inactivation of tnfr1 signaling in ad transgenic mice prevented intraneuronal accumulation of a,35 whereas genetic deletion of tnfr2 exacerbated ad pathology in a transgenic mouse model of ad.36 in ad transgenic mice, tnf- increased a production through upregulation of both -secretase expression37 and -secretase activity,38 and chronic neuronal tnf- expression resulted in extensive neuronal cell death.19 excess tnf- levels in the brain can disrupt clearance of a by inhibiting microglial clearance of a,39 cause synaptic dysfunction,40 and accelerate disease progression and cognitive decline.41 overall, tnf-driven processes are involved at multiple stages of ad pathophysiology and disease progression (figure 1). the most potent tnfis are biologic drugs that are food and drug administration (fda) approved for the treatment of peripheral inflammatory conditions including crohn disease, rheumatoid arthritis, and psoriatic arthritis. these biologic tnfis include tnf-specific monoclonal antibodies (mabs) (infliximab, adalimumab, golimumab, and certolizumab), and recombinant fusion proteins (etanercept).15 tnf-specific mabs bind to either mtnf- or stnf- thus preventing tnf- signaling mediated by both tnfrs. however, recombinant fusion protein tnfi (etanercept) is comprised of the extracellular domain (ecd) of the tnfr2 fused to the c - terminal of the fc domain of human igg1. the fusion protein also binds to both mtnf- and stnf- with high affinity preventing tnf- signaling mediated by both tnfrs.23 the protective effects of tnf- inhibition using these biologic tnfis have been demonstrated both in clinical and experimental ad (table 1). inhibition of stnf- signaling by intracerebral infusion of a dominant negative tnfi prevented preplaque a-associated pathology in a transgenic mouse model of ad.35 intracerebroventricular injection of the anti - tnf- mab, infliximab, reduced tnf- levels, a plaques and tau - phosphorylation,42 and cognitive deficits43,44 in mouse models of ad. in a small open - label 6-month study in humans, perispinal etanercept administration (dose range : 2550 mg per week) resulted in significant improvement in 3 standard measures of cognition : the ad assessment scale - cognitive subscale, the severe impairment battery, and the mini - mental state examination in patients with ad.45 these observed clinical improvements are hypothesized to be due to rapid penetration of etanercept into the csf following perispinal administration and mediated by nonsynaptic signal transduction mechanisms of etanercept. however, in a recent study, perispinal injection of etanercept resulted in perispinal drug accumulation, but consistent intracranial delivery of the drug was not observed.46 in a recent case report, intrathecal administration of infliximab resulted in rapid cognitive improvement in a female patient with ad,47 and larger controlled trials are warranted. collectively, both clinical and preclinical evidence underscore the therapeutic potential of biologic tnfis for ad therapy. one hindrance to the further development of the fda - approved biologic tnfis for ad is their limited blood - brain barrier (bbb) penetration,48 and invasive delivery approaches may not be practical or safe for chronic dosing for ad. recently, modulation of peripheral tnf- activity by biologic tnfis has shown promise in transgenic mouse models of ad49 ; however, in a randomized double - blind phase 2 clinical trial, weekly subcutaneous injections of etanercept did not improve cognition, global function, or behavior in a small group of subjects with mild - to - moderate ad dementia.50 further studies will be needed to determine whether peripheral modulation of tnf- offers clinical improvement in ad. biologic drugs such as etanercept, ecd of the tnfr2, can be reengineered to enable bbb penetration by fusion of the biologic drug to a bbb molecular trojan horse (mth).51 a bbb mth is an endogenous peptide or peptidomimetic monoclonal antibody that undergoes receptor - mediated transport across the bbb via endogenous peptide receptors, such as the transferrin receptor (tfr) or the insulin receptor. this binding triggers receptor - mediated transport of the mab across the bbb, which ferries the biologic drug into the brain.52 one such bbb - penetrating tnfr, specific for the mouse, has been engineered and tested in mouse models of neurological diseases (figure 2). the amino terminus of the human tnfr2 ecd is fused to the carboxyl terminal of the heavy chain of the chimeric tfrmab to engineer a fusion protein designated as ctfrmab - tnfr.53 this orientation frees the amino terminus of the ctfrmab that is involved in binding to the bbb tfr and places the tnfr ecd in a dimeric configuration. because both human and mouse monomeric tnf- share high amino acid identity, and the human tnfr binds mouse tnf- with high affinity,54 the ctfrmab - tnfr fusion protein involves the human tnfr and not the mouse tnfr. the ctfrmab - tnfr fusion protein binds to both the mouse tfr and tnf- with high affinity evident from the low binding constants (kd = 2.6 0.3 nm for mouse tfr and 96 34 pm for tnf-). the ctfrmab - tnfr is thus a bifunctional molecule that allows (a) rapid noninvasive transvascular delivery of the tnfr across the bbb using a receptor - mediated transcytosis approach and (b) once in the brain, the tnfr can sequester excess tnf-. a complete pharmacokinetic analysis of this bbb - penetrating biologic tnfi, the ctfrmab - tnfr fusion protein, showed that systemic routes (intraperitoneal, subcutaneous, or intravenous) of administration result in brain concentrations of the ctfrmab - tnfr fusion protein that are 20-fold to 50-fold greater than the concentration of tnf- in pathologic conditions of the brain.55 the intravenous route of administration is preferred for acute neurological conditions, such as ischemic stroke, whereas the intraperitoneal or subcutaneous routes of administration are more practical for dosing during chronic conditions including ad. the ctfrmab - tnfr fusion protein was found to be protective in both acute (ischemic stroke) and chronic (parkinson disease) mouse models of neurological diseases5658 and is currently being investigated in a mouse model of ad. for use in humans, a fusion protein of the ecd of the tnfr2 and a chimeric or humanized antibody against the human insulin receptor (hirmab), designated as the hirmab - tnfr fusion protein, has been engineered.48 the hirmab is the most potent mth engineered for the human brain and cross - reacts with the insulin receptor of the old world primates. in rhesus monkeys, the brain uptake of a 0.2 mg / kg dose of the hirmab - tnfr fusion protein after intravenous injection was 3 0.1% id/100 g brain compared with the brain uptake of the tnfr : fc fusion protein which was 0.23 0.06% id/100 g brain. furthermore, chronic administration of a high dose (20 mg / kg) of a hirmab - based fusion protein was found to be safe in rhesus monkeys.59 a plethora of clinical and animal studies strongly suggest an involvement of tnf- in the pathophysiology of ad. the fda - approved biologic tnfis are thus a potential treatment for ad ; however, these large molecules have limited bbb penetration. clinical studies using the perispinal route of administration for biologic tnfis have shown encouraging results in small open - label trials ; however, larger controlled trials are required to confirm these results. another approach currently under investigation is to target peripheral tnf- rather than brain tnf-. however, this approach has not shown any cognitive improvement in a clinical setting so far, and further studies are required to determine the effect of peripheral tnf- modulation on ad pathology. novel drug delivery strategies, such as the mth technology, enable noninvasive delivery of biologic tnfis to the brain and target both peripheral and brain tnf-. considering the multifactorial role of brain tnf- in ad pathophysiology, such noninvasive drug delivery strategies may be a reasonable approach to deliver biologic tnfis to the brain for ad treatment. | tumor necrosis factor (tnf-) plays a central role in the pathophysiology of alzheimer s disease (ad). food and drug administration approved biologic tnf- inhibitors are thus a potential treatment for ad, but they do not cross the blood - brain barrier. in this short review, we discuss the involvement of tnf- in ad, challenges associated with the development of existing biologic tnf- inhibitors for ad, and potential therapeutic strategies for targeting tnf- for ad therapy. |
paradoxical enlargement of lymph nodes in tuberculosis is well established in the early phase of treatment.[13 ] this is more common in hiv co - infection. mediastinal lymphadenopathy resulting in recurrent laryngeal nerve palsy, pulmonary artery occlusion and superior vena caval obstruction has been described. we report a case of bronchial obstruction in a patient with fully sensitive organism in the late phase of the treatment. a 26-year - old somalian woman was diagnosed with mycobacterium tuberculosis infection in january 2008. she presented with cervical and mediastinal lymph node enlargement without any obvious lung parenchymal involvement. acid fast bacilli (afb) were seen on fine needle aspiration of the cervical lymph node. after initial problems with compliance, full standard quadruple therapy was effectively started in april 2008. maintenance treatment was commenced in june 2008. the chest x - ray (cxr) on first presentation did show a narrowed left main bronchus in the absence of any clinical signs or symptoms. the only relevant past medical history was of childhood asthma, for which she had no hospitalizations and did not use any regular inhaled therapy. she presented in august 2008 to the accident and emergency department with a one - week history of worsening shortness of breath. inhaled steroids and bronchodilators were started for a possible asthma exacerbation and she was discharged home. her symptoms deteriorated and when seen in the tb clinic four weeks later, her exercise tolerance was limited to about 100 yards. on examination, a repeat cxr showed marked narrowing of the left main bronchus, confirmed on ct scan which showed almost complete occlusion of the left main stem bronchus. spirometry showed fev1 - 1.09 l / min (36.3% of predicted), fvc 1.44 (41.7% of predicted) [table 1a ]. the flow - volume loop had a characteristic shape of large airway obstruction [figure 1 ]. spirometry at presentation flow - volume loop of the patient which is characteristic of large airway obstruction she was commenced on oral prednisolone 30 mg once daily. there was no concern with compliance at this time. a bronchoscopy also showed complete occlusion of left main bronchus with white necrotic material [figure 2 ]. white necrotic matter seen occluding the left main bronchus at bronchoscopy her treatment was continued with both anti - tuberculous therapy and steroids. three days prior to the clinic visit she said she had coughed up a large lump of white material and her breathing had returned to near normal since. 2.81(81.6%) [table 1b ] with normalization of the flow volume loop [figure 3a ]. bronchoscopy was repeated and this showed a patent left main bronchus with some necrotic material on the lateral wall [figure 3b ]. spirometry after treatment (a) flow - volume loop improved with less evidence of airway obstruction, (b) patent left main bronchus with only a small amount of necrotic matter remaining at repeat bronchoscopy she continued with anti - tuberculous treatment for a full one year and steroids were slowly tapered off. her cxr showed marked improvement in the narrowing of the left main bronchus towards the end of treatment. endobronchial tuberculosis is less common since the advent of effective anti - tuberculous chemotherapy, but mediastinal lymphadenopathy due to mycobacterial infection is still common in developing countries. paradoxical reactions on the whole are more common with hiv co - infection (28%) than without (10%). lymph node enlargement is the most common presentation and bronchial obstruction due to mediastinal lymphadenopathy is more common in the pediatric age group. in adults, patients with tuberculous mediastinal lymphadenopathy presenting with recurrent laryngeal nerve palsy, pulmonary artery occlusion and superior vena caval obstruction have been described. these presentations may be due to either external compression or erosion into the surrounding structure. in this particular case, the bronchoscopy showed caseating material eroding into the left main bronchus. nakvi., described this in children, in whom the obstruction was relieved by either aspiration or surgical excision. response to steroids has not been consistent in many series described but generally paradoxical reactions, unlike endobronchial tb, are thought to be more steroids responsive. our patient responded well to steroids. even after coughing up necrotic debris and relieving endobronchial obstruction late paradoxical reactions should be considered in any patient on anti - tuberculous chemotherapy presenting with stridor. | we present here a case of bronchial obstruction secondary to late paradoxical reactive enlargement and erosion by mediastinal lymph nodes into the left main bronchus in a 26-year - old woman with tuberculosis lymphadenitis. bronchial obstruction due to paradoxical reactions, especially in the late phase of treatment, has not been described in adults before. |
here we report the preparation of poly(oligonucleotide) brush polymers and amphiphilic brush copolymers from nucleic acid monomers via graft - through polymerization. we describe the polymerization of pna - norbornyl monomers to yield poly - pna (poly(peptide nucleic acid)) via ring - opening metathesis polymerization (romp) with the initiator, (imesh2)(c5h5n)2(cl)2ruchph.1 in addition, we present the preparation of poly - pna nanoparticles from amphiphilic block copolymers and describe their hybridization to a complementary single - stranded dna (ssdna) oligonucleotide. |
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many potential human health effects have been hypothesized to result either directly or indirectly from global climate change. changes in the prevalence and spread of infectious diseases are some of the most widely cited potential effects of climate change, and could have significant consequences for human health as well as economic and societal impacts. these changes in disease incidence would be mediated through biologic, ecologic, sociologic, and epidemiologic processes that interact with each other and which may themselves be influenced by climate change. although hypothesized infectious disease effects have been widely discussed, there have not yet been thorough quantitative studies addressing the many processes at work. in part this is because of the complexity of the many indirect and feedback interactions or mechanisms that bear on all aspects of the climate issue. it also results from the difficulty of including the multitude of always - changing determinants of these diseases. this paper proposes a framework for an integrated assessment of the impacts of climate change on infectious diseases. the framework allows identification of potentially important indirect interactions or mechanisms, identification of important research gaps, and a means of integrating targeted research from a variety of disciplines into an enhanced understanding of the whole system.imagesfigure 1figure 2 |
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contact investigation around tuberculosis (tb) patients is considered the most effective strategy in low prevalence settings for early detection of infection and disease, and for prevention of secondary tb cases. the aims of contact investigation are primarily to find and treat secondary active tb disease to disrupt ongoing transmission. screening for latent tb infections (ltbi) is also performed enabling preventive treatment or follow - up screening to prevent future tb disease in contacts. in addition, source tracing investigations are performed around cases of extra - pulmonary tb (etb) to find undiagnosed pulmonary tb (ptb) cases. in the netherlands, active tb is a notifiable disease, and source and contact investigation is included in the national tb control program. contacts are differentiated according to the ' stone in the pond ' principle, a concentric circle or ring approach based on their closeness to the index, as a proxy for exposure. based on the yield of screening in the respective rings, starting with the closest ring, and the risk of transmission as estimated by the public health nurse, the investigation can be stopped or extended to include more distant contacts. factors such as the age of the index patient and contact, duration and intensity of the contact, bacteriological status of the index patient, and ethnic background of the index patient are all know to be important, but the relative importance of these factors has not been established for contact investigations in the netherlands. improved knowledge of risk factors determining the chance of contacts being infected with tb would strengthen contact investigations by improving prioritization so high - risk contacts can be targeted first. the municipal public health service (mphs) of rotterdam, this has resulted in a unique database that, unlike other known registries, includes information on all screened contacts, not just those who were found to have ltbi or tb. this database of index patients and their contacts offers an excellent opportunity for an in - depth evaluation of contact investigations in the control of tb. in this study, we set out to identify risk factors for tb transmission among contacts of tb patients, in order to strengthen the evidence base of the current national contact investigation policy in the netherlands. the mphs in rotterdam is responsible for tb control in the larger rotterdam - rijnmond area, with a population of 1.2 million., data on source tracing and contact investigations have been stored systematically in a database. our study includes data on the index cases and their contacts over the period 2001 - 2006. contact investigations are conducted according to indications of infectiousness of the index case, such as the anatomical location of tb disease, the bacteriological status, and estimated patient and/or doctor 's delay. a public health nurse interviews the index to determine which (if any) contacts should be investigated according to the ' stone in the pond ' principle of concentric rings, where the ring can be seen as a proxy for exposure to the index patient in the case of ptb. the first ring (ring 1) contains close contacts, the second ring (ring 2) contains regular contacts, while the third ring (ring 3) contains community contacts. this classification is based on the frequency and duration of exposure, and physical aspects of the area where the exposure takes place (in particular, compartment size and ventilation). the mphs databases on the index patients and contacts were combined and linked to create a new database of screened contacts to which the recorded characteristics of the index patient were added. for the contacts we included age, gender, date of screening, tuberculin skin test (tst) and/or chest x - ray (cxr). for the index patients we included the type of tb disease, age, ethnic background, sensitivity of the tb strain and the diagnostic steps that were taken. a positive tst was defined as an induration of 10 mm or more to 0.1 ml of purified protein derivative, measured 48 and 72 h after tst. positive tst results were followed up by cxr to screen for abnormalities indicating pulmonary tb. contacts with a history of tb, a previous tst induration of 10 mm or more, evidence of bcg vaccination or birth before 1945 were only screened by cxr, as a tst would be unable to provide evidence of a recent infection in these contacts. in the netherlands, universal bcg vaccination for the general population has never been implemented, and is only given to travellers to endemic countries or children of parents from endemic countries. when tst was positive, but clinical and radiological signs of active tb disease were absent, the final ltbi diagnosis was made by a tb physician. the first part determined the risk factors for being diagnosed with ltbi using a tst. the second part aimed at determining the risk factors for being diagnosed with active tb. the outcome of interest in the first analysis regarding tst tested contacts was a positive tst. in the second analysis, the outcome of interest was defined as an abnormal cxr with, if possible, bacteriological confirmation of active tb. both analyses were performed with the contacts as the unit of analysis and stratified by ring. univariate logistic regression was performed to estimate the effect of the characteristics on the individual contact investigation outcomes. variables with p0.05. exceptionally large contact investigations were excluded from the dataset, after which the analysis was performed again to ensure these large contact subpopulations did not weigh too heavily in the regression analyses. between 2001 and 2006, a total of 1,165 index patients with active tb were registered. of these, 731 led to investigation of at least one contact (range 1 - 1,810 contacts ; median 6 ; iqr=3.22). together, these 731 index patients had 21,540 contacts participating in source tracing or contact investigations (figure 1). their general characteristics are shown in table 1 and in more detail in the appendix (supplementary tables 1 - 5). of the contacts, 12,698 (59%) were eligible (e.g. not previously vaccinated with bcg) and tested for latent tb infections with a tst. the database contained 20,494 unique contacts, indicating that 1,046 of the investigations (4.9%) were performed in contacts who had participated in an earlier contact investigation between 2001 and 2006. the percentage of tb infected contacts among those tested with tst was almost halved when we compared regular contacts with close contacts (56%) and community contacts with regular contacts (50%). for active tb among all contacts, the decrease is even stronger ; 21% for regular contacts compared with close contacts, and 11% comparing community contacts with regular contacts. figure 1cumulative number of contacts screened over the period 2001 - 2006 in source tracing or contact investigations in the rotterdam - rijnmond area plotted against the size of the individual contact investigations. cumulative number of contacts screened over the period 2001 - 2006 in source tracing or contact investigations in the rotterdam - rijnmond area plotted against the size of the individual contact investigations. table 1characteristics of the contacts included in the source tracing and contact investigations 2001 - 2006 performed by the municipal public health service rotterdam - rijnmond, the netherlands. contacts are stratified by ring according to a concentric circle approach, as specified in the national guidelines for contact investigations in the netherlands. ring 1 contains close contacts, ring 2 contains regular contacts, while ring 3 contains community contacts.ring 1, tst testedring 2, tst testedring 3, tst testedring 1 allring 2 alltotal n. contacts169762324769329610967postives16.0%9.0%4.3%9.6% (1.5%)5.7% (0.3%)male gender50.1%51.1%50.1%51.9%50.7%age of contact, median (interquartile range)25 (14 - 39)32 (21 - 43)28 (20 - 40)28 (15 - 42)34 (23 - 46)relationship between contact and index residential / family27.7%18.5%1.0%29.7%20.7% work / education64.4%56.8%48.8%61.6%54.6% leisure time / other4.4%21.8%29.2%5.0%21.9%unknown / censored3.5%2.9%21.0%3.7%2.7%age of index, median (interquartile range)31 (23 - 47)34 (25 - 44)33 (19 - 44)33 (23 - 47)34 (25 - 47)contacts investigated by location and bacteriological status of index ptb afb+75.0%87.5%94.0%67.6%87.2% ptb afb15.5%9.7%5.9%18.8%9.4% etb9.5%2.8%0.1%13.6%3.4%contacts investigated by index cases and specific ethnic background dutch63.6%51.8%73.5%52.0%47.2% western0.8%1.1%0.1%0.8%1.2% non - western35.7%47.1%26.4%47.2%51.7%tst, tuberculin skin test ; ptb, pulmonary tubercolosis ; etb, extra - pulmonary tubercolosis.percentage of people found positive in the diagnostic test, confirmed by the tubercolosis (tb) physician. percentage includes the number of latent tb infections (ltbi) cases in tuberculin skin test - tested persons, the percentage of tb and ltbi for the column with all contacts. for columns including all ring 1 and 2 contacts, the percentage in brackets represents active tb percentage. tst, tuberculin skin test ; ptb, pulmonary tubercolosis ; etb, extra - pulmonary tubercolosis. percentage of people found positive in the diagnostic test, confirmed by the tubercolosis (tb) physician. percentage includes the number of latent tb infections (ltbi) cases in tuberculin skin test - tested persons, the percentage of tb and ltbi for the column with all contacts. for columns including all ring 1 and 2 contacts, the percentage in brackets represents active tb percentage. of the 12,698 contacts tested with tst, 1,091 were diagnosed with ltbi while the tst follow up with cxr revealed signs of active tb in 49. table 2 shows the multivariate regression results for the three rings (details are to be found in the appendix, supplementary tables 1 - 3). older age (10-year intervals) of the contact increases the risk of having a positive tst in all rings, with odds ratios (or) of 1.12, 1.17 and 1.32 for the first, second and third ring, respectively. the relationship between the index and the contacts indicates that residential or family relationships are at the highest risk of having a positive tst, while work or education related contact results in a lower chance of transmission, although the differences are not always statistically significant among the types of relationships or for every ring. the diagnosis of the index is a strong predictor for contacts with positive tst : smear positive ptb (ptb afb+) results significantly more often in the diagnosis of ltbi in contacts compared to smear negative ptb (ptb afb) or etb in ring 1, but the differences between ptb+ and ptb decrease in ring 2 and there is no significant difference between them in ring 3. additionally, the ethnic background of the index patient is statistically significant in rings 1 and 3, but not in ring 2. as far as investigation characteristics are concerned, the infection fraction of rings closer to the index is a strong predictor for the more remote rings. for ring 2, or is 3.19 (95% ci:2.4 - 4.2) in the multivariate analysis, with the fraction positives within an investigation as risk factor. the correlation is more pronounced in ring 3, in which the positive fraction of ring 2 gives an or of 9.22 (95% ci:3.37 - 25.2). table 2odds ratios and their corresponding 95% confidence intervals for all significant factors in source tracing and contact investigations among contacts of tb patients from the five multivariate logistic regression models. gender did not reach significance in any of the models and is not, therefore, shown. ring 1 contains close contacts, ring 2 contains regular contacts, ring 3 contains community contacts.tst tested contactstst and/or cxr screened contactsring 1 ltbiring 2 ltbiring 3 ltbiring 1 tbring 2 tbage of contact (10-year interval continuous)1.12 (1.03 - 1.22)1.17 (1.09 - 1.24)1.32 (0.12 - 0.55) leisure time / other0.57 (0.28 - 1.17)0.77 (0.60 - 0.99)0.63 (0.29 - 1.41) unknown / censored0.57 (0.27 - 1.19)1.59 (1.05 - 2.41)0.61 (0.08 - 4.68)age of index (10-year interval)0.83 (0.76 - 0.92)1.28 (1.14 - 1.44)0.78 (0.61 - 1.00)ethnic background dutch1.001.00 western2.08 (1.40 - 3.09)2.41 (1.29 - 4.51) non - western1.70 (1.28 - 2.27)3.44 (2.41 - 4.90)bacteriological status index ptb afb+1.001.001.00 ptb afb0.31 (0.19 - 0.50)0.98 (0.72 - 1.32)0.21 (0.09 - 0.58) etb0.49 (0.29 - 0.85)0.25 (0.09 - 0.68)0.09 (0.02 - 0.39)drug resistance susceptible1.00 resistant4.61 (1.90 - 11.2)bal performed yes0.080 (0.64 - 0.99) no1.00investigation size (100-person interval)1.05 (1.01 - 1.08)0.19 (0.06 - 0.58)quarter of starting the investigation first1.00 second2.26 (0.84 - 6.07) third4.20 (1.60 - 11.0) fourth3.92 (1.54 - 9.97)fraction infected in ring 1na3.19 (2.40 - 4.23)nana3.67 (1.44 - 9.38)fraction infected in ring 2nana9.22 (3.37 - 25.2)nanatst, tuberculin skin test ; cxr, chest x - ray ; ltbi, latent tubercolosis infections ; tb, tubercolosis ; ptb, pulmonary tubercolosis ; etb, extra - pulmonary tubercolosis ; bal, benzaldehyde.na, not applicable, as only more proximal fraction of infected contacts within a source and contact investigation was entered into the multivariate regression analysis. tst, tuberculin skin test ; cxr, chest x - ray ; ltbi, latent tubercolosis infections ; tb, tubercolosis ; ptb, pulmonary tubercolosis ; etb, extra - pulmonary tubercolosis ; bal, benzaldehyde. na, not applicable, as only more proximal fraction of infected contacts within a source and contact investigation was entered into the multivariate regression analysis. among the 21,540 contacts, 91 cases of active tb were diagnosed. of these 91 cases, 2 were found by source tracing, meaning that these 2 screen - detected ptb cases were likely the first patients in the cluster. the correlation observed for the type of relationship between index and contact in the ltbi screening analysis was also seen for contacts diagnosed with active tb ; work or education related contact has a lower relative risk of being diagnosed with tb, compared to residential, family, leisure time, or other contacts. for ring 1, the bacteriological status of the index is a strong predictor, with an or of 0.21 for ptb and 0.09 for etb, compared to ptb+. the infection fraction in ring 1 is also a predictor for being diagnosed with tb in ring2, with an or of 3.67 (95% ci:1.4 - 9.4). the age of the contacts showed an inverse relationship to active tb, compared to the association found for ltbi, as 40% of the tb cases were in children aged 14 years or under with an odds ratio of 0.70 in the multivariate model in ring 1, with 16 cases of active tb disease in young children aged 5 years or under. it was not possible to analyze the community contacts because only 2 cases of active tb were found among the 7,277 contacts in this ring. after exclusion of the largest 5 contact investigations (26% of all contacts in the study) the direction of the associations did not change (data not shown). details of the analysis of the close and regular contacts are shown in the appendix (supplementary tables 4 and 5). between 2001 and 2006, at least one contact was screened of 731 index patients, resulting in a total of 21,540 contacts. the screening of 12,698 contacts who were eligible for tst testing led to the detection of 1,091 ltbi cases. important risk factors were age of the contact, age of the index patient and the relationship to the index. infections among regular (ring 2) or incidental and community (ring 3) contacts were strongly associated with a higher fraction of infected close (ring 1) or regular (ring 2) contacts, respectively. our study is based on data from routine investigations, which has implications for the interpretation of the findings. the records of all index patients and their contacts (if any) were available, enabling comparison between those with positive and those with negative contact investigation outcomes. although all contacts who participated and all investigations were documented, it is difficult to determine how many contacts were missed. a fraction of contacts will be missed by the mphs, despite the extensive effort to screen all reported or possibly exposed contacts. some groups are not included in routine contact investigations, such as the homeless or illicit substance users, because there is a separate regular screening program for these subpopulations. a recent study using data from the dutch tb surveillance register showed that contacts of immigrant cases were less often investigated. despite possible incompleteness of the study population, this database provides an excellent opportunity to determine risk factors in contact investigations because the netherlands is one of few countries where contact investigations are fully documented, out of the thirteen countries known to investigate contacts of all possibly infectious index patients. firstly, close contacts (ring 1) had a higher risk of infection and tb disease than regular (ring 2) and community (ring 3) contacts. although more refined proxies for exposure are possible, the currently used ring approach has proven to be adequate in discriminating between contact levels. it is unknown how much could be gained by using data underlying this current proxy, such as actual duration and frequency of contact, and the environmental characteristics of the place of contact. unfortunately, a more precise registration of such indicators of contact level is not practical under routine control conditions. secondly, the contagious status of the index patient, in this study recorded as bacteriological status of the index patient in combination with the infection fraction in closer contact rings, was strongly associated with the number of infections among distant contacts. it is unclear whether this effect is fully attributable to recent transmission or is because the screened contacts are part of a subpopulation with a higher risk of infection. this is one of the limitations of using the tst in contact investigations. finally, increasing age of the contacts this is understandable from the perspective that with age, the time that an individual has been at risk of coming into contact with tb (and other cross - reacting non - tb mycobacteria) accumulates. young children (age < 5 years) are more at risk for progression to active tb, which is also recognized in this study, but in our analysis this was only proven in ring 1. these three factors have already been well established as risk factors for tb in contacts and are included in the national guidelines for source tracing and contact investigations in the netherlands. similar results have been reported in evaluations of contact investigations in comparable settings and one systematic review, while one study did not find an association between the closeness to the index and symptomatic ptb after confirming transmission through dna fingerprinting. the study was carried out before the application of the interferon- assay blood test (igra) in the netherlands. later studies in comparable settings show that the igra test has a higher specificity than tst and thereby reduces the number of persons who would have received prophylactic treatment in case of a false - positive tst. we do not expect that the results would change drastically if the igra test were applied instead of the tst. there is no indication that the igra test responds differently in the risk groups that we established in this study, other than detecting additional old infections in bcg vaccinated persons from endemic countries. in this respect, we would like to point out that the goal of contact investigations is primarily to prevent large clusters by stopping ongoing transmission. this study focused on a population of contacts participating in source tracing and contact investigations. we identified important risk factors for the diagnosis of tb among contacts of tb patients, based on routine practice in a low prevalence setting, and established that the findings basically match the current national guidelines. the age of the patients and their contacts, the number of infections among close contacts, the type of contact relationship, and the diagnosis of the index patient are all associated with ltbi and tb in contacts. these risk factors emphasize the importance of including personal and interpersonal factors in decision making regarding contact investigations. | contact investigations around tuberculosis patients enable early detection of infection and disease, and prevention of secondary tuberculosis cases. we aim to identify risk factors for m. tuberculosis transmission to contacts of tuberculosis patients, based on unique data from routine contact investigations by the public health service in rotterdam, the netherlands, collected between 2001 and 2006. through logistic regression analysis, we determined the effect of various risk factors on the chance of finding a latent tuberculosis (tb) infection or overt tuberculosis case among contacts. a total of 1165 index patients with active tuberculosis were registered and at least one contact was investigated in 731, resulting in 21,540 contacts overall. altogether, the contact investigations led to 91 cases of active tuberculosis. of the 12,698 contacts eligible for screening by tuberculin skin test, 1091 (9%) were diagnosed with latent tuberculosis infections. risk factors were old age of the contact, old age of the index patient, and the relationship to the index. a larger fraction of infected close contacts was strongly associated with infections among more distant contacts.our findings emphasize the importance of including these personal and interpersonal risk factors in decision making in contact investigations. |
adolescence is a vulnerable period for the development of obesity and also appears to be a critical period for establishing risk factors for some chronic diseases in adulthood. available statistics indicate that an alarming proportion of people in most arab countries suffer from obesity. studies have revealed that the prevalence of overweight and obesity among adolescents in arab countries ranges from 18% to 44%. in general, overweight has been found to be more prevalent than obesity in both boys and girls. however, the prevalence of obesity by gender at the adolescent stage (1018 years) does not indicate the same trend. in countries such as bahrain, egypt, tunisia, kuwait, and qatar obesity is also higher among adolescent girls than boys in bahrain, egypt, and kuwait. in some countries such as lebanon and the united arab emirates (uae), obesity management is an important step in the prevention and control of chronic noncommunicable diseases, such as cardiovascular disease, diabetes, hypertension, and some kinds of cancer. therefore, understanding the prevalence of obesity among schoolchildren is essential to any strategy for combating obesity in the community. although, there is information about obesity among schoolchildren in some arab countries, there is a lack of such data for other countries such as algeria, djibouti, libya, somalia, sudan, palestine, and yemen. however, there are two main reservations about using the available data on obesity among schoolchildren : (a) the use of such a wide age range (618 years) does not take into consideration the prepuberty and puberty stages which have a significant effect on weight gain ; (b) the use of different reference standards and cutoffs to determine obesity in turn provide different rates of overweight and obesity. in bahrain, for example, al - sendi and colleagues used three different sets of criteria to determine the prevalence of obesity among adolescents aged 1217 years, (iotf, who, and cdc references). they found that there was a considerable variation in the prevalence of obesity between these standards. in general, the who reference standard gave a higher prevalence of obesity, but a lower prevalence of overweight than other standards. the objectives of this study, therefore, were to provide the prevalence of overweight and obesity among adolescents within a narrower age range of 1518 years using the iotf reference standard, and to find out the cross - cultural differences in the prevalence of obesity in seven arab countries, namely, algeria, jordan, kuwait, libya, palestine, syria, and uae. it is a part of the arab - eat project, which aims to study obesity and eating disorders, and barriers to healthy eating and physical activity in large cities in arab countries. the target population of this study was adolescents in secondary schools (1518 years). the minimum sample size in each city calculated as the sample size within 0.05 of the population proportion with a 95% confidence level. a multistage stratified random sampling procedure was used to select the subjects. at the first stage, each city was divided into administrative regions, which varied from two to five regions, depending on the countries. were included, due to the difficulty in obtaining permission from private schools and the lack of data regarding these schools. at the second stage, classes were selected for each secondary level (levels 10, 11, and 12) in each school using a simple random method. therefore, the number of adolescents selected varied, based on the number of students in each class and the number of selected schools, which in turn depended on the total secondary school population in each city. to assure the accuracy and consistency of the methodology (sampling procedure, measurements, and collection of the data), a standardized protocol was prepared and distributed to all the participating centres in the seven countries. each centre was responsible for training its research team and obtaining ethical authorization and approval from the governmental institutes (generally the ministry of education). the total sample size in the seven arab countries was 4698 adolescents (2240 boys, 2458 girls), ranging from a total of 459 students in algeria to 1062 in syria. it is worth noting that due to the difficulty in obtaining permission from one administrative region in an algiers city, the number of selected schools in that case represented two geographical regions instead of three. height was measured to the nearest centimeter with the subject in full standing position, using a calibrated measuring rod. data were first entered in an excel file, the entered data were then sent to the central processing station (bahrain), along with the questionnaires for cleaning the data and carrying out the analysis, using the spss statistical package. the international obesity task force (iotf) reference standard was used to classify the adolescents into three categories : nonobese, overweight, and obese. data for age, weight, height, and bmi were presented as means and standard deviations. one way anova was used to determine the differences in anthropometric measurements between countries, and between males and females. the sample sizes, means, and standard deviations for age, weight, height, and body mass index of adolescents (1518 years) according to gender in seven arab cities are presented in table 1. the mean age is very close among the seven countries, ranging from 16.15 years in libya to 16.96 years in algeria. significant differences in age, between sexes and among countries were revealed using one way anova. in general, males were significantly heavier than females in all countries. kuwaiti adolescents showed the highest mean weight among both males (82.7 23.1 kg) and females (65.1 20.5 kg) ; whereas, the lowest mean weight for males was observed in algeria (64.0 10.9 kg), and for females was found in uae (54.6 14.2 kg). algerian adolescent males showed the highest mean height (173.4 7.2 cm), while the highest mean for females was observed in jordan (162.1 5.7 cm). the lowest mean height was found in libya for both males (167.4 7.4 cm) and females (157.9 5.5 cm). as for bmi, the highest mean was found in kuwait for both males (28.1 7.5 kg / m) and females (25.6 7.7 kg / m). the differences in weight, height, and bmi were analysed using one way anova and were statistically significant between the seven countries for both genders (p < 0.000). the prevalence of overweight and obesity among adolescents in seven arab cities using the iotf standard according to gender is shown in table 2. among males, overweight was most prevalent among kuwaiti adolescents (25.6%), followed by jordanian (21.6%), and syrian (19.7%) adolescents ; while among females, the highest prevalence was found in libya (26.6%), followed by kuwait (20.8%), and syria (19.7%). regarding obesity, kuwaiti adolescents showed the highest prevalence among both males (34.8%) and females (20.6%). in general, obesity was more prevalent among males than females in all countries included in this study. figures 1 and 2 illustrate the comparison of the prevalence of overweight and obesity among adolescent males and females in the seven arab countries, respectively. kuwaiti males showed the highest prevalence of overweight and obesity (60.4%), while the lowest prevalence was reported in algeria (13.4%). as for females, the highest prevalence was also found in kuwaiti females (41.4%) and the lowest prevalence was in palestinian females (16%). a comparison between the iotf and cdc reference standard for the prevalence of overweight and obesity among adolescents (1518 years) in seven arab countries is presented in table 3. in general, the cdc reference provided a lower prevalence of overweight and higher prevalence of obesity than the iotf reference. for example, the prevalence of overweight in algerian males was 9.3% based on the iotf reference, compared with 4.6% based on the cdc reference. this paper has presented for the first time a comparison of the prevalence of overweight and obesity in seven arab countries using a similar methodology and reference standard (iotf). the findings have revealed a relatively high percentage of overweight among adolescents (1518 years) in all countries studied, ranging from 9.3% in algeria to 25.6% in kuwait. as for obesity, the prevalence was higher in males than females, and ranged from 4.1% in algeria to 34.8% in kuwait. it is difficult to compare the findings of this study with many previously published studies because of the differences in the standard used and in the age range. moreover, there has been an absence of published data on adolescent obesity in some arab countries included in this study, such as algeria, libya, and sharjah. however, when the results of this study are compared with those reported in kuwait for 1419 year olds using the iotf standard, we noticed that the present data showed a higher prevalence of obesity among boys (34.8% versus 24.8%), and a similar finding among girls (20.6% versus 20.0%). as for overweight, the findings of this study were 25.6% for boys, compared with 18.8% in the previous study, and the equivalent proportions for girls were 20.8% and 25.3%, respectively. it is important to mention that there was a 610 years gap between the two studies, and we can observe that obesity is increasing among male kuwaitis, but not among females. similar findings were seen when we compared the present data for sharjah (uae) with the national data for uae adolescents aged 1019 years, using same reference standard (iotf). there was an increase in proportion of obesity among males (19.1% versus 13.2%), but a decrease in females (6.6% versus 11.0%). for overweight nevertheless, it is difficult to reach a concrete conclusion, since we are comparing national data with regional data and a different age group. therefore, it is necessary in any national information on obesity to show the regional differences in the obesity proportion. the high prevalence of overweight and obesity among some arab countries, especially in kuwait, could be due to rapid nutrition transition, which started earlier in kuwait than in other countries. in addition, kuwait has the highest socioeconomic status among these countries, which may contribute to the increase in the prevalence of obesity. in general, the difference in the prevalence of obesity between the seven arab countries can be attributed to several factors such as ethnicity, economic status, physical activity, dietary habits, timing of puberty, and genetic admixtures [12, 13 ]. overweight and obesity were more prevalent among males in jordan, kuwait, and uae, than among females. bmi is an expression of weight related to height, not adiposity, and there is no standard definition of adiposity or excess body fat in childhood against given bmi levels in the arab countries. the higher prevalence of obesity among males compared to females in some arab countries may be related to the difference in timing of puberty, muscular tissue, and dietary habits between boys and girls. the association between obesity and the timing of puberty differs among populations [13, 15 ]. the prevalence of overweight and obesity among adolescents (using different reference standards) in previous reports in jordan, kuwait, palestine, syria, and uae is relatively high, which is also confirmed by this study. this study has also suggested a high prevalence of overweight and obesity in middle - income countries, such as libya, and in poor countries, such as palestine. a plan of action to prevent and control obesity should be urgently established in the arab countries, especially among children and adolescents to combat the comorbidities associated with obesity. this study provides comparable and useful data to build on for this plan of action. we hope that this study will stimulate researchers in other arab countries to carry out similar surveys to compare obesity in this region. | objective. the aim of this study was to find out the prevalence of overweight and obesity among adolescents in seven arab countries using similar reference standard. methods. a school - based cross - sectional study was carried out in seven cities in arab countries, namely, algeria, jordan, kuwait, libya, palestine, syria, and united arab emirates. a multistage stratified random sampling technique was used. the total sample included was 4698 adolescents aged from 15 to 18 years (2240 males, 2458 females). the international obesity task force (iotf) reference standard was used to classify the adolescents as nonobese, overweight, and obese. results. among males, overweight was highest among kuwaiti adolescents (25.6%), followed by jordanian (21.6%), and syrian (19.7%) adolescents. among females, the highest prevalence of overweight was reported in libyan adolescents (26.6%), followed by kuwaiti (20.8%), and syrian (19.7%) adolescents. as for obesity, kuwaiti adolescents showed the highest prevalence of obesity for both males (34.8%) and females (20.6%). conclusion. there is an urgent need to establish a plan of action to combat obesity in schoolchildren in these countries. |
various types of dermatological manifestations have been reported due to hepatitis c virus (hcv) infection and antihcv therapy. some of them have been described during ifn - based therapies. peg - ifn--2a / rbv combination is used as the international standard of treatment for hcv infection for a long time. the combination therapy yields an adverse - event profile similar to standard interferon (ifn) therapy. we reported a 43-year - old woman with dystrophic anagen effluvium (dae), rheumatoid arthritis and hashimoto thyroiditis, which were developed under the combination therapy for chronic hcv infection. although some cases of alopecia areata (aa) and telogen effluvium (te) were reported in literature, no case of dae associated with peg - inf--2a /rbv combination therapy was reported previously. peg - ifn--2a / rbv combination therapy is used as the international standard of treatment for hcv infection for a long time. the combination therapy yields an adverse - event profile similar to the standard interferon (ifn) therapy. these adverse effects include hematological, cardiovascular, endocrinological, rheumatologic, neuropsychiatric, digestive and dermatological manifestations, which are sometimes so severe able to restrain the therapy (1). some adverse effects are also suspected to be an autoimmune character such as alopecia and thyroid disorders (1). hair disorders including reversible hair discoloration, hypertrichosis, straight hair, effluvium (alopecia), diffuse thinning of the hair, hair curling, repigmentation of grizzled hair, trichomegaly of the eyelashes have been described during ifn therapy (1, 2). a 43-year - old woman was admitted with chronic hcv infection approved by positive anti - hcv antibody and hcv - rna and two - fold increase in maximum transaminase levels for previous six months. according to family and personal history of the patient, anti - hcv treatment started in august 2012 with 180 mcg of peg - ifn -2a subcutaneously once a week in combination with rbv 1000 mg / daily for a 48-week course. she had an early virological response (evr), because hcv rna level was undetectable and the serum aminotransferase levels returned to normal in the second month of therapy. in the seventh month, the patient complained of insomnia and joint - pains on her wrists and ankles. in physical examination, there were mild swellings on the back of the both hands, wrists, metatarsophalangeal (mtf) and proximal inter phalangeal (pif) joints, and tenderness in the related joints. ranges of motion were normal. in laboratory tests, anti - cyclic citrullinated peptide (anti - ccp) and c reactive protein (crp) levels had positive results (59.76 u / ml and 8.54 mg / l), but rf had negative finding. after the treatment with prednisolone 5 mg / day, naproxen 750 mg / day (15.04.2013) for one month, the symptoms regressed by 50%, and after ceased the inf and rbv treatment the symptoms were alleviated. treatment with methotrexate 2,5 mg / week, folic acid 5 mg / week and prednisolone 5 mg / day was changed in 22.07.2013. on the ninth month of therapy, v shaped 16 14 cm on the vertex and occiput and ovoid and 33 cm on the right parieto - occipital region. on alopecic areas, there were no vellus or miniaturized hairs and no exclamation mark hairs. no scaling, erythema, scarring, atrophy or induration was seen (figure 1 a, 1b). fe, ferritine, vitamin e, zn, 25-hydroxy vitamin d3, 1 - 25-dihydroxy vitamin d3, igg subtypes, tsh, thyroid function tests, fsh, lh, progesterone, dhea - s, shbg, prolactin, testosterone, parathormone and complement levels (c3, c4) all had normal findings. rose - bengal, wright agglutination, rf, vdrl, antinuclear, anti - mitochondrial and anti - smooth - muscle antibodies, ena profile and antithyroglobulin antibodies had negative results, but antibodies against thyroid peroxidase (tpo) were elevated (263.3 iu / ml) and e2 was low (11.8 pg / ml). anti - ccp and crp were still at high levels (48.72 u / ml, 6.44 mg / l). ultrasonography showed enlargement of thyroid gland, hypoechoic and heterogeneous appearance and two nodules on the right lobe. she had been received no therapy for it and decided to get follow - ups. in the trichogram, the ratio of anagen, dystrophic anagen, telogen and catagen hairs were 32%, 50%, 14% and 4%, respectively. histopathological examination of excisional scalp biopsy showed a normal number of catagen - follicles in the dermis. there were no anagen - hairs on the alopecic patches and no inflammation in the periphery of the follicles (figure 2 a, 2 b). based on clinicopathological and trichological findings, dae was diagnosed. because of severe alopecic adverse effect and early virological response, the treatment was stopped after getting an informed consent from the patient at 42 week. although inf therapy was discontinued, anti - ccp level increased and rf became positive (145 u / ml, 31.7iu / ml), elevated anti - tpo levels remained high (239.3 iu / ml) and virological response persisted for seven months after the termination of therapy. four months later, she had a sustained virological and biochemical response and hair regrowth was completed (figure 1 f). hcv rna level was undetectable during the whole treatment period and the patient had still sustained virological response 14 months after the cessation of therapy (21.07.2014). at the time of writing the manuscript, she was still under follow - up and hcv rna level was controlled every six months. her anti - ccp level and rf still had positive results (> 300 u / ml, 29 iu / ml), therefore she was under control of our rheumatology department (21.07.2014). a, b, ninth month of the combine therapy ; c, d, 3 weeks later ; e, 7 weeks later, and f, 4 months later cessation of the therapy. a, b, histopathological examination of an excisional scalp biopsy showed a slight orthokeratosis in the epidermis and a normal number of catagen follicles in the dermis. there was no anagen hair on the alopecic patches and no inflammation on the periphery of the catagen follicles. alopecia is a frequent adverse effect of peg - ifn--2a / rbv combination therapy in 19% of reported patients (2). drug - induced hair loss can be te, dae, dystrophic - telogen effluvium (dte) or anagen effluvium (ae) according to the degree of toxicity in the hair molecules and the types of affected hair cycles (3, 4). inf - therapy related alopecia types are te, localized alopecia (la) at the injection site, alopecia areata (aa) and alopecia universalis (au) (1). it usually occurs in some metabolic deficiencies or diseases such as iron, vitamin b12, folic acid deficiencies, high fever, hypothyroidism and hyperthyroidism (3, 5) in approximately 30% of patients treated with ifn (1, 6). hair pull test has negative results. the percentage of telogen - hairs is abnormally high compared to the percentage of anagen - hairs. the a / t ratio (normally is 80 - 85/15 - 20) is low. the percentage of dystrophic - hairs is normal and the percentage of catagens sometimes increases (3, 5). au is a severe form of aa, which hair loss extends over the whole skin (1). there are published cases of aa during or after treatments with warfarin, cyclosporine, rifampicin, zotepine and zidovudine (6). hair pull test has positive results and exclamation mark hairs are shown at alopecic patches (3, 5). ae is a prominent adverse effect of antineoplastic agents, which is caused by acute damage of rapidly dividing hair matrix cells. the best - known drugs are antimetabolites, alkylating agents and mitotic inhibitors such as adriamycin, cyclophosphamide and etoposide (7). ae occurs after any insult to the hair follicle that impairs its mitotic or metabolic activity. inhibition of cell division in the hair matrix can lead to a weakened segment of hair shaft susceptible to fracture. the follicles should show no signs of inflammation, dystrophic changes of the inner sheath or traction. these features allow distinction of ae from aa, androgenetic alopecia and traction alopecia. only actively growing anagen follicles are subject to these processes. this form of alopecia is more common and severe in combination chemotherapy than monotherapy (8). in ae and dae, the shedding course starts within days or weeks, which is much earlier than te. the growth of anagen hairs usually ends prematurely due to the absence of mitoses within the matrix. the distal part of the hair takes on a characteristic pointed, discoloured aspect with no matrix or shaft. the number of dystrophic anagen hairs is normally observed below 2%. in dae, the percentage of dystrophic hairs is higher compared to the percentage of normal anagen hairs due to acute alteration of the function of the matrix. moreover, yun. reported that chemotherapy - induced ae could be diffuse or patterned. they also found no significant differences in hair loss patterns according to age, associated symptoms or combination of chemotherapeutic agents (7). trueb stated that the chemotherapy - induced alopecia presents with different clinical patterns of hair loss. when an arrest of mitotic activity occurs, obviously numerous and interacting factors influence the shedding pattern (8). because hair - shedding of our patient was a patterned alopecia and hair pull test had positive results, we firstly thought that the alopecia could be an aa. however, due to absence of exclamation mark hairs and perifollicular inflammation and presence of a large number of dystrophic - anagen hairs, this diagnosis was ruled out. te was excluded, because the clinical presentation of alopecic lesions was not similar to a te, also absence of previously mentioned significant metabolic causes, positivity of hair pull test, normal number of telogen hairs and increased dystrophic - anagen hairs. ae can be distinguished from te by means of hair pull test (5). the pull test of our patient had positive result ; therefore, we secondly thought that the alopecia could be an ae. although, hair loss usually occurs within days to weeks of drug administration in ae (9), the effluvium of our patient began nine months after beginning inf therapy and this starting time of alopecia was not compatible with an usual ae. moreover, because the a / t ratio of our patient was significantly decreased, and a normal anagen - to - telogen ratio is characteristic finding of ae (5, 8) the diagnosis of ae was excluded, and lesions were diagnosed as dae. the prevalence and severity of alopecia usually depend on the drug as well as individual predisposition (2, 9). upon the cessation of drug therapy ; follicles resume their normal activity within a few weeks. mitotic inhibition apparently stops the reproduction of matrix cells, but it does not permanently destroy the hair follicles. hair regrowth of our patient began within three weeks of completion of combined therapy and completed four months later. because peg - ifn--2a / rbv therapy has primarily an immune - modulating effect, we thought that late starting time and comparatively faster regrowing time might be related to less toxic follicular effect of peg - ifn--2a / rbv therapy compared to very toxic chemotherapeutic agents such as antimetabolites, alkylating agents and mitotic inhibitors. it has been stated that peg - ifn does not increase the incidence of alopecia compared to standard ifn therapy (36% vs. 32%). because most of reported au cases were caused by pegylated ifn (1, 10), this alopecic effect might be related to the pegylated form of ifn. the alopecic effects have been seen in approximately one - third of patients receiving peg - inf / rbv and particularly in cases receiving peg - inf--2b (10). the authors stated that some of them were reversible, while some cases were irreversible. patients with reversible au received long - term treatments, while irreversible cases received very short - term therapies less than fifteen weeks (10). therefore, we think that the alopecic effects of therapy are unrelated to the cumulative doses or treatment duration. occurrence of this type of alopecia is considered unpredictable ; however, some factors such as psychiatric, endocrine or metabolic disorders may increase the risk of developing this condition (1, 10). on the other hand, neuropsychiatric side effect is one of the relative contraindications of inf therapy (10) ; therefore, from when the patient was diagnosed with psychiatric disorder, we continued therapy closely monitoring her. the association between alopecia and thyroid disorders or thyroid autoantibodies had been documented previously, especially in aa in a ratio of 8 - 28% (1). our patient exhibited a positivity of anti - tpo antibody associated with initiation of alopecia and the positivity continued to persist after stopping the treatment and even after the patient completely recovered from the alopecia. on the other hand, ifn- treatment has been clearly linked to the exacerbation or occurrence of several types of autoantibodies or autoimmune diseases (thyroid disorders, hematological disorders and insulin - dependent diabetes mellitus) or diseases involving altered cell - mediated immune functions (inflammatory dermatological diseases, nephritis, pneumonitis and colitis) (1). moreover, it has been stated that peg - inf might induce immunological modulation (shift from a t helper-2 immune - driven response to t helper-1) and might stimulate synthesis of th1-cytokines such as il-1, il-2 and inf - gamma and increased cytotoxic t cell activity (1, 3). furthermore, in all reported cases, alopecic lesions developed after the viral clearance (10). similarly, in our patient, ra and ht developed after the viral clearance. therefore, we think that accompanying disorders might have been caused by peg - inf/ induced - immunological modulation. additionally, except for our patient, there is no example of peg - ifn 2a/ rbv induced dae and enough information to describe its clinical or histopathological features, and even about its pathogenesis in literature. however, in the light of previously mentioned immunological effects of peg - ifn--2a / rbv therapy, we think that our patient s dae might have been caused by insult of increased cytotoxic t cells to anagen hair follicles. peg - inf a-2b /rbv induced alopecic effect is reversible, but regrowth of hair can take one year after completion of treatment (1). we think and speculate that the regrowing time can be related to affected hair - cycle, severity of damaging effect of the therapy within the follicular matrix, the subtypes of peg - ifn and even individual predisposition, similarly to the shedding time. additionally, in the light of clinicopathological and trichogram findings of our patient, we think that dae cases can be incorrectly assessed as te, dte, androgenetic alopecia or aa according to their clinical appearances. therefore, the diagnosis of dae must be kept in mind when facing with any alopecic condition, especially in patients receiving antiviral therapy. however, further investigations are needed to clarify this issue. in conclusion, alopecia is one of the adverse effects of peg - ifn treatment, independently subtypes. to the best of our knowledge, our case was the first reported one in the literature for peg - inf -2a/ rbv induced dae. | introduction : various types of dermatological manifestations have been reported due to hepatitis c virus (hcv) infection and antihcv therapy. some of them have been described during ifn - based therapies. peg - ifn--2a / rbv combination is used as the international standard of treatment for hcv infection for a long time. the combination therapy yields an adverse - event profile similar to standard interferon (ifn) therapy. some of these adverse effects are rheumatologic, neuropsychiatric and dermatological manifestations including alopecia.case presentation : we reported a 43-year - old woman with dystrophic anagen effluvium (dae), rheumatoid arthritis and hashimoto thyroiditis, which were developed under the combination therapy for chronic hcv infection.conclusions:although some cases of alopecia areata (aa) and telogen effluvium (te) were reported in literature, no case of dae associated with peg - inf--2a /rbv combination therapy was reported previously. |
chromium is an environmentally significant human carcinogen with complicated metabolism and an unknown mechanism of mutagenesis. chromium(vi) is taken up by cells as the chromate anion and is reduced intracellularly via reactive intermediates to stable cr(iii) species. chromium(iii) forms tight complexes with biological ligands, such as dna and proteins, which are slow to exchange. in vitro, crcl3.6h2o primarily interacts with dna to form outer shell charge complexes with the dna phosphates. however, at micromolar concentrations, the cr(iii) binds to a low number of saturable tight binding sites on single - stranded m13 dna. additional chromium interacts in a nonspecific manner with the dna and can form intermolecular dna cross - links. although high concentrations of cr(iii) inhibit dna replication, micromolar concentrations of cr(iii) can substitute for mg2 +, weakly activate the klenow fragment of e. coli dna polymerase i (pol i - kf), and act as an enhancer of nucleotide incorporation. alterations in enzyme kinetics induced by cr(iii) increase dna polymerase processivity and the rate of polymerase bypass of dna lesions. this results in an increased rate of spontaneous mutagenesis during dna replication both in vitro and in vivo. our results indicate that chromium(iii) may contribute to chromate - induced mutagenesis and may be a factor in the initiation of chromium carcinogenesis.imagesfigure 1. |
|
patients with higher levels of postoperative pain tend to have more postoperative complications, longer hospital stays leading to higher medical costs, and lower levels of patient satisfaction. the purpose of this retrospective study was to test the hypothesis that patients undergoing open hepatic resection require less opioid to manage postoperative pain than those undergoing open pancreaticoduodenectomy (whipple procedure). this hypothesis was based on the fact opioids are subjected initially to either oxidation or hydrolysis by cytochrome p450 enzymes and then conjugated to hydrophilic molecules for excretion. any reduction in hepatic volume, therefore, can lead to an increased opioid concentration in the blood resulting in greater opioid bioavailability. the study population included all adult patients who underwent open hepatic resection or open pancreaticoduodenectomy at our institution between january 01, 2010 and june 30, 2013. patients included in the study received a combination of general and neuraxial (epidural) anesthesia. their medical record numbers were then used to access the patients ' information from the electronic health record (ehr). the records contained : the patient 's medical history, diagnoses, procedures performed, medications administered, patient condition, pain scores, and laboratory values. doses of preoperative and post - intensive care unit (icu) medications administered to patients were acquired from the electronic medication administration record. all intraoperative information, including medication dosing and type of surgical procedure, was obtained from the electronic anesthesia record. patient 's pain scores were documented in the postanesthesia care unit (pacu)/icu nursing electronic record. information regarding icu care, including the length of stay (los) and medications administered, was acquired from the icu medical record. opioid doses were converted to morphine equivalents using an online calculator (globalrph, http://www.globalrph.com/narcoticonv.htm). statistical analysis was performed using sas9.3 software (sas institute, cary, nc). the student 's t - test was used to analyze differences in continuous variables between the two groups : age, body mass index (bmi), total hospital los, total icu los, total length of surgery, total opioid administered during entire hospitalization, total preoperative opioid administered, total intraoperative opioid administered, total postoperative opioid administered during the first 48 h, and total postoperative acetaminophen administered during the first 48 h. a chi - square test was used to test for differences in categorical variables between the two groups : gender and american society of anesthesiologists (asa) physical status classification. to determine predictors of opioid requirement, a multivariate analysis was performed by adjusting a linear regression model of total opioid (in morphine equivalents [mg ]) administered postoperatively during the first 48 h against gender, age, asa status, bmi, and type / extent of liver resection. univariate summary statistics and frequencies were analyzed for all variables of interest. the value of p < 0.05 was used to indicate significance. statistical analysis was performed using sas9.3 software (sas institute, cary, nc). the student 's t - test was used to analyze differences in continuous variables between the two groups : age, body mass index (bmi), total hospital los, total icu los, total length of surgery, total opioid administered during entire hospitalization, total preoperative opioid administered, total intraoperative opioid administered, total postoperative opioid administered during the first 48 h, and total postoperative acetaminophen administered during the first 48 h. a chi - square test was used to test for differences in categorical variables between the two groups : gender and american society of anesthesiologists (asa) physical status classification. to determine predictors of opioid requirement, a multivariate analysis was performed by adjusting a linear regression model of total opioid (in morphine equivalents [mg ]) administered postoperatively during the first 48 h against gender, age, asa status, bmi, and type / extent of liver resection. univariate summary statistics and frequencies were analyzed for all variables of interest. the value of p < 0.05 was used to indicate significance. data from 159 patients were included in the final analysis in our study, eighty patients having had open pancreaticoduodenectomy and 79 patients having had open hepatic resection [figure 1 ]. the study population in the pancreaticoduodenectomy group included 62 patients with primary pancreatic carcinoma, three patients with pancreatic metastases, eight patients with a cystic pancreatic lesion, four patients with neuroendocrine tumors, and three patients with duodenal carcinoma. the study population in the hepatic resection group included 22 patients with primary hepatic / cholangiocarcinoma, 38 patients with liver metastases, nine patients with a hepatic cyst, two with echinococcal cysts, and eight patients with an adenoma or hemangioma. three patients in the resection group had early stage cirrhosis with normal coagulation that did not preclude use of neuraxial anesthesia. of the 79 patients who underwent a hepatic resection, three had wedge resections, 41 had resections greater than a wedge but less than hemihepatectomy, five had a left hemihepatectomy, 16 had a right hemihepatectomy, and 12 had resections greater than a hemihepatectomy. open pancreaticoduodenectomy and the majority of hepatic resections were performed primarily through an upper midline incision. a small number of hepatic resections were performed through a right subcostal incision. for both types of cases, fixed retraction was achieved with the use of a thompson retractor (thompson surgical instruments, traverse city, mi, usa). before induction of anesthesia, 0.51 mg) and fentanyl (50100 mcg) were administered intravenously prior the procedure. ropivacaine 0.2% only or bupivacaine 0.125% + 10 mcg hydromorphone / ml was administered using a cadd (smits medical asd inc., st. a bolus of infusion medication (46 ml) was administered before surgical incision and followed by a continuous epidural infusion of the same solution at 48 ml / h. epidural anesthesia was managed intraoperatively by the anesthesiologist and postoperatively by members of the acute pain team. the intraoperative bupivacaine : ropivacaine ratio was 13:67 in the pancreaticoduodenectomy group and 14:65 in the hepatic resection group. the postoperative bupivacaine : ropivacaine ratio was 33:47 in the pancreaticoduodenectomy group and 29:50 in the hepatic resection group. the most common reason that, postoperatively, ropivacaine was changed to bupivacaine + hydromorphone was hypotension. in the combination infusion, the bupivacaine concentration was lower when compared to when ropivacaine was used as the sole agent (0.125% vs. 0.2%). fentanyl was administered intraoperatively (50 mcg increments) only if patients demonstrated signs of pain (tachycardia or hypertension). hydromorphone was administered to all patients at the end of the procedure (0.40.8 mg based on patient weight). postoperatively, in either the pacu or icu, hydromorphone boluses (0.40.8 mg based on patient weight) were administered by the nursing staff. all study participants were managed with an epidural infusion at rate of 48 ml / h for 3 days. after removal of the epidural catheter, patient - controlled analgesia (pca) using an alaris infusion pump (carefusion corporation, san diego, ca, usa) was initiated. pca settings included 0.2 mg of hydromorphone with a lockout time of 6 min and a maximal dose of 2 mg in 1 h. the pca pump was set without a basal rate. pain intensity was evaluated with an 11 point numerical rating scale (scores 010). pain scores were recorded postoperatively at multiple time points by the bedside nurse in the patient care setting (pacu, icu, and floor). hour 0 was defined as the time of patient admission to the pacu or when the patient was extubated. postoperative pain scores at 0, 3, 6, 24, and 48 h were obtained from the ehr. for statistical evaluation, all opioid doses administered were converted to morphine equivalents. the liver resection group was younger and had more females (p = 0.003 and 0.03, respectively). fourteen patients (17.7%) in the liver resection group and 15 patients (18.8%) in the pancreaticoduodenectomy group received preoperative opioids, but no statistically significant difference was demonstrated after dose conversion to morphine equivalents (p = 0.8). length of surgery for pancreaticoduodenectomy was significantly longer than for hepatic resection (p = 0.0001) [table 1 ]. demographic and perioperative data : comparison between groups no statistically significant differences were found in postoperative pain scores at 0, 3, 6, 24, and 48 h between the two groups [figure 2 ]. postoperative pain in patients undergoing liver resection and those undergoing pancreaticoduodenectomy a significantly larger amount of opioids was administered to patients having pancreaticoduodenectomy than for patients having hepatic resection. this difference was demonstrated intraoperatively (p = 0.006), in the first 48 h after surgery (p = 0.001), and for the entire hospital stay (p = 0.002) [table 2 ]. the difference in opioid consumption in the first 48 h remained statistically significant after adjustment for age, sex, bmi, and asa classification (adjusted p = 0.006). twenty - three patients (28.8%) in the pancreaticoduodenectomy group and 9 (11.4%) in the hepatic resection group received intravenous acetaminophen postoperatively in the first 48 h. the overall acetaminophen dose was significantly lower in the hepatic resection group (p < 0.0001) [table 1 ]. the pancreaticoduodenectomy group had a significantly longer average total hospital los as compared to the hepatic resection group (8.7 vs. 7.0 days, respectively [p = 0.03 ]). the lower opioid consumption in the hepatic resection group in the first 48 h postoperatively was confirmed in the multivariate linear regression analysis (p = 0.03). there was no statistically significant difference between the amount of opioids used for the different types of hepatic resections [table 3 ]. total opioids in morphine equivalents given intra- and post - operatively in the postanesthesia care unit / intensive care unit and during total hospital stay in liver resection and pancreaticoduodenectomy cohorts factors associated with opioids requirement multivariate linear regression analysis open pancreaticoduodenectomy and the majority of hepatic resections were performed primarily through an upper midline incision. a small number of hepatic resections were performed through a right subcostal incision. for both types of cases, fixed retraction was achieved with the use of a thompson retractor (thompson surgical instruments, traverse city, mi, usa). before induction of anesthesia, all patients received a thoracic epidural catheter placed at the t6t8 level. midazolam (0.51 mg) and fentanyl (50100 mcg) were administered intravenously prior the procedure. ropivacaine 0.2% only or bupivacaine 0.125% + 10 mcg hydromorphone / ml was administered using a cadd (smits medical asd inc., st. a bolus of infusion medication (46 ml) was administered before surgical incision and followed by a continuous epidural infusion of the same solution at 48 ml / h. epidural anesthesia was managed intraoperatively by the anesthesiologist and postoperatively by members of the acute pain team. the intraoperative bupivacaine : ropivacaine ratio was 13:67 in the pancreaticoduodenectomy group and 14:65 in the hepatic resection group. the postoperative bupivacaine : ropivacaine ratio was 33:47 in the pancreaticoduodenectomy group and 29:50 in the hepatic resection group. the most common reason that, postoperatively, ropivacaine was changed to bupivacaine + hydromorphone was hypotension. in the combination infusion, the bupivacaine concentration was lower when compared to when ropivacaine was used as the sole agent (0.125% vs. 0.2%). fentanyl was administered intraoperatively (50 mcg increments) only if patients demonstrated signs of pain (tachycardia or hypertension). hydromorphone was administered to all patients at the end of the procedure (0.40.8 mg based on patient weight). postoperatively, in either the pacu or icu, hydromorphone boluses (0.40.8 mg based on patient weight) were administered by the nursing staff. all study participants were managed with an epidural infusion at rate of 48 ml / h for 3 days. after removal of the epidural catheter, patient - controlled analgesia (pca) using an alaris infusion pump (carefusion corporation, san diego, ca, usa) was initiated. pca settings included 0.2 mg of hydromorphone with a lockout time of 6 min and a maximal dose of 2 mg in 1 h. the pca pump was set without a basal rate. pain intensity was evaluated with an 11 point numerical rating scale (scores 010). pain scores were recorded postoperatively at multiple time points by the bedside nurse in the patient care setting (pacu, icu, and floor). hour 0 was defined as the time of patient admission to the pacu or when the patient was extubated. postoperative pain scores at 0, 3, 6, 24, and 48 h were obtained from the ehr. for statistical evaluation, all opioid doses administered were converted to morphine equivalents. the liver resection group was younger and had more females (p = 0.003 and 0.03, respectively). fourteen patients (17.7%) in the liver resection group and 15 patients (18.8%) in the pancreaticoduodenectomy group received preoperative opioids, but no statistically significant difference was demonstrated after dose conversion to morphine equivalents (p = 0.8). length of surgery for pancreaticoduodenectomy was significantly longer than for hepatic resection (p = 0.0001) [table 1 ]. no statistically significant differences were found in postoperative pain scores at 0, 3, 6, 24, and 48 h between the two groups [figure 2 ]. postoperative pain in patients undergoing liver resection and those undergoing pancreaticoduodenectomy a significantly larger amount of opioids was administered to patients having pancreaticoduodenectomy than for patients having hepatic resection. this difference was demonstrated intraoperatively (p = 0.006), in the first 48 h after surgery (p = 0.001), and for the entire hospital stay (p = 0.002) [table 2 ]. the difference in opioid consumption in the first 48 h remained statistically significant after adjustment for age, sex, bmi, and asa classification (adjusted p = 0.006). twenty - three patients (28.8%) in the pancreaticoduodenectomy group and 9 (11.4%) in the hepatic resection group received intravenous acetaminophen postoperatively in the first 48 h. the overall acetaminophen dose was significantly lower in the hepatic resection group (p < 0.0001) [table 1 ]. the pancreaticoduodenectomy group had a significantly longer average total hospital los as compared to the hepatic resection group (8.7 vs. 7.0 days, respectively [p = 0.03 ]).. the lower opioid consumption in the hepatic resection group in the first 48 h postoperatively was confirmed in the multivariate linear regression analysis (p = 0.03). there was no statistically significant difference between the amount of opioids used for the different types of hepatic resections [table 3 ]. total opioids in morphine equivalents given intra- and post - operatively in the postanesthesia care unit / intensive care unit and during total hospital stay in liver resection and pancreaticoduodenectomy cohorts factors associated with opioids requirement multivariate linear regression analysis in this study, we were able to confirm our hypothesis that patients undergoing open hepatic resection have significantly reduced postoperative opioid requirements in comparison with patients undergoing open pancreaticoduodenectomy. both procedures, pancreaticoduodenectomy and hepatic resection, are major abdominal surgery with identical or very similar surgical incisions and perioperative anesthetic management. the majority of patients in both groups had malignancies which are associated with significant pain and frequently accompanied with chronic opioid administration. our findings demonstrated lower opioid use in the hepatic resection group coincident with no statistically significant difference in pain scores between groups for the first 48 h postoperatively. the explanation for this reduced postoperative opioid use is most likely multifactorial, but increases in blood opioid concentration due to a reduction in liver volume, and therefore metabolic capacity, likely plays an important role. in fact, hepatic volume reduction has been demonstrated to result in increased opioid bioavailability. a capacity for compensation by increased metabolism in the kidney and intestine has been demonstrated. this compensation can be expected in patients with a chronic liver disease but not acutely after hepatic resection. this study also demonstrates that the intraoperative opioid requirement of patients having hepatic resection was significantly lower in comparison to the pancreaticoduodenectomy group. this finding is most likely related to the fact that the duration of hepatic resections, on average, was significantly shorter. there are two primary mechanisms for opioid metabolism : oxidation via the cytochrome p450 system and glucuronidation. it has been demonstrated that despite the fact that glucuronidation is less affected by liver disease, the final bioavailability of substances undergoing glucuronidation can be very high and thus, the use of opioids that undergo oxidative metabolism is potentially safer in patients with liver dysfunction. fentanyl, a short - acting opioid, is metabolized by oxidation to norfentanyl and other inactive metabolites. morphine, on the other hand, is metabolized by glucuronidation to morphine-3-glucuronide (55%) (an inactive compound), morphine-6-glucuronide (10%) (an active compound), normorphine (4%) (a compound with minimal activity), and codeine. hydromorphone is also metabolized by glucuronidation and although its metabolites do not have significant analgesic activity, they are responsible for central effects similar to those produced by morphine-6-glucuronide. the effects of all opioids can be more pronounced in patients with concomitant renal impairment. our study also demonstrates a decreased use of acetaminophen in the liver resection group. considering the hepatic metabolism of this medication, a higher bioavailability of acetaminophen the decreased use of acetaminophen in the liver resection group might also be due to decreased prescribing due to the concern for hepatic toxicity. it has been recommended to reduce the maximum dose of acetaminophen in patients with impaired hepatic function to 2 g / day. in general, the use of nonsteroidal anti - inflammatory drugs is not recommended in patients with cirrhosis or renal insufficiency. it is important to note that only three patients in the hepatic resection group had early stage cirrhosis that was unlikely to have significantly affected the metabolism of medications. in this study, three of 79 patients in the hepatic resection group had either a wedge resection or multiple wedge resections, resulting in minimal liver volume reductions. the majority of patients in the hepatic resection group (96%) had larger resections, most likely resulting in significant alterations in opioid metabolism. rudin., in a prospective study, compared morphine pharmacokinetics in patients undergoing either hepatic or colon resection. in the liver resection group, they demonstrated that postoperatively, patients having a hepatic resection had a significantly higher plasma morphine concentration (p < 0.01) and significantly lower morphine clearance (p < 0.01) than patients in the colon resection group. in addition in their study, blood morphine concentrations on postoperative day 1 correlated significantly with the volume of liver resected (spearman 's r = 0.6, p < 0.02). the authors also demonstrated an inverse correlation between the ratios of morphine-6-glucuronide (active metabolite) : morphine (r = 0.45, p = 0.03) and morphine-3-glucuronide (inactive metabolite) : morphine (r = 0.52, p = 0.04) with the volume of liver resected. this study also demonstrated that hepatic resection patients more frequently experienced sedation and respiratory depression, particularly if more than 50% of liver volume was removed. beebe. described opioid - related respiratory depression in two donors after living donation of the right hepatic lobe. two patients in the hepatic resection group and none in the pancreaticoduodenectomy group were excluded from data collection due to the need for mechanical ventilation for more than 24 h postprocedure. both patients had significant hepatic resections : (extended right hemihepatectomy and resection of segments 7 and 8). both patients also had a relatively low bmi (20 kg / m and 29 kg / m) and received significant doses of opioids in the first 24 h (436 mg and 268 mg of morphine equivalents). after extensive review of the medical records, we concluded that relative opioid - related respiratory depression was the reason for prolonged ventilation. although our study demonstrated a significant difference in opioid use between hepatic and nonhepatic (whipple) resection, our investigation did not show a relationship between the size of hepatic resection and amount of opioids used. it has been demonstrated that female, obese, and younger patients report both greater postsurgical pain after abdominal surgery and chronic pain in general. there was no statistical difference in bmi between groups in our study, but the pancreaticoduodenectomy cohort had more males and was, on average, older. despite these demographic differences, the hepatic resection group in our study demonstrated decreased total opioid use. this generally indicates less perceived pain. in this study, patients undergoing pancreaticoduodenectomy had longer total hospital los in comparison to patients after hepatic resection. one possible explanation is that increased opioid use is associated with increased adverse effects related to relative opioid overdose. they likely needed longer to return to their normal eating habits as compared to hepatic resection patients. effective perioperative pain management is extremely important not only in regards to a patient 's quality of life, but also as it can affect their outcome. inadequate pain control after major surgery is associated with a number of significant complications as well as increased mortality. high quality pain management helps prevent the surgical stress and organ dysfunction seen with major surgery. the single center retrospective nature of this evaluation can be associated with selection bias and unrecognized confounders. in addition, the level of evidence obtained in a retrospective evaluation is inferior compared to prospective randomized trials. despite the fact that our patient 's preoperative pain scores were not available, we were able to determine that their preoperative opioid consumption did not differ between groups. our investigation has demonstrated that hepatic resection is associated with reduced opioid requirement. aggressive use of opioids in this patient population can potentially lead to significant complications, primarily related to relative opioid overdosing. taking into consideration the retrospective design of our study, further multicenter prospective pharmacokinetic studies should be performed to determine the association between the amount of liver tissue resected and opioid bioavailability. | background and aims : postoperative pain can significantly affect surgical outcomes. as opioid metabolism is liver - dependent, any reduction in hepatic volume can lead to increased opioid concentrations in the blood. the hypothesis of this retrospective study was that patients undergoing open hepatic resection would require less opioid for pain management than those undergoing open pancreaticoduodenectomy.material and methods : data from 79 adult patients who underwent open liver resection and eighty patients who underwent open pancreaticoduodenectomy at our medical center between january 01, 2010 and june 30, 2013 were analyzed. all patients received both general and neuraxial anesthesia. postoperatively, patients were managed with a combination of epidural and patient - controlled analgesia. pain scores and amount of opioids administered (morphine equivalents) were compared. a multivariate lineal regression was performed to determine predictors of opioid requirement.results:no significant differences in pain scores were found at any time point between groups. significantly more opioid was administered to patients having pancreaticoduodenectomy than those having a hepatic resection at time points : intraoperative (p = 0.006), first 48 h postoperatively (p = 0.001), and the entire length of stay (los) (p = 0.002). statistical significance was confirmed after controlling for age, sex, body mass index, and american society of anesthesiologists physical status classification (adjusted p = 0.006). total hospital los was significantly longer after pancreaticoduodenectomy (p = 0.03). a multivariate lineal regression demonstrated a lower opioid consumption in the hepatic resection group (p = 0.03), but there was no difference in opioid use based on the type of hepatic resection.conclusion:patients undergoing open hepatic resection had a significantly lower opioid requirement in comparison with patients undergoing open pancreaticoduodenectomy. a multicenter prospective evaluation should be performed to confirm these findings. |
pilocytic astrocytomas (pas), which are most common in children, are world heath organization grade i tumors with a good prognosis. this neoplasm can occur at all levels of the neuraxis but occurs most frequently in the cerebellum and the optic pathways2). calcification in pas is not a common finding and is reported more frequently in the optic nerve, hypothalamic / thalamus and superficially located cerebral tumors9). massive calcification is extremely rare, and the treatment modality for pas is surgery ; the recurrence rate after total excision is very low. the recurrence time can be as long as four or five years even in the case of residual tumors7). here, we present a case of cerebellar pa with polar spongioblastoma - like cells revealed upon histopathological examination. this pa had massive calcification and a rapid recurrence rate from the residual tumor that led to re - operation. a three - year - old male patient presented complaints of vomiting and imbalance that had been present for approximately three months. a computerized tomography (ct) examination revealed 533triventricular hydrocephalus and a left cerebellar mass with hypodense cystic components surrounded by hyperdense area. magnetic resonance imaging (mri) revealed a lesion, approximately 64.5 cm in size with cystic central part and peripheral contrast enhancement, which was hypointense on t1-weighted images and heterogeneously enhancing on t2-weighted images (fig. the tumor was accessed by left suboccipital craniectomy following an external ventricular drainage catheter being placed in the right lateral ventricle. the cystic tumor components were evacuated, and the solid part, which had a gray - white color with moderate vascularity and minimal stiffness and could not be clearly distinguished from the normal parenchyma, was excised with the help of an ultrasonic aspirator. the tumor had a biphasic pattern with pilocytic cells and oligo - like cells in the focal areas ; no necrosis or mitotic figures were observed. no ema staining was seen, and the tumor cells were diffusely stained positive for gfap. the ki67 index was less than 1% (fig. the age of the patient, the localization of the tumor, the lack of mitosis and necrosis that was indicative of high - grade tumors and the focal biphasic and pilocytic features hinted at pa with massive calcifications. residual calcified areas were found on post - operative ct scans ; no adjuvant therapy was initiated. post - operative ct and mri at six months revealed growth of calcified tissue, but no contrast enhancement and perfusion increase was observed. calcified areas were found to have grown more at the 12-month post - operative ct ; areas that showed contrast enhancement were present on the mri (fig. macroscopically, the tumor was gray - white in color with moderate vascularity and medium stiffness, and it was totally excised with the help of an ultrasonic aspirator. a microscopic examination revealed extensive calcification and prominent oligo - like and pilocytic cells as well as the previous tumor. in addition, polar spongioblastoma - like cells with palisadic sequence were detected in the focal areas. they are the most common gliomas in children and the majority (67%) tend to be locate in the cerebellum9). the cystic portion is hypodense on ct, hypointense on t1wi, hyperintense on t2wi, and the solid components are highly enhancinglesions10). treatment for pas is surgery ; radiotherapy and chemotherapy treatment can be added for tumors that recur and suffer a malignant transformation. after the total excision of pas, the 10-year survey is 94%, and the 20-year survey is 79%10). rosenthal fibers and bipolar cells constitute the compact part ; loose array multipolar cells, microcysts and granular bodies constitute the biphasic view10). oligo - like cells rarely have mitosis, hyperchromatic cells, pleomorphism, glomeruloid vascular proliferation and leptomeningeal infiltration, but they do not indicate malignancy110). diffuse astrocytomas, ganglion cell tumors and pleomorphic xanthoastrocytomas are present in the morphological differential diagnosis of pas. a pilocytic appearance, a biphasic pattern, rosenthal fibers and eosinophilic granular bodies are important in the differential diagnosis. however, none of these features are characteristic of pas. in cases with prominent glomeruloid vascular proliferation the presence of marked oligo - like cells, as in our case, can lead to confusion with oligodendriogliomas. age, radiological appearance and the presence of the pilocytic pattern, even focal, are important for differential diagnosis. the term " primitive polar spongioblastoma " is used for tumors not included in the new who classification and composed of cells with a specific palisading sequence. this pattern can be seen in pas (e.g., the second resection specimen of our patient)4). tihan and burger13) have defined pilomixoid astrocytoma, which is an aggressive subtype of pas. pilomyxoid astrocytoma is typically seen in early childhood ; most cases settle in the chiasm and hypothalamic region and metastasis through recurrence and cerebrospinal fluid is high. the reduced metabolism of degenerative tissue stops carbon dioxide production, and the lesion becomes more alkaline than the surrounding tissues as a result. additionally, increased alkaline phosphatase in degenerative tissues leads to pathologic mineralization such as dystrophic calcification9). up to 25% of pas have intratumoral calcification6). three intraventricular pa cases with massive calcification have been reported in the literature, but no case involving the cerebellar region has been published911). massive calcified pa cases in the literature include patients ages 7, 15, and 58 years ; our case involved a 3 year old, which we believe is an early age for massive calcification. in our patient, the residual calcified areas began to grow in the third month ; cystic and contrast - enhancing solid components with giant calcification emerged again at the end of the patient 's first year. normally ; calcification and recurrence development can take long period of time. it takes 45 years for recurrence to occur even after partial resection, which is interesting in terms of a pathology such as pa. as a result, extensive massive calcification is not a common feature of pas, and it can lead to difficulties in radiological and pathological differential diagnoses. the gross total excision of pas, including calcified areas, is important to prevent recurrence. | pilocytic astrocytomas (pas) are world heath organization grade i tumors and are most common in children. pa calcification is not a common finding and has been reported more frequently in the optic nerve, hypothalamic / thalamus and superficially located cerebral tumors. we present a cerebellar pa in a 3-year - old male patient with cystic components and massive calcification areas. the residual tumor grew rapidly after the first operation, and the patient was operated on again. a histopathological examination revealed polar spongioblastoma - like cells. massive calcification is not a common feature in pas and can lead to difficulties in radiological and pathological differential diagnoses. |
supplementary material is available for this article at 10.1007/s13659 - 011 - 0032 - 6 and is accessible for authorized users. | the reduction of geraniol to citronellol is the first step for the synthesis of natural phytol in the production of tocopherols and natural vitamin k. baker s yeast was used in the bioreduction described above as a whole - cell biocatalyst. however, the enzyme responsible for the reduction of geraniol to citronellol is not yet known. four old yellow enzyme (oye) genes were cloned from yeast and plants, and expressed in escherichia coli for a high level of recombinant proteins. the recombinant protein displayed a catalytic activity of converting geraniol to citronellol as a sole product verified by gc - ms analyses. the recombinant oye2 intact cells were found to show 3.7 and 1.9-fold higher activity than that of yeast cells and the recombinant crude extracts, respectively. compared to the recombinant fusion enzyme, the entrokinase - cleaved enzyme displayed nearly identical activity for geraniol reduction. to our knowledge, this is the first enzyme identified to catalyze the formation of citronellol from geraniol by reducing the allylic alcohol double bond, which is normally known as inactivating group for the old yellow enzymes. electronic supplementary materialsupplementary material is available for this article at 10.1007/s13659 - 011 - 0032 - 6 and is accessible for authorized users. |
the precise measurement of corneal power after myopic laser refractive surgery is still currently an issue under debate. several methods have been proposed during the last years which are classified as methods requiring historical clinical data and methods not requiring historical data. among those methods requiring previous clinical data, some of them are based on a correction of the corneal power using the refracting change achieved [13 ] and others on performing such correction by adjusting the keratometric index [47 ]. the main disadvantage of all these methods is that they are infeasible if previous clinical patient 's data are not available. for this reason, other methods that do not require patient 's historical data have been developed [3, 5, 811 ]. in this line, our research group has recently proposed a new method for estimating with enough accuracy the corneal power using the keratometric approach that has been found to be valid in both healthy and post - lasik eyes. this algorithm based on a variable keratometric index was named adjusted keratometric index (nkadj) and it has been prevalidated clinically in a sample of 32 eyes that had undergone previously myopic lasik surgery. the aim of the current study is to validate clinically this algorithm for the estimation of the corneal power in eyes with previous myopic lasik in a larger population including also a larger range of intended refractive corrections. this retrospective study comprised 62 eyes of 62 patients that had undergone previous correction of a myopic refractive error by means of laser in situ keratomileusis (lasik) surgery. all lasik surgeries had been performed using the pulzar z1 solid - state laser (customvis laser pty ltd., osborne park, australia, currently cv laser pty ltd.) at the department of ophthalmology (oftalmar) of the vithas medimar international hospital (alicante, spain). all surgeries had been performed by one experienced surgeon (aa) between october 2012 and december 2013. for this study, only one eye from each subject was chosen according to a random number sequence (dichotomic sequence, 0 and 1). a comprehensive ophthalmologic examination was performed in all cases at least 3 months after surgery, which included refraction, corrected distance visual acuity (cdva), slit lamp biomicroscopy, goldman tonometry, fundus evaluation, and the analysis of the corneal structure by means of a scheimpflug photography - based tomographer, the pentacam system (oculus optikgerte gmbh, germany, software version 1.14r01). all patients were informed after surgery about this retrospective study and signed an informed consent document in accordance with the helsinki declaration. our research group recently proposed the use of a variable keratometric index (nkadj) for the estimation of the corneal power (pc) using the keratometric approach in patients with previous myopic lasik surgery. the following expression was defined for nkadj considering the ocular conditions of the gullstrand eye model and the range of anterior and posterior curvature that is commonly found in this kind of patients : (1)nkadj=0.0064286r1c+1.37688,where r1c is the postoperative anterior corneal radius. furthermore, adjusted keratometric corneal power (pkadj) was defined as follows : (2)pkadj = nkadj1r1c. for comparison purposes, the keratometric corneal power was also calculated using the classical keratometric index nk = 1.3375 (pk(1.3375)). the gaussian corneal power was calculated using the following expression:(3)pcgaussp1c+p2cp1cp2c = ncnar1c+nhancr2cecncncnar1cnhancr2c, where pcgauss is the gaussian total corneal power, p1c is the anterior corneal power, p2c is the posterior corneal power, r1c is the anterior corneal radius, r2c is the posterior corneal radius, na is the refractive index of air, nc is the refractive index of the cornea, nha is the refractive index of the aqueous humour, and ec is the central corneal thickness. likewise, the true net power (tnp) was also recorded, which is the corneal power provided by the pentacam system (oculus) based on the anterior (r1c) and posterior (r2c) corneal radius and calculated by using the gaussian equation (pcgauss) with the gullstrand eye model, but neglecting the corneal thickness (ec):(4)true net power tnp=1.3761r1c1000 + 1.3361.376r2c1000. besides this, corneal power was also estimated by using other methods described previously for such purpose in eyes with previous myopic laser refractive surgery:(1)methods requiring previous clinical data:(a)awwad method : (5)pcawwad = pc0.23se;(b)camellin method : (6)pccamellin=1.3319 + 0.00113se1r1cpost/1000;(c)clinical history method:(7)pcpost = pcpre+se;(d)jarade method : (8)pcjarade=1.3375 + 0.0014se1r1cpost/1000;(e)savini method [5, 6 ] : (9)pcsavini=1.338 + 0.0009856se1r1cpost/1000.(2)methods not requiring previous data:(a)haigis - l method:(10)pchaigisl=5.1625r1cpost+82.26030.35;(b)shammas method : (11)pcshammas=1.14pc6.8;(c)seitz method : (12)pcseitz=1.114pc4.98,where se = sepre sepost, sepre and sepost being the pre- and postsurgery spherical equivalents, r1cpost is the postsurgery anterior corneal radius, and pcpre is the presurgery keratometric corneal power. methods requiring previous clinical data : (a)awwad method : (5)pcawwad = pc0.23se;(b)camellin method : (6)pccamellin=1.3319 + 0.00113se1r1cpost/1000;(c)clinical history method:(7)pcpost = pcpre+se;(d)jarade method : (8)pcjarade=1.3375 + 0.0014se1r1cpost/1000;(e)savini method [5, 6 ] : (9)pcsavini=1.338 + 0.0009856se1r1cpost/1000. awwad method : (5)pcawwad = pc0.23se ; camellin method : (6)pccamellin=1.3319 + 0.00113se1r1cpost/1000 ; clinical history method:(7)pcpost = pcpre+se ; jarade method : (8)pcjarade=1.3375 + 0.0014se1r1cpost/1000 ; savini method [5, 6 ] : (9)pcsavini=1.338 + 0.0009856se1r1cpost/1000. methods not requiring previous data : (a)haigis - l method:(10)pchaigisl=5.1625r1cpost+82.26030.35;(b)shammas method : (11)pcshammas=1.14pc6.8;(c)seitz method : (12)pcseitz=1.114pc4.98, haigis - l method:(10)pchaigisl=5.1625r1cpost+82.26030.35 ; shammas method : (11)pcshammas=1.14pc6.8 ; seitz method : (12)pcseitz=1.114pc4.98, for the clinical validation of pkadj, it was compared with pcgauss and tnp. likewise, the different methods mentioned above were also compared with pkadj and pcgauss in order to demonstrate which was the most accurate approach. statistical analysis was performed using the software spss version 19.0 for windows (spss, chicago, illinois, usa). normality of all data distributions was first confirmed by means of the kolmogorov - smirnov test. specifically, the paired student t - test or wilcoxon test was used for comparing the different methods of pc calculation depending on whether the normality condition could be assumed or not. the bland - altman analysis was used for evaluating the agreement and interchangeability of the different methods for obtaining the corneal power. this study comprised 62 eyes of 62 patients (34 women [54.8% ]), with a mean age of 33.42 7.16 years (range 21 to 52 years) and with preoperative myopia between 0.25 and 6.8 d. the sample comprised 31 left eyes (50%). mean ocular features of the eyes evaluated in the current study can be seen in table 1. table 2 shows the values of corneal power estimated with the previously published methods (awwad, camellin, clinical history, haigis - l, jarade, savini, seitz, and shammas methods). as shown in table 3, there were significant differences (p 0.5 d). only differences between pkadj and pchaigisl (mean : 0.04 0.17 d) did not reach clinical significance (loa : [0.37 to 0.29 d ]). table 4 summarizes the results of the comparison between the corneal power calculated considering the curvature of the two corneal surfaces as well as corneal thickness (pcgauss) and those values obtained with the other previously published methods for corneal power estimation in corneas with previous myopic laser refractive surgery. as shown, all differences between pcgauss and pc values obtained with such methods were statistically significant (p < 0.01). considering the bland - altman analysis, pkadj and pchaigisl provided the lower mean differences with pcgauss (0.14 0.24 d and 0.18 0.21 d, resp.) and the smaller ranges of agreement ([0.6 to 0.33 ] and [0.60 to 0.23 ], resp.). pccamellin was also close to pcgauss (mean difference : 0.42 0.14 d), but the range of agreement was larger than that found for pkadj and pchaigisl ([0.15 to 0.68 d ]). in the current study, the use of pkadj as a method to estimate the corneal power in corneas with previous myopic laser refractive surgery has been validated clinically. furthermore, the results show that previously reported methods for this estimation are not better than the calculation of pkadj. we have confirmed the clinical prevalidation we conducted in 32 eyes that had undergone myopic lasik surgery where no statistically significant differences between pkadj and tnp were found, with a mean difference value of 0.00 d and limits of agreement of 0.45 and + 0.46 d. in the current series, a mean difference of 0.03 0.24 d between tnp and pkadj (p = 0.319) and a range of agreement from 0.50 to 0.44 d have been found. besides this, the comparison of pkadj with pcgauss has been also performed. it should be considered that pcgauss takes into account neither the curvature of the two corneal surfaces nor the corneal thickness. in this comparison, the mean difference between pkadj and pcgauss was 0.14 0.24 d (p < 0.01), with a range of agreement from 0.33 to 0.60 d. there were only 4 cases out of 66 that showed differences between pkadj and pcgauss of more than 0.5 d. all these results are consistent with the theoretical predictions reported previously that estimated maximum differences between pkadj and pcgauss of 0.7 d. therefore, pkadj is an acceptable method for estimating the corneal power of corneas with previous myopic laser refractive surgery as 100% of estimations were within 0.7 d if pcgauss is taken as reference. when pkadj was compared with other different methods of corneal power estimation, statistically significant differences were found (p < 0.01), except for the comparison between pkadj and pchaigisl (p = 0.09). the comparison between pchaigisl and pkadj showed a mean of difference of 0.04 0.17 and a range of agreement from 0.37 to 0.29 d, confirming that pkadj and pchaigisl were interchangeable. considering that pcgauss is the most exact method of calculation of the central corneal power in paraxial optics and that pkadj, pchaigisl, and pcgauss can be considered interchangeable according to the results of our study, pkadj and pchaigisl can be considered appropriate methods for estimating the corneal power in corneas with previous myopic laser refractive surgery when posterior corneal surface data are unknown in clinical practice. the remaining methods (independently if previous historical data are required or not) were found to overestimate pcgauss (table 4). the classical keratometric approach for corneal power estimation based on calculations performed only considering the anterior corneal radius (pk(1.3375)) induces significant overestimations, ranging from 1.07 to 1.97 d. however, this was not the method providing the poorest performance. the clinical history method was the most variable procedure, providing corneal power estimations that differed from pcgauss in a range going from 1.55 to 3.76 d. among methods requiring previous historical clinical data, pccamellin was the method providing lower overestimations of corneal power, with differences compared to pcgauss ranging from 0.04 to 0.78 d. furthermore, this corneal power estimation method could be clinically valid by using a correction factor. the rest of the methods evaluated led to overestimations of more than 1 d. among methods requiring previous historical clinical data, pccamellin, pcsavini, and pcjarade methods are based on a variation of the keratometric refractive index depending on the induced refractive change (se). this keratometric approach for corneal power estimation is more appropriate than the use of a single value of keratometric index in all cases, but it is still associated with some level of limitation, as has been shown in the current study. a specific keratometric index value for each cornea must be calculated if the keratometric approach is intended to be used for an estimation of corneal power, as demonstrated by our research group in a previous study. this keratometric index was named as exact keratometric index (nkexact) and its calculation requires the measurement of anterior and posterior corneal curvature. as devices measuring the posterior corneal curvature are not available in all clinical settings, our group developed the concept of adjusted keratometric index (nkadj), which is a clinically valid method for estimating an appropriate keratometric index allowing calculation of the corneal power with enough accuracy. this method only requires the measurement of the anterior corneal radius postoperatively, which can be easily obtained in any clinical setting. among methods not requiring previous historical clinical data, the methods evaluated in the current study led to overestimations of more than 1 d except for pchaigisl, as commented previously. these findings confirm the relevance of the curvature of the posterior corneal surface in the error associated with the corneal power estimation when the keratometric approach is used. this is consistent with previous scientific evidence remarking that there is an error in considering the k = r2c / r1c ratio as a constant when corneal power is estimated in eyes after refractive surgery using the keratometric approach. this is also the reason explaining the inaccuracy of methods of corneal power estimation based on considering the refractive change induced. our results also confirm that the use of an inappropriate method for corneal power estimation in eyes with previous laser refractive surgery mainly leads to an overestimation of corneal power and consequently to an underestimation of the iol power required when cataract surgery is planned in this type of cases, resulting in hyperopic residual refractive errors postoperatively. in conclusion, the use of the adjusted keratometric index (nkadj) is a valid and easy method to estimate the central corneal power in corneas with previous myopic laser refractive surgery, improving the accuracy of methods described previously for such purpose. only pchaigisl has been found to be comparable to our method and therefore leading to differences compared to the gaussian corneal power which are clinically acceptable. the advantage of the use of pkadj method is that the estimation of corneal power can be performed with the only requirement of measuring the postoperative anterior corneal radius and with an associated error within 0.7 d and only 6% of cases showing differences with the gaussian power out of the range of 0.5 d. with this paper, a new method for calculating corneal power after myopic refractive surgery was validated clinically, with the advantage that only postoperative refractive surgery parameters are required. | purpose. to validate clinically a new method for estimating the corneal power (pc) using a variable keratometric index (nkadj) in eyes with previous laser refractive surgery. setting. university of alicante and medimar international hospital (oftalmar), alicante, (spain). design. retrospective case series. methods. this retrospective study comprised 62 eyes of 62 patients that had undergone myopic lasik surgery. an algorithm for the calculation of nkadj was used for the estimation of the adjusted keratometric corneal power (pkadj). this value was compared with the classical keratometric corneal power (pk), the true net power (tnp), and the gaussian corneal power (pcgauss). likewise, pkadj was compared with other previously described methods. results. differences between pcgauss and pc values obtained with all methods evaluated were statistically significant (p < 0.01). differences between pkadj and pcgauss were in the limit of clinical significance (p < 0.01, loa [0.33,0.60 ] d). differences between pkadj and tnp were not statistically and clinically significant (p = 0.319, loa [0.50,0.44 ] d). differences between pkadj and previously described methods were statistically significant (p < 0.01), except with pchaigisl (p = 0.09, loa [0.37,0.29 ] d). conclusion. the use of the adjusted keratometric index (nkadj) is a valid method to estimate the central corneal power in corneas with previous myopic laser refractive surgery, providing results comparable to pchaigisl. |
jaffe, lichestein, and portis first defined the criteria for diagnosing gct in 1940 to differentiate it from other giant cell containing lytic lesions of bones. giant cell tumor (gct) of the spine is a rare entity and has been known as a benign aggressive tumor in the literature. it has been known to affect mobile as well as rigid and immobile segments of spine. the incidence of gct is estimated to be 1.4 - 9.4% in the mobile segment of spine. it affects females slightly more than males and affects people in their most productive years in a third or fourth decade. treatment of gct has been variable with good results being shown by intralesional excision, en bloc spondylectomy, recurrent embolization, and also medical management with denosumab and bisphosphonates. we report a case of gct involving three vertebral bodies at the cervicothoracic region with weakness in the left upper limb, which was treated with a posterior approach based corpectomy and anterior reconstruction from posterior approach itself followed by radiotherapy. a 38-year - old lady presented with complaints of pain over the neck radiating to left upper limb for last 4 years with progressively increasing weakness in her left hand for last 2 years. she also complained of numbness in the left hand, left axilla, and on left side of chest. there was significant wasting of intrinsic muscles of the hand. on clinical examination, motor strength of her left long flexors and intrinsic muscles of hand were weak (2/5 as per mrc). imaging studies were done which showed a lobulated tumor arising from c7, d1, and d2 compressing the left c7, c8, d1, and d2 roots [figure 1 ]. intraoperative neuromonitoring was used to detect deficit due to the handling of cord and nerve roots. posterior stabilization using lateral mass screws in c4, c5, and c6 and pedicle screws in d3, d4, and d5 was done. laminectomy extending from c6 to d2 was done, and a tapered rod was applied on one side. facetectomies were done on bilateral sides, and roots were isolated and separated from the tumor mass. roots were freed to the most far lateral extent possible to allow a greater space for insertion of cage among roots [figure 2 ]. it also allows roots to be free and allows for some manipulation, which can occur while inserting cage. burr was used to decorticate the remaining anterior shell of bone from a posterior approach. roots were slightly retracted under neuromonitoring to allow insertion of the cage from a posterior approach and anterior reconstruction with mesh titanium cage was done from a posterior approach. d2 root was also preserved as sometimes intrinsic muscles of hand get a partial nerve supply from d2 if it is a postfixed plexus. patient recovered well and had improved grip strength 1-year postsurgery [figures 3 and 4 ]. (a) t2-weighted magnetic resonance imaging showing tumor involving vertebral bodies at cervicothoracic region. (c) computed tomography scan of cervical spine intraoperative picture showing c6, c7, d1 and d2 roots exposed bilaterally postoperative magnetic resonance imaging at follow - up postoperative lateral view of cervical spine it is a slow growing tumor and can persist as a mass long before it gets diagnosed. common presenting complaints are pain over the affected site, weakness, and paresthesias due to compression of nerve roots. eckardt and grogan have recommended intralesional curettage with adjuvant therapy for stage i and ii enneking lesions and en bloc should be reserved for most stage iii lesions. however, this recommendation was for gct involving long extremities. moreover, surgeon should remember gct is primarily a benign lesion to begin with which can behave aggressively sometimes. en bloc spondylectomy is reserved for tumors, which involve a part of vertebral body and do not involve at least one pedicle. en bloc spondylectomy can be done at cervical region but requires careful isolation of vertebral arteries, which involves combined anterior, posterior approach, and can be a prodigious task at cervicothoracic region and may not be technically feasible in hands of all oncosurgeons. literature also does not show clearly the superiority of en bloc resection over intralesional resection and adjuvant treatment in these difficult to assess regions [table 1 ].. demonstrated acceptable recurrence rates and good clinical outcome after intralesional resection of gct in sacrum.. also suggested that en bloc spondylectomy may not be feasible in the cervical spine and intralesional resection followed by radiation gives acceptable results. hart. reported that en bloc removal was more difficult to achieve and recurrence higher if the tumor extended into the posterior elements or into soft tissues. sanjay. reported en bloc resection in 10 patients and intralesional resection in 14 patients with gcts in mobile spine. 50% of the patients with en bloc resection had a recurrence while 38% of those who had intralesional resection had a recurrence. authors suggested intralesional resection as a minimum treatment modality for the treatment of gct. in this case, tumor had already breached capsule and was compressing nerve roots of c7, t1-weighted, and t2-weighted on left side. gross total resection was done with an intralesional margin from posterior approach and patient was started on postoperative radiotherapy. patient has been kept at regular follow - up and has been recurrence free at 1-year follow - up. this is a very short follow - up, as gct has been known to recur as late as 60 months after index surgery. the aim of the case report is to emphasize the technique and feasibility of doing triple corpectomy with anterior stabilization from the posterior only approach at the not so easily accessible cervicothoracic region. gct has got a depraved standing of recurrence after surgery, and high recurrence has been reported in clinical series. adjuvant treatment in the form of cryotherapy, phenol, recurrent embolization, and radiation has been described in the literature. there exists around ten percent risk of conversion of gct to sarcoma after radiation, but potential benefits of radiation have also been described in preventing recurrence after intralesional excision of gct. combined anterior and posterior approach have been described in the literature for removal of the tumor but these approaches are morbid, time - consuming. anterior approach at the cervicothoracic junction is difficult and requires sternal splitting approach and requires mobilization of great vessels. there is a lot less literature about management of gct at cervicothoracic regions and no report involving three or more vertebra at this location to the best of our knowledge. reported a case of triple cervical corpectomy, which required ligation of bilateral vertebral arteries, and was done from combined anterior, posterior approach. bilateral vertebral artery ligation may not be possible in all patients and requires combined approach. intralesional removal of the tumor can be done at these difficult to resect regions, but it leaves a definite risk of recurrence of gct, which needs to be addressed with radiation or other adjuvant treatment and careful follow - up. posterior - only approach allows for complete tumor removal along with circumferential stabilization in a single setting. there are numerous case reports, which also suggest the role of recurrent embolization, as solo treatment of gct, and also role of agents such as denosumab and bisphosphonates are being explored. aggressive en bloc resection of the tumor though gold standard might not be possible in all circumstances. intralesional resection with adjuvant therapy from posterior only approach may be a suitable alternative for gct involving multiple vertebra at the cervicothoracic region. radiation is a double edge sword in gct, and its pros and cons should be discussed with the patient. with the stereotactic radiosurgery available now, it might be possible to decrease the hazards of radiation to a minimum while piling the maximal gains. | giant cell tumor (gct) is a benign aggressive tumor, which affects axial as well as a peripheral skeleton. it affects epiphysis of long bones and can result in pathological fractures. gct affects cervical spine rarely and has been known to affect almost all vertebra in the human body. it has a predilection for fixed spine, that is, sacrum though it can affect mobile spine as well. gct of cervicothoracic region poses a challenge for the surgeon because of the difficulty in approaching this region anteriorly. this situation is further compounded when gct involves multiple contiguous vertebral bodies in this region and has already spread beyond the confines of its capsule. we report a case of gct involving three vertebral bodies c7, d1, and d2 at cervicothoracic region who presented to us and was treated with triple corpectomy from the posterior only approach. this is the first ever case report of triple corpectomy and anterior reconstruction by a posterior only approach for gct at the cervicothoracic junction to the best of author 's knowledge. |
chest x - ray is one of the most widely used methods for population screening and patient evaluation in virtually all cardiac diseases.[14 ] the american college of cardiology / american heart association (acc / aha) and the european guidelines for the diagnosis and treatment of heart failure advocate looking for the presence of appropriate symptoms and signs and the objective evidence of cardiac dysfunction, as provided by electrocardiogram (ecg), chest x - ray, and cardiac imaging. in this respect, chest x - ray represents a firm element of the diagnostic process in this group of patients. however, the value of chest x - ray in detecting left ventricular (lv) size and function is still controversial, and it has been suggested that less than half of the patients with lv systolic dysfunction show a cardiothoracic ratio > 55%. although comparison with other techniques has shown only a weak correlation between cardiothoracic ratio and radionuclide, echocardiographic, or magnetic resonance imaging (mri)-derived volumes and ejection fraction (ef), heart size on the chest x - ray is still of considerable prognostic value in the patients with low lv ejection fraction (lvef).[1012 ] moreover, the measurement of the transverse chest dimensions could represent a source of error in the evaluation of the cardiothoracic ratio, which is the parameter considered for the assessment of heart size in most of the studies published so far. an index derived from cardiac silhouette, which does not take into account measures of the chest, could therefore provide more reliable parameters for definition of lv dimension and function. cardiac mri due to its good accuracy and superior reproducibility is now considered as the gold standard for in vivo quantification of left and right ventricular volumes and ef.[1318 ] mri equipment, however, is not always available and its costs are considerable. on the other side, chest x - ray is fast, cheap, relatively safe in terms of radiation exposure, totally non - invasive, and is an easily available form of investigation. with these considerations in mind, the aim of this study was to evaluate in an unselected population undergoing cardiac mri for clinical reasons the ability of a simple measurement derived from chest x - ray (transverse diameter of the heart shadow [tdh ]) in detecting lv dilatation and systolic dysfunction, independently measured by cardiac mri. a group of 101 consecutive patients admitted to our institute and undergoing cardiac mri and chest x - ray for clinical reasons were studied : 69 patients were males, mean age was 6214 years. the indications for cardiac mri were lv ischemic dysfunction, idiopathic cardiomyopathy, acute myo - pericarditis, valvular heart disease, hypertrophic cardiomyopathy, or arrhythmogenic right ventricular dysplasia in 50, 26, 6, 5, 2, and 12 patients, respectively. chest x - rays (postero - anterior and lateral projections), of patients in the erect position during an inspiratory breath - hold, with rib position on the 6 rib anteriorly and the 10 rib posteriorly, with focus - to - detector distance of approximately 2 m and very short exposure times, were analyzed. the digital system imix - thorax (oy imix ab, tampere, finland), comprising a detector, scintillation camera, optical system, a system for converting x - ray photons, reconstructions computer, and post - processing computer, is capable of displaying the image in about 10 s. in the imix - thorax, the detector system is strictly related to the arco comb column (arcoma ab, vaxjo, sweden). it includes an automatic exposure control system connected to the x - ray generator medira 2064 (medira ab, tampere, finland), which ensures the constant and correct emission of radiation as a result of the automatic setting of the anode current at the x - ray tube. the chest x - rays were obtained within 2 days of the cardiac mri study. the films were independently read by two expert radiologists who were blinded to the patient 's clinical and functional status and level of severity of cardiac disease. for the analysis, tdh was measured by drawing a line near the middle of the heart shadow and the spine with a line from the right heart border to that line. another line from the left heart border farthest from the line in the middle of the heart shadow was added. the two lengths were added together to derive the transverse diameter of the heart [figure 1 ]. the cardiothoracic ratio (ctr) was also calculated as the ratio between the widest portion of the heart and the diameter of the chest, determined by drawing a line at the level of right leaf of the diaphragm and extending to the inner border of the rib cage on the right and on the left [figure 1 ]. postero - anterior chest x - ray projection where the measure of the transverse diameter of heart shadow (tdh) is reported. the measure was taken by drawing a line near the middle of the heart shadow and the spine and a line from the right border to that line. another line from the left heart border, drawn to the middle, was added. cardiac mri was performed using a 1.5 tesla whole body scanner (ge medical systems, milwaukee, wisconsin, usa). a breath - hold segmented - gradient fast - imaging echo, employing steady - state acquisition, triggered with the ecg, was used to evaluate global lv function according to standard parameters. in each patient, a total of 9 - 12 short - axis views (depending on the lv volumes) and 2 long - axis views (one vertical and one horizontal) were acquired, with a minimum of 30 cine frames for each slice. to measure lv volumes, the endocardial borders were manually drawn in all the short axis end - diastolic and end - systolic images, excluding papillary muscles. end - diastolic and end - systolic lv volumes were calculated and lvef was derived. cut - off values of lv end - diastolic volume (lvedv) were calculated on the basis of normal values reported in our mri lab. therefore, an absolute lvedv > 210 ml was considered as abnormal in males and > 150 ml as abnormal in females. the procedures followed were in accordance with the ethical standards of the institutional committee on human experimentation and with the declaration of helsinki of 1974, as revised in 2000. approval of the institutional committee was obtained and each patient gave informed consent for undergoing clinical examination and chest x - ray after cardiac mri. quantitative data were expressed as mean standard deviation, qualitative data as percentage. the relationship between tdh and lv volume or lvef at mri was evaluated by least square linear regression analysis and the pearson linear correlation (r). the cut - off value of the tdh utilized to identify volumes > 150 ml in females and > 210 ml in males was computed using the receiver operating characteristics (roc) curve and jouden 's j. the performance indices of the cut - off value were used to determine sensitivity, specificity, positive and negative likelihood ratio (lr+, lr-), and the area under the roc curve (auc). the comparison between females and males for the auc was made using the method for independent sample. inter - observer reliability in chest x - ray measurements of two observers was evaluated using the correlation coefficient of lin, the intra - class correlation coefficient (icc), and the bland and altman method. the icc was calculated using a two - way random effects model where people and measure effects were random. a p value 210 ml was considered as abnormal in males and > 150 ml as abnormal in females. the procedures followed were in accordance with the ethical standards of the institutional committee on human experimentation and with the declaration of helsinki of 1974, as revised in 2000. approval of the institutional committee was obtained and each patient gave informed consent for undergoing clinical examination and chest x - ray after cardiac mri. the relationship between tdh and lv volume or lvef at mri was evaluated by least square linear regression analysis and the pearson linear correlation (r). the cut - off value of the tdh utilized to identify volumes > 150 ml in females and > 210 ml in males was computed using the receiver operating characteristics (roc) curve and jouden 's j. the performance indices of the cut - off value were used to determine sensitivity, specificity, positive and negative likelihood ratio (lr+, lr-), and the area under the roc curve (auc). the comparison between females and males for the auc was made using the method for independent sample. inter - observer reliability in chest x - ray measurements of two observers was evaluated using the correlation coefficient of lin, the intra - class correlation coefficient (icc), and the bland and altman method. the icc was calculated using a two - way random effects model where people and measure effects were random. a p value 150 ml with a sensitivity of 82%, a specificity of 69%, a lr+ of 2.63, and a lr- of 0.26. a cut - off value of 15.5 mm identified absolute lv volumes > 210 ml with a sensitivity of 84%, a specificity of 74%, a lr+ of 2.97, and a lr- of 0.22. no significant difference in the auc between females and males was observed (chi - square=0.37, p=0.545). when ctr was considered, a less significant relation was reported with absolute values of lvedv (r=.46, p 150 ml with a sensitivity of 82%, a specificity of 69%, a lr+ of 2.63, and a lr- of 0.26. a cut - off value of 15.5 mm identified absolute lv volumes > 210 ml with a sensitivity of 84%, a specificity of 74%, a lr+ of 2.97, and a lr- of 0.22. no significant difference in the auc between females and males was observed (chi - square=0.37, p=0.545). when ctr was considered, a less significant relation was reported with absolute values of lvedv (r=.46, p 15 mm ; this result may suggest that an increased tdh by itself may address the diagnosis of a reduced global lv function. the study suffers from some limitations : first, a conventional digital x - ray was adopted. probably an ecg - gated digital chest x - ray could have helped on timing the x - ray shot more precisely during the filling phase of the heart, so improving the relation observed between tdh and of lv volumes by mri. secondly, patients with significant pericardial effusion or severe pulmonary hypertension were not present in the studied population. enlargement of the heart silhouette unrelated to lv dimensions like in the presence of significant diffuse pericardial effusion could have lead to poorer relations between tdh and lv volumes. finally, for the present study, tdh only was used to estimate lv dimensions and function. other x - ray - derived parameters such as peribronchial cuffing or vascular pedicle have been reported in literature in heart failure patients, mostly for the assessment of intravascular volume status, although results are still not univocal. the study suffers from some limitations : first, a conventional digital x - ray was adopted. probably an ecg - gated digital chest x - ray could have helped on timing the x - ray shot more precisely during the filling phase of the heart, so improving the relation observed between tdh and of lv volumes by mri. secondly, patients with significant pericardial effusion or severe pulmonary hypertension were not present in the studied population. enlargement of the heart silhouette unrelated to lv dimensions like in the presence of significant diffuse pericardial effusion could have lead to poorer relations between tdh and lv volumes. finally, for the present study, tdh only was used to estimate lv dimensions and function. other x - ray - derived parameters such as peribronchial cuffing or vascular pedicle have been reported in literature in heart failure patients, mostly for the assessment of intravascular volume status, although results are still not univocal. in conclusion, the results of this study show that chest x - ray, a widely available, low - cost, and low - risk technique since radiological exposure is almost negligible (around 0.01 msv, equivalent to the dose absorbed for 10 days due to the natural background radiation) should still be considered as the first approach for a rapid and reliable screening of cardiac dimensions. the use of highly technological, expensive, and much less available techniques should be devoted to specific clinical questions, which would be otherwise unanswered. this is particularly true when a properly executed and interpreted chest x - ray examination is integrated with clinical data collection and a careful physical examination. | objectives : the development of technologically advanced, expensive techniques has progressively reduced the value of chest x - ray in clinical practice for the assessment of left ventricular (lv) dilatation and dysfunction. although controversial data are reported on the role of this widely available technique in cardiac assessment, it is known that the cardio - thoracic ratio is predictive of risk of progression in the nyha class, hospitalization, and outcome in patients with lv dysfunction. this study aimed to evaluate the reliability of the transverse diameter of heart shadow [tdh ] by chest x - ray for detecting lv dilatation and dysfunction as compared to magnetic resonance imaging (mri) performed for different clinical reasons.materials and methods : in 101 patients, tdh was measured in digital chest x - ray and lv volumes and ejection fraction (ef) by mri, both exams performed within 2 days.results:a direct correlation between tdh and end - diastolic volumes (r =.75, p 150 ml (sensitivity : 82%, specificity : 69%) ; in males a cut - off value of 15.5 mm identified lv end - diastolic volumes > 210 ml (sensitivity : 84% ; specificity : 72%). a negative relation was found between tdh and lvef (r = -.54, p<0.0001). the above cut - off values of tdh discriminated patients with lv systolic dysfunction lvef < 35% (sensitivity and specificity : 67% and 57% in females ; 76% and 59% in males, respectively).conclusions : chest x - ray may still be considered a reliable technique in predicting lv dilatation by the accurate measurement of tdh as compared to cardiac mri. technologically advanced, expensive, and less available imaging techniques should be performed on the basis of sound clinical requests. |
bone remodeling occurs throughout life via synthesis of bone matrix through the action of two major bone cells : osteoblasts and osteoclasts [1 - 3 ]. the proper functioning of these cells is necessary for the maintenance of bone mass as well as bone mineral density (bmd). during old age and especially postmenopause there is excessive bone resorption relative to bone formation due hormone deficiencies, which reduces bone mass and ultimately causes bone diseases and osteoporosis. osteoporosis is a widespread health dilemma and its occurrence is projected to rise in the upcoming decades due to the aging of many societies. it is a bone disease that is thought to cause stumpy bone mass and micro - architectural weakening of bone materials, enhance bone brittleness, decrease bone strength, and subsequently increase the threat of fracture. osteoporosis causes distress throughout the united states, europe, china, japan and the rest of the world. according to the world health organization, almost 75 million people in the united states, europe, and japan are affected by osteoporosis. primary osteoporosis is usually associated with aging and low levels of reproductive hormones, especially estrogen, which leads to reduced gonadal activity in the elderly. secondary osteoporosis has other causes, including deficiencies of calcium, vitamin d, and parathyroid hormone. an understanding of the molecular mechanisms and anabolic and catabolic activity of bone is important in the development of new drugs for treating bone diseases, especially osteoporosis. for osteoporosis, the search for new therapeutic agents is mostly concerned with the control of bone resorption and the induction of bone formation by osteoclasts and osteoblasts, respectively. the main molecular factors involved in osteoporosis are the receptor activator of nuclear factor kappa - b ligand (rankl), osteoprotegerin (opg) and the receptor activator of nuclear factor kappa - b (rank). osteoclasts are multinucleate and highly specialized giant cells that have the ability to work in perfect synchronization with osteoblasts to retain the skeletal system.. the discoveries of the rankl and opg have altered our understanding of the primary mechanisms involved in osteoclast differentiation and activity. rankl, a transmembrane protein, belongs to the tumor necrosis factor superfamily member 11, and a ligand for opg is produced by osteoblast and stromal cells [15 - 17 ]. rankl binds with the membrane bound protein rank that is expressed by osteoclasts (oc) with the combination of tumor necrosis factor (tnf)-receptor - associated factor-6 (traf-6) and activates different downstream signaling pathways (fig. consequently, it plays an important role in osteoclast differentiation and bone resorption, which leads to osteoporosis. opg belongs to tumor necrosis factor receptor superfamily member 11b, which has the ability to restrain osteoclast differentiation / formation by preventing rankl from binding to rank. the most important factor in osteoclast activation is a protein known as rankl, which brings these osteoclasts and osteoblasts in contact by inserting itself into the external membrane of osteoclasts, thereby triggering its receptor rank, which is then responsible for bone resorption. the coupling of rankl to rank is facilitated by the nuclear factor (nf)-b signaling pathway. the traf family of proteins (traf, 2 - 6) comprises the most significant factors involved in osteoclastogenesis when rankl binds to rank. a pivotal role is played by traf6 via the regulation and activation of downstream signaling pathways, such as the nf-b pathway, the inhibitor of nf-b kinase pathway, the c - jun n - terminal kinase (jnk) pathway, and the p38 pathway. these pathways ultimately prop - up osteoclast differentiation and bone resorption by stimulating different transcriptional factors such as activator protein-1 and the nf-b pathways. however, it is not clear how these factors are activated by traf6 or how they lead to bone resorption by activating osteoclast - specific markers such as tartrate - resistant acid phosphatase (trap), cathepsin, 3 integrin, and calcitonin receptors [21 - 23 ]. recently it has been suggested that, among the proteases, matrix metallopeptidases (mmps) are responsible for the deprivation of bone extracellular matrix. in particular, mmp-9 is strongly expressed in osteoclasts since it is stimulated by the action of the rankl signaling pathway and it plays a vital role in bone resorption. at the molecular level, however, little is known about how rankl induces mmp-9 gene stimulation. osteoblasts are mononucleate cells that are derived from mesenchymal stem cells, which have the ability to suppress osteoclast activity and increase bone formation by secreting a bone mineralization organic matrix such as collagen - i (col - i) in the osteoid. in osteoblast differentiation, the wnt canonical signaling pathway and the bone morphogenetic protein (bmps) pathways are the most pivotal. the bmps belong to the transforming growth factor (tgf)- superfamily and bmp-2 plays an especially significant role in osteoblastogenesis. when bmp-2 binds to a transmembrane protein such as a bone morphogenetic protein receptor ii, it phosphorylates type i receptor, and hence activates the smad complex (smad 1, 4, 5, and 8) signaling pathways [26 - 29 ], which can aid in the activation of osteoblast - specific transcriptional regulation genes involved in bone formation, such as osteocalcin (ocn), collagen type i, osteonectin, osteopontin, osterix, and bone sialoprotein (bsp) (fig. recently it has been suggested that retinoblastoma binding protein 1 may be the co - activator of runt - related transcription factor 2 (runx2). different therapeutic agents have been used for the treatment, management, and prevention of osteoporosis, including denosumab, bisphosphonate, raloxifene, calcitonin, teriparatide, strontium ranelate, and hormone replacement therapy, but these drugs may have side effects. recently, it has been found that bisphosphonate causes esophagitis, esophageal cancer, and atypical femur fractures when it is used for more than five years. similarly, it has been suggested that hormone replacement therapy may cause breast cancer, coronary heart disease, stroke, and venous thromboembolism [32 - 35 ]. modern pharmacological therapy is costly and produces many side effects, resulting in significant patient non - compliance. thus, there is a strong need to explore alternative therapies, particularly from herbal sources as these are cost - effective and have minimal side effects. over the past decades, herbal medicine has become a topic of global importance, making an impact on both world health and international trade. medicinal plants continue to play a central role in the healthcare of much of the world population. as much as 2000 years ago the roots of american ginseng (p. quinquefolius) and korean ginseng (p. ginseng meyer) were traditionally used for the treatment of many diseases. among the 15 panax species, 4 different species include p. ginseng, p. notoginseng, p. japonicus, p. quinquefolius were used commercially. the ginseng plant contains many active ingredients, including steroidal saponins with a dammarane skeleton as well as protopanaxadiol and protopanaxatriol, commonly known as ginsenosides, which is the energetic part of glycoside plus an aglycone. although ginsenosides have been reported to possess antioxidant, anti - cancer, anti - diabetic, anti - adipocyte, and sexual enhancement properties, very few studies have been conducted on their anti - osteoporotic activity [56 - 60 ]. cell and animal studies of the anti - osteoporotic activities of different ginsenosides have been conducted. protopanaxadiol - type saponins (rb1, rg3, rd, and rh2), protopanaxatriol - type saponins (re and rg1), p. notoginseng saponins (pns ; rg1, rb1, and r1), and ginseng mixtures have been reported to show anti - osteoporotic activity in these studies (table 1). effects of ginseng on different molecular pathways related to osteoporosis in cell line and animal studies alp, alkaline phosphatase ; ocn, osteocalcin ; opn, osteopontin ; osx, ostrex ; col - i, collagen i ; oc, osteoclast ; pkd, protein kinase d ; rankl, receptor activator of nuclear factor kappa - b ligand ; nf-b, nuclear factor kappa - b ; jnk, c - jun n - terminal kinases ; ampk, amp - activated protein kinase ; nfatc1, nuclear factor of activated t - cells, cytoplasmic 1 ; bmp-2, bone morphogenetic protein 2 ; pns, panax notoginseng saponins ; opg, osteoprotegerin ; cbfa1, core binding factor alpha-1 ; ppar 2, peroxisome proliferator - activated receptors 2 ; erk, extracellular - signal - regulated kinases ; bmd, bone mineral density ; camp, cyclic adenosine monophosphate ; tnf-, tumor necrosis factor - alpha ; il-6, interleukin-6 ; trap, tartrate - resistant acid phosphatase ; nk, natural killer cells ; inos, isoform nitric oxide synthases ; no, nitric oxide ; ovx, ovariectomised rats ; dpd / cr, urinary deoxypyridinoline / creatinine ; bv / tv, trabecular bone volume over total bone volume ; conn.d, connectivity density ; tb.n, trabecular number ; tb.th, trabecular thickness ; bmscs, bone marrow stromal cells ; runx 2, runt - related transcription factor 2 ; bsp, bone sialoprotein. even though the exact mechanisms of ginseng s anti - osteoporotic effects are not fully understood, in vitro and in vivo data suggest three possibilities : 1) modulation of osteoblastogenesis and bone formation, 2) modulation of osteoclastogenesis and bone resorption, and 3) modulation of osteoporosis. it is obvious from the table 1 that rh2, rd, rh2(s), and (tr) ginsenosides stimulate the secretion and phosphorylation of alkaline phosphatase (alp), ocn, osteopontin, ostrex, col - i, bmp-2, protein kinase d (pkd)/amp - activated protein kinase, pkd / p38, and camp and increase bone mineral density. li. demonstrated that p. notoginseng has the ability to affect the mrna level of alp, bsp, core - binding factor subunit alpha-1, and opg, increases phosphorylation of extracellular - signal - regulated kinases and p38, and inhibits the secretion of peroxisome proliferator - activated receptors 2 (ppar2) and rankl. on the other hand, ginsenosides rh2(r), rb1, rh2, and rg1 may have the ability to suppress the secretion and activity of osteoclasts, rankl, nf - kb, jnk, c - fos, nfatc1, and tnf-, interleukin (il)-6, and lipopolysaccharide. monroe., kim., and yovel. suggest that red ginseng acidic polysaccharide can increase the tumoricidal activity of isoform nitric oxide synthases of natural killer cells in ovariectomised rats (ovx), while it may increase tnf-, nitric oxide, and il-1 expression by peritoneal macrophages. however, calcium and hydroxyproline secretion in the urine of male wistar rats was significantly suppressed by treatment with a liquid extract from siberian ginseng (eleutherococcus senticosus), while also increasing the strength of femoral diaphyses and vertebrae. according to kim., the ginseng mixture (her - s) has the ability to induce the deposition of bmd, increase femoral trabecular width and estrogen levels, and decrease trap activity in ovx. and li. reported that pns decreased the loss of bmd, ppar2, microstructure corrosion in trabecular bone, and urinary deoxypyridinoline / creatinine, while increasing trabecular bone volume over total bone volume, connectivity density, trabecular number, trabecular thickness, alkaline phosphatase, runx2, oc, and bsp levels in bone marrow stromal cells. kim. found clinically that treatment of postmenopausal osteoporosis patients with korean red ginseng together with maltose capsules had no significant consequences. ginseng that is used as a folk and conventional medicine for the treatment of different diseases for decades has been recognized as one of the valuable source in drug discovery and development. many studies demonstrate that ginsenosides can decrease osteoporosis by inhibiting production of nf - kb, stimulate alp, col - i, runx2, increase blood circulation, and enhance memory. furthermore many studies indicate that ginsenosides have a huge number of activities in both pathological and physiological circumstances concerning with bone disease. however the effects and mechanisms of action of ginsenosides are still not yet entirely understood. data from in vitro and in vivo studies have revealed that ginseng saponins (ginsenosides) have beneficial effects in the treatment of osteoporosis and may increase the osteogenesis of bone marrow stromal cells and pre - osteoblast cells. even though some ginsenosides have been studied for the treatment of osteoporosis, their functions at the pharmacokinetic and pharmacodynamic levels are not well understood, and are important for understanding the inhibition of bone resorption and osteoclastogenesis. future studies about osteoporosis with ginsenosides should include detailed mode of action and mechanisms both in vitro and in vivo. | the ginseng plant (panax ginseng meyer) has a large number of active ingredients including steroidal saponins with a dammarane skeleton as well as protopanaxadiol and protopanaxatriol, commonly known as ginsenosides, which have antioxidant, anticancer, antidiabetic, anti - adipocyte, and sexual enhancing effects. though several discoveries have demonstrated that ginseng saponins (ginsenosides) as the most important therapeutic agent for the treatment of osteoporosis, yet the molecular mechanism of its active metabolites is unknown. in this review, we summarize the evidence supporting the therapeutic properties of ginsenosides both in vivo and in vitro, with an emphasis on the different molecular agents comprising receptor activator of nuclear factor kappa - b ligand, receptor activator of nuclear factor kappa - b, and matrix metallopeptidase-9, as well as the bone morphogenetic protein-2 and smad signaling pathways. |
progression through the eukaryotic cell cycle is controlled by the sequential activation and inactivation of cyclin - dependent kinases (cdks). the kinase activity of cdks is regulated by cyclin synthesis and destruction, phosphorylation of the kinase and cyclin, binding to inhibitory polypeptides and subcellular localization. in metazoa there are four cyclin types (a, b, d and e) known to regulate cell - cycle transitions. these four cyclins have multiple isotypes, the precise number varying between organisms. in mammals and amphibians, members of the a- and d - cyclin families in contrast, b - type cyclins are ubiquitous, being co - expressed in most, if not all, proliferating cells. both a- and b - type cyclins have been shown to have an essential role in progression into, through and out of m - phase. in vertebrates, cyclin a1 is expressed during meiosis, in early cleaving embryos and in some transformed cell lines. cyclin a2 is present in all proliferating cells from the beginning of s - phase until mitosis, with the exception of cells undergoing the meiotic division in males. the reason for this restricted expression pattern is unclear but male mice lacking the cyclin a1 gene are sterile owing to an arrest in meiosis. however, female mice lacking the cyclin a1 are fertile, possibly as a result of rescue by cyclin a2 which is expressed during female meiosis. deletion of murine cyclin a2 is lethal ; embryos are able to undergo the cleavage divisions but die immediately after implantation and appear unable to undergo proliferative growth ablation of expression of cyclin a2 in cultured cells leads to an arrest in s - phase or at the end of g2. the best characterized vertebrate b - type cyclins, b1 and b2, are co - expressed in the majority of dividing cells, but are differentially localized within the cell (reviewed in). gene - deletion experiments have shown that mice lacking the cyclin b1 gene die in utero whereas mice lacking the cyclin b2 gene develop normally and have no immediately apparent phenotype. this suggests that loss of cyclin b2 can be compensated for by another cyclin, probably cyclin b1. cyclin b3 is less well characterized, with a mammalian homolog only recently recognized. in drosophila redundancy between members of each cyclin class is probably responsible for the non - essential phenotypes described above. this effect is best understood in budding yeast, saccharomyces cerevisiae, which has six b - type cyclins that display functional redundancy, yet fulfill distinct roles. to be better able to interpret the phenotypes of a- and b - type cyclin mutants in animals and the degree of redundancy that has to be taken into consideration, we have carried out a comprehensive search to identify a- and b - type cyclin genes in the completed caenorhabditis elegans, drosophila melanogaster and homo sapiens genomes. the search has identified two novel b - type cyclin genes in c. elegans and has shown that there is only a single functional a - type cyclin gene in both invertebrates. on the basis of hybridization of a cyclin a cdna to a southern blot of genomic dna it has been reported that c. elegans has multiple a - type cyclin genes. to investigate this, the c. elegans genome sequence was queried with cyclin a sequences. in addition to the previously characterized c. elegans cyclin a gene (zk507.6) another 15 regions were found with 80 to 95% sequence identity to the query sequence at the nucleotide level. the cyclin a pseudogenes can be divided into six types based on identity to parts of the known cyclin a gene, although all could be derived from a single precursor (figure 1). the pseudogenes are all derived from the central part of the cyclin a gene and span both exons and introns and are thus non - processed pseudogenes. however, it is worth noting that in several of the pseudogenes some of the conserved splice - site consensus sequences are missing, such that if the message was transcribed it would not be correctly spliced. in addition, all contain deletions and mutations relative to the cyclin a gene, resulting not only in the loss of sizeable regions of coding sequence, but also in frameshifts, point mutations and premature stop codons, and thus can not encode a functional protein (figure 1). the pseudogenes are found on all six chromosomes, and none is present in a tandem array. this contrasts with the arrangement of the major sperm protein (msp), another c. elegans gene found in multiple copies. these genes (both transcribed and pseudogenes) are found in loose clusters of between 3 and 13 copies. the cyclin a pseudogenes contain regions of sequence identity to each other which lie outside the region of identity to the cyclin a gene. these regions of identity extend over approximately 4.5 kilobase pairs (kbp) in total, and include direct and inverted repeats, some of which occur at other locations in the genome. this observation supports the possibility that the pseudogenes had a single origin and it is possible that the repeats have had a role in the transposition of the cyclin a pseudogenes around the genome. a search of the genome with several b - type cyclin query sequences identified a total of four genes : the previously characterized cyclins b (zc168.4) and b3 (t06e6.2) and two genes which appear to encode novel b - type cyclins (h31g24.4 and y43e12a.1). the latter two encode polypeptides that have 87% identity to each other ; the main difference between the two is the presence of an insert of 24 amino acids in h31g24.4 that is absent in y43e12a.1. both are more similar to cyclin b than to cyclin b3, and have the same exon and intron structure as the cyclin b gene. at the amino - acid level ests derived from both genes are present in the database : be228087 from h31g24.4 and c44218 from y43e12a.1. on the basis of the available est sequences and homology to the known b - type cyclin, conceptual translations of the two newly identified genes were aligned with the known b - type cyclin (figure 2). this alignment illustrates the high degree of sequence conservation between the b - type cyclins, particularly over the cyclin box. however, all four b - type cyclins differ in the size and sequence composition of their amino termini, and it may be this region which determines their individual cellular functions. the d. melanogaster genome sequence was queried with the complete cyclin a protein sequence. equivalent searches were performed using d. melanogaster cyclin b and cyclin b3 protein sequences. using all search permutations, therefore, it is highly probable that there is one a - type cyclin gene, a single cyclin b and a single cyclin b3 gene within the available sequence. no pseudogenes derived from any of these three cyclin genes were identified. the available human genome sequence (version 5.28.1) the most significant matches were the two known a - type cyclins and the next most significant were the known b - type cyclins. thus, there are only two a - type cyclin genes in the available sequence. searches of the available human genome sequence using b - type cyclin queries revealed three b - type cyclins - b1, b2 and b3 - but no other genes identifiable as encoding a functional b - type cyclin. specifically, no homologs of the xenopus cyclins b4 and b5 were found. although the human genome sequence is not quite complete it seems unlikely that additional human b - type cyclins will be revealed. further evidence for this is provided by the lack of additional b - type cyclin sequences in the est databases, in which cyclins b1, b2 and b3 were clearly present. in addition to these three b - type cyclin genes, a region of high identity to cyclin b2 was found at chromosome 7p22.1. this region had the equivalent of 84% identity over 241 of the 398 amino acids of cyclin b2 and no identity outside this region. thus the predicted open reading frame (orf) contains only a fragment of the cyclin b2 gene and probably represents a pseudogene. on the basis of hybridization of a cyclin a cdna to a southern blot of genomic dna it has been reported that c. elegans has multiple a - type cyclin genes. to investigate this, the c. elegans genome sequence was queried with cyclin a sequences. in addition to the previously characterized c. elegans cyclin a gene (zk507.6) another 15 regions were found with 80 to 95% sequence identity to the query sequence at the nucleotide level. the cyclin a pseudogenes can be divided into six types based on identity to parts of the known cyclin a gene, although all could be derived from a single precursor (figure 1). the pseudogenes are all derived from the central part of the cyclin a gene and span both exons and introns and are thus non - processed pseudogenes. however, it is worth noting that in several of the pseudogenes some of the conserved splice - site consensus sequences are missing, such that if the message was transcribed it would not be correctly spliced. in addition, all contain deletions and mutations relative to the cyclin a gene, resulting not only in the loss of sizeable regions of coding sequence, but also in frameshifts, point mutations and premature stop codons, and thus can not encode a functional protein (figure 1). the pseudogenes are found on all six chromosomes, and none is present in a tandem array. this contrasts with the arrangement of the major sperm protein (msp), another c. elegans gene found in multiple copies. these genes (both transcribed and pseudogenes) are found in loose clusters of between 3 and 13 copies. the cyclin a pseudogenes contain regions of sequence identity to each other which lie outside the region of identity to the cyclin a gene. these regions of identity extend over approximately 4.5 kilobase pairs (kbp) in total, and include direct and inverted repeats, some of which occur at other locations in the genome. this observation supports the possibility that the pseudogenes had a single origin and it is possible that the repeats have had a role in the transposition of the cyclin a pseudogenes around the genome. a search of the genome with several b - type cyclin query sequences identified a total of four genes : the previously characterized cyclins b (zc168.4) and b3 (t06e6.2) and two genes which appear to encode novel b - type cyclins (h31g24.4 and y43e12a.1). the latter two encode polypeptides that have 87% identity to each other ; the main difference between the two is the presence of an insert of 24 amino acids in h31g24.4 that is absent in y43e12a.1. both are more similar to cyclin b than to cyclin b3, and have the same exon and intron structure as the cyclin b gene. at the amino - acid level ests derived from both genes are present in the database : be228087 from h31g24.4 and c44218 from y43e12a.1. on the basis of the available est sequences and homology to the known b - type cyclin, conceptual translations of the two newly identified genes were aligned with the known b - type cyclin (figure 2). this alignment illustrates the high degree of sequence conservation between the b - type cyclins, particularly over the cyclin box. however, all four b - type cyclins differ in the size and sequence composition of their amino termini, and it may be this region which determines their individual cellular functions. the d. melanogaster genome sequence was queried with the complete cyclin a protein sequence. equivalent searches were performed using d. melanogaster cyclin b and cyclin b3 protein sequences. using all search permutations, therefore, it is highly probable that there is one a - type cyclin gene, a single cyclin b and a single cyclin b3 gene within the available sequence. the available human genome sequence (version 5.28.1) was searched with cyclin a1 and a2 query sequences. in both cases the most significant matches were the two known a - type cyclins and the next most significant were the known b - type cyclins. thus, there are only two a - type cyclin genes in the available sequence. searches of the available human genome sequence using b - type cyclin queries revealed three b - type cyclins - b1, b2 and b3 - but no other genes identifiable as encoding a functional b - type cyclin. specifically, no homologs of the xenopus cyclins b4 and b5 were found. although the human genome sequence is not quite complete it seems unlikely that additional human b - type cyclins will be revealed. further evidence for this is provided by the lack of additional b - type cyclin sequences in the est databases, in which cyclins b1, b2 and b3 were clearly present. in addition to these three b - type cyclin genes, a region of high identity to cyclin b2 was found at chromosome 7p22.1. this region had the equivalent of 84% identity over 241 of the 398 amino acids of cyclin b2 and no identity outside this region. thus the predicted open reading frame (orf) contains only a fragment of the cyclin b2 gene and probably represents a pseudogene. the main findings of this study are first, that invertebrates have a single a - type cyclin whereas vertebrates have two, and second, that the number of b - type cyclins varies from two in drosophila to three in humans and four in c. elegans, but all have at least one cyclin b1-like gene and a cyclin b3 gene. in vertebrates, cyclin a1 is expressed predominantly in the germline during the meiotic divisions and early cleavage divisions of the embryo, whereas cyclin a2 is present in all proliferating cells. near - completion of the human genome sequence has revealed that there are only two a - type cyclins. in support of this finding an analysis of the extensive mouse and zebrafish est databases and the draft pufferfish genome database identified cyclin a1 and a2 cdnas and genes but no further a - type cyclins (data not shown). these sequences were used to construct a phylogenetic tree for all known a - type cyclins (figure 3). from this tree it is clear that all known vertebrate a - type cyclins fall into one of two groups (a1 and a2) and there is no evidence for multiple invertebrate a - type cyclins. the analysis of the number of a - type cyclin genes was initiated by the observation that mouse embryos lacking a cyclin a2 gene complete the normal number of cleavage divisions to produce an embryo of more than 100 cells before ceasing to proliferate further. there are four possible explanations : first, cyclin a is not necessary for the cleavage divisions ; second, there is persistence of maternal cyclin a mrna ; third, the cleavage divisions are rescued by cyclin a1 expression or (a fourth possibility) by expression of another unidentified a - type cyclin. the intriguing question now is why vertebrates have evolved a distinct a - type cyclin with a limited role within the germline. one possible explanation is that cyclin a1 is part of the larger phenomenon of the occurrence of germline isoforms of a large number of genes. a number of enzymes are already known to have testis - specific isoforms (reviewed in), including glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. in each case, testis - specific isoforms have been identified only in the vertebrate lineage (data not shown), suggesting that the second isoforms arose after the divide between invertebrates and vertebrates. it is clear that in invertebrates a single a - type cyclin can carry out all the functions of the mitotic and meiotic cell cycles. our finding of 15 cyclin a pseudogenes was unexpected, as the c. elegans genome is generally considered to contain a limited number of pseudogenes. however, the observation does explain the multiple restriction enzyme fragments that hybridize with a cyclin a probe on a southern blot. in each organism we identified a single b3-type cyclin, distantly related to both the a- and b - type cyclin families. the precise cellular role of cyclin b3 remains to be determined, although it appears to function late in mitosis. in drosophila there is functional redundancy between the b and b3 cyclins in mitosis ; however, deletion of both genes results in embryonic lethality. in contrast, both cyclins b and b3 are required for female fertility and cyclin b, but not b3, is required for male fertility. therefore, it is possible that the mammalian b3 cyclins could also be partially functionally redundant with b - type cyclins. while drosophila has only one more b - type cyclin gene in addition to cyclin b3, c. elegans has a further three genes which encode near - identical b - type cyclins. it is worth noting that these multiple nematode b - type cyclins are more closely related to each other than they are to any other b - type cyclins (figure. 4). the b - type cyclin phylogenetic tree provides evidence for multiple gene - duplication events. such an event early in animal evolution may have given rise to the b- and b3-type cyclins identified in all animals to date. more recent duplication events can explain the variation in number of b - type cyclins between organisms. the finding of three closely related c. elegans b - type cyclins has some significance in the interpretation of previously published data. rna interference however, gene product ablation using rnai may not distinguish between such high levels of identity and consequently the function of all three b - type cyclins will be lost, so the best interpretation possible is that at least one of these b - type cyclins is required. additionally, three c. elegans cyclin b mrnas of different lengths were observed and it was reported that they may arise from the use of different polyadenylation sites. an alternative interpretation, in the light of this study, is that each gene produces a single message so similar (approximately 80% nucleotide identity over the orf) that the probes did not distinguish between them. one of the probes (a 3 ' utr fragment) used by kreutzer. should have been specific for the previously identified cyclin b and did detect a single band. this allows us to predict that this gene gives rise to the largest (1.7 kbp) of the three transcripts. interestingly, kreutzer. observed that the three cyclin b transcripts are differentially expressed in the maternal and paternal germlines. therefore, it is possible that the three c. elegans cyclin b genes have distinct expression patterns through development. the remaining vertebrate b - type cyclins fall into two evolutionarily divergent groups, one containing the b1 cyclins and the recently identified xenopus cyclin b4 and the other the b2 cyclins and the xenopus cyclin b5. the genes encoding cyclins b1, b2 and b3 were readily identifiable within the draft human genome sequence ; there are no other b - type cyclin genes present. furthermore, homologs of the mitotic cyclins a1, a2, b1, b2 and b3 were readily identified in the extensive zebrafish est database and the draft pufferfish genome database, but no cyclin b4 or b5 homologs were found. together, this suggests that cyclins b4 and b5 are unique to amphibians, perhaps, as suggested by hochegger. in conclusion we suggest that there are three conserved families of mitotic cyclins in animals : the a - type cyclins with one gene in invertebrates and two genes in vertebrates ; the b3-type cyclins with a single gene in all animals ; and the b - type cyclins with different numbers of genes in different organisms, possibly as an adaptation to their distinct developmental strategies. in vertebrates, cyclin a1 is expressed predominantly in the germline during the meiotic divisions and early cleavage divisions of the embryo, whereas cyclin a2 is present in all proliferating cells. near - completion of the human genome sequence has revealed that there are only two a - type cyclins. in support of this finding an analysis of the extensive mouse and zebrafish est databases and the draft pufferfish genome database identified cyclin a1 and a2 cdnas and genes but no further a - type cyclins (data not shown). these sequences were used to construct a phylogenetic tree for all known a - type cyclins (figure 3). from this tree it is clear that all known vertebrate a - type cyclins fall into one of two groups (a1 and a2) and there is no evidence for multiple invertebrate a - type cyclins. the analysis of the number of a - type cyclin genes was initiated by the observation that mouse embryos lacking a cyclin a2 gene complete the normal number of cleavage divisions to produce an embryo of more than 100 cells before ceasing to proliferate further. there are four possible explanations : first, cyclin a is not necessary for the cleavage divisions ; second, there is persistence of maternal cyclin a mrna ; third, the cleavage divisions are rescued by cyclin a1 expression or (a fourth possibility) by expression of another unidentified a - type cyclin. the intriguing question now is why vertebrates have evolved a distinct a - type cyclin with a limited role within the germline. one possible explanation is that cyclin a1 is part of the larger phenomenon of the occurrence of germline isoforms of a large number of genes. a number of enzymes are already known to have testis - specific isoforms (reviewed in), including glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. in each case, testis - specific isoforms have been identified only in the vertebrate lineage (data not shown), suggesting that the second isoforms arose after the divide between invertebrates and vertebrates. it is clear that in invertebrates a single a - type cyclin can carry out all the functions of the mitotic and meiotic cell cycles. our finding of 15 cyclin a pseudogenes was unexpected, as the c. elegans genome is generally considered to contain a limited number of pseudogenes. however, the observation does explain the multiple restriction enzyme fragments that hybridize with a cyclin a probe on a southern blot. in each organism we identified a single b3-type cyclin, distantly related to both the a- and b - type cyclin families. the precise cellular role of cyclin b3 remains to be determined, although it appears to function late in mitosis. in drosophila there is functional redundancy between the b and b3 cyclins in mitosis ; however, deletion of both genes results in embryonic lethality. in contrast, both cyclins b and b3 are required for female fertility and cyclin b, but not b3, is required for male fertility. therefore, it is possible that the mammalian b3 cyclins could also be partially functionally redundant with b - type cyclins. while drosophila has only one more b - type cyclin gene in addition to cyclin b3, c. elegans has a further three genes which encode near - identical b - type cyclins. it is worth noting that these multiple nematode b - type cyclins are more closely related to each other than they are to any other b - type cyclins (figure. 4). the b - type cyclin phylogenetic tree provides evidence for multiple gene - duplication events. such an event early in animal evolution may have given rise to the b- and b3-type cyclins identified in all animals to date. more recent duplication events can explain the variation in number of b - type cyclins between organisms. the finding of three closely related c. elegans b - type cyclins has some significance in the interpretation of previously published data. rna interference however, gene product ablation using rnai may not distinguish between such high levels of identity and consequently the function of all three b - type cyclins will be lost, so the best interpretation possible is that at least one of these b - type cyclins is required. additionally, three c. elegans cyclin b mrnas of different lengths were observed and it was reported that they may arise from the use of different polyadenylation sites. an alternative interpretation, in the light of this study, is that each gene produces a single message so similar (approximately 80% nucleotide identity over the orf) that the probes did not distinguish between them. one of the probes (a 3 ' utr fragment) used by kreutzer. should have been specific for the previously identified cyclin b and did detect a single band. this allows us to predict that this gene gives rise to the largest (1.7 kbp) of the three transcripts. interestingly, kreutzer. observed that the three cyclin b transcripts are differentially expressed in the maternal and paternal germlines. therefore, it is possible that the three c. elegans cyclin b genes have distinct expression patterns through development. the remaining vertebrate b - type cyclins fall into two evolutionarily divergent groups, one containing the b1 cyclins and the recently identified xenopus cyclin b4 and the other the b2 cyclins and the xenopus cyclin b5. the genes encoding cyclins b1, b2 and b3 were readily identifiable within the draft human genome sequence ; there are no other b - type cyclin genes present. furthermore, homologs of the mitotic cyclins a1, a2, b1, b2 and b3 were readily identified in the extensive zebrafish est database and the draft pufferfish genome database, but no cyclin b4 or b5 homologs were found. together, this suggests that cyclins b4 and b5 are unique to amphibians, perhaps, as suggested by hochegger. in conclusion we suggest that there are three conserved families of mitotic cyclins in animals : the a - type cyclins with one gene in invertebrates and two genes in vertebrates ; the b3-type cyclins with a single gene in all animals ; and the b - type cyclins with different numbers of genes in different organisms, possibly as an adaptation to their distinct developmental strategies. blast 2.0 was used to carry out similarity searches against the available sequence databases with protein or dna query sequences. for searches against the c. elegans and human genome sequences pileup program was used for creating multiple sequence alignments (using progressive pairwise alignments) ; alignments were further refined using the clustal w program version 1.8 and manually. blast 2.0 was used to carry out similarity searches against the available sequence databases with protein or dna query sequences. for searches against the c. elegans and human genome sequences the gcg wisconsin package version 10.0 pileup program was used for creating multiple sequence alignments (using progressive pairwise alignments) ; alignments were further refined using the clustal w program version 1.8 and manually. the alignments used for the construction of the phylogenetic trees of a- and b - type cyclins is available with the online version of this paper. the alignments used for the construction of the phylogenetic trees of a- and b - type cyclins click here for additional data file we thank tim hunt, bill colledge, howard baylis, helena webb and catherine french for critical reading of the manuscript and/or helpful discussions. the intron / exon structure of cyclin a (cosmid zk507) is shown with the cyclin box shaded. below this are shown the six different cyclin a pseudogene structures : regions with homology to exons are represented by boxes and regions homologous to introns by lines (with deletions also marked). the cosmid clones containing each of the genes are shown on the right with the genes actually represented on the schematic in bold. note that cosmids b0019 and w02a11 overlap and represent only one pseudogene. for each schematic, potential open reading frames (orfs) black bars represent orfs with high identity to cyclin a1 (> 70%) and gray bars represent orfs with no significant identity to cyclin a1. orfs were predicted by assuming that each pseudogene was fully transcribed and spliced in a manner analogous to the cyclin a gene. the largest orf was identified in the pseudogene on clone c02b8 and would result in a 75- or 113-amino - acid protein depending on whether the first initiation codon within the pseudogene was used (asterisk) or an initiation codon just 5 ' of the pseudogene. frameshifts and point mutations limit the extent of the identity and frequently result in termination codons. alignment of c. elegans b - type cyclins. the known b - type and b3 cyclins (zc168 and t06e6) were aligned to the two novel b - type cyclins (h31g24 and y43e12a) using the gcg pileup program and refined using the clustalw program. below the sequences are marked identical (), highly similar (:) and similar (.) vertebrate a - type cyclins can be divided into two groups : somatic (a2) and germline (a1), whereas completion of the c. elegans and d. melanogaster genome - sequencing projects shows that there is only one a - type cyclin in these organisms. the alignment used for the construction of this phylogenetic tree is given in the additional data files. a. pectinifera, asterina pectinifera ; h. pulcherrimus, hemicentrotus pulcherrimus ; h. robusta, helobdella robusta. the b3 cyclins form an evolutionarily conserved family distinct from all the other b - type cyclins. there are two evolutionarily divergent groups ; one includes the b1 and xenopus (frog) b4 cyclins and the other the b2 and xenopus b5 cyclins. the alignment used for the construction of this phylogenetic tree is given in the additional data files. | multiple a- and b - type cyclins have been identified in animals, but their study is complicated by varying degrees of functional redundancy. a single functional a - type cyclin gene was found in invertebrates and two were found in vertebrates. the number and relationship of b - type cyclins varies between caenorhabditis elegans, drosophila melanogaster and homo sapiens but all contain at least one cyclin b1-like gene and a cyclin b3 gene. |
amniocentesis for prenatal karyotyping is today the most frequently performed invasive intrauterine procedure (eisenberg and wapner, 2002), but still carries a procedure related risk for amniotic fluid leakage estimated to be approximately 1 - 2% (reece, 1997 ; tabor., 1986). postprocedural amniotic fluid leakage is the strongest predictor of subsequent fetal loss (saltvedt and almstrom, 1999). instrument size influences the created defect in the fetal membranes and the subsequent risk for amniotic fluid leak and miscarriage. traditionally, 20 and 22 g spinal needles have been used for amniocentesis, although remarkably, this was not based on experimental data. more recently, a new needle was designed and marketed specifically for invasive intrauterine procedures (23 g : cook echotip, spencer, indiana, usa). this smaller 23 g needle potentially represents less trauma to the uterus and fetal membranes. additionally, the specifically designed tip (echotip, fig. 1) is designed to enhance ultrasound visualisation and could reduce the sampling time, and therefore the discomfort for the patient and risks of the procedure. the aim of this study was to compare this new needle type with a standard 22 g spinal needle in terms of defect size created in the fetal membranes and time needed to perform an invasive intrauterine procedure. fetal membranes were freshly obtained from four women delivered by elective caesarean section at term (37 - 40 weeks) following uncomplicated pregnancies. membrane patches of 100 50 mm were excised and divided in two equal parts. each membrane patch was wrapped around the bottom of a plastic tube and secured with an elastic ring. the tube was fixed in a vertical position and then filled with hartmann s solution or amniotic fluid at 37c (fig. the fluid pressure was kept constant at 15 cm h2o representing a physiologic value for amniotic pressure in pregnancy (fisk., 1992). the membranes were punctured by introduction of the needle at an angle of 90 1 to 1.5 cm deep through the bulging membranes using either a 22 g or 23 g needle. after the experiment, the membrane was detached and the wound edge stained with india ink to facilitate recognition during microscopic evaluation. we calculated the size (mm) of the defect using a microscope (zeiss, oberkauchen, germany), at magnification (20) and adapted software (ks 100 3.0, carl zeiss, oberkochen, germany). in cases where sliding of the amnion and chorion against each other had resulted in a smaller net defect, this net defect through both the amnion and chorion, was measured. 22 g : vygon, valkenswaard, the netherlands 23 g : cook echotip, spencer, indiana, usa calculated as the cross - sectional area, based on the outer diameters provided by the manufacturer. as a second part of the study, we determined the performance of six operators with different levels of clinical experience [novice (n = 2), intermediate (n = 2), expert (n = 2) ], using either a 22 g or 23 g needle for cordocentesis in a randomised video - controlled setting (silver., 1998). all procedures were performed on a cordocentesis simulator (limbs & things ltd, bristol, united kingdom). this simulation model allows for the combination of realistic ultrasound visualisation as well as mock blood sampling from an anterior or posterior placenta with umbilical cord (fig. ultrasonography was performed using the same machine we use for all our invasive intrauterine procedures, an acuson sequoia (mountain view, ca) with a 2.5 - 6 mhz transducer. each operator performed four cordocentesis procedures (single operator, two hands technique) with each needle. in total, 24 procedures were performed with each needle. each operator performed only one procedure per day. for each procedure, we measured the procedure time, defined as the time from puncturing the cordocentesis simulator to successfully collecting 2 ml of artificial blood, in seconds. after completion of the project, measurement of the procedure - times was performed from the videotapes by two co - workers blinded to the type of needle that was used during the experiment. results were stored and analysed using spss 10 (spss inc., chicago, illinois) software and are expressed as mean sd. the expected and actual defects created by both 22 g and 23 g needles are shown in table 1. the defect sizes created by 22 g needles were slightly larger than those created by 23 g needles : 0.66+/-0.12 versus 0.59+/-0.13 mm. in accordance to previous observations, the defect size created by both needles was larger than the expected defect size based on the cross - sectional diameter of the needle (devlieger., 2002). the mean sampling duration for all operators pooled was similar : 144 188 sec with a 22 g needle versus 144 120 sec with a 23 g needle, p = 0.99. the mean duration of sampling for the different study groups is shown in table 2. there were no significant differences in duration of sampling between the two needles in the three study groups, however, 4 of the 6 operators required less time for cordocentesis with the 22 g needle as compared to the 23 g needle. despite the increasing number of pregnant women undergoing invasive intrauterine procedures and the not infrequent, potentially dramatic complications, few experimental studies have focused on the technical aspects of these procedures. as a general rule, to reduce the risks of intrauterine procedures, it seems logical to use the least traumatic needle that combines fast sampling and easy ultrasound visualisation. in today s clinical practice, most fetal medicine specialists use 22 g spinal needles to perform amniocentesis (eisenberg and wapner, 2002). we have shown that this needle creates a small defect in the fetal membranes reducing subsequent fluid leakage, as compared to bigger needles (devlieger., 2002). smaller needles with a diameter of 24 g and 26 g are associated with increased sampling time, which results in relatively larger defects, probably as a result of increased manipulation and physiologic tremor (devlieger., 2002). additionally, these very thin needles can be blocked by particles in amniotic fluid leading to unsuccessful sampling. larger needle diameters are associated with slightly shorter sampling time, but exponentially larger defects in the fetal membranes (devlieger., 2002). therefore, a 22 g needle appears to provide a good compromise between sampling time and defect size. only recently, needle diameters, shape and ultrasonographic properties the 23 g needle with echo - tip has several theoretical advantages, but these were not confirmed in this experimental study. first, the specially designed echotip was expected to give a better ultrasonographic image than the needles currently used. this should help the operator in determining the location of the needle inside the amniotic sac and lead to a decrease in sampling time. however, most operators did not have shorter sampling times with the 23 g needle, on the contrary : 4 out of 6 operators performed better with the 22 g needle. in general, the oblique tip of a standard needle sufficiently enhances sonographic visualisation. the additional reflections created by the 23 g echotip not only seem unnecessary, but may even have a negative effect on precisely localizing the needle tip. a possible limitation of our study could be the use of both an excellent ultrasound machine and a good model ; we can not exclude that the echotip could be of more value with the use of less advanced equipment or in procedures done in obese patients. secondly, the defect created by the 23 g needle was not significantly smaller than the fetal membrane defect created with a 22 g needle. theoretically, the cross - sectional area of the needle determines the minimal size of the created defect. this cross - sectional area relates to the needle diameter according to the equation r. therefore, the relationship between needle diameter and defect size can be expected to be exponential (fig. defects created by both 22 g and 23 g needles are in the horizontal part of this exponential curve (devlieger., 2002), explaining the small differences measured. of course, the relatively small sample size used in this study could also contribute to the fact that no significant difference was noted in our experiments. using the cordocentesis model also, procedures performed on the model are not complicated by a moving fetus and complications like prom do not occur. nonetheless, with the blinded randomised design, we would not expect the results of our study to be different if performed in the actual clinical setting. in summary, a 23 g needle with adapted tip for enhanced ultrasound visualisation did not, in comparison with a 22 g spinal needle, result in decreased defect size in the fetal membranes or decreased sampling time in a randomised experimental setting. therefore, the claimed advantages of the more expensive 23 g needle over the 22 g needle are not supported by our study. | objectives : to experimentally compare the standard 22 g spinal needle with a newer 23 g needle with specific ultrasound visualisation tip (cook echotip, spencer, indiana, usa) in the setting of ultrasound - guided invasive intrauterine procedures.materials and methods : we first determined the size of the defects created in human fetal membranes using light microscopy and adapted area calculating software by both needles in an in vitro model performing 20 paired experiments on 4 term membrane specimens. secondly, we determined the performance of 3 groups of operators, with different levels of experience in invasive intrauterine procedures during cordocentesis on a simulator model, using either a 22 g or 23 g needle. for each procedure, we measured the time required to successfully obtaining 2 ml of artificial blood in 24 paired experiments.results : the mean sd defect sizes created was 0.66 0.12 mm2 with the 22 g needle and 0.59 0.13 mm2 with the 23 g needle (p = 0.11). the mean duration of sampling was 144 188 sec with the 22 g needle versus 140 120 sec with the 23 g needle (p = 0.99) for all operators pooled but four out of six operators showed shorter sampling times with a 22 g needle.conclusion : this experimental study shows that the use of a 23 g needle compared to a 22 g needle was not associated with significantly different procedure duration or defect size. |
atlantoaxial instability can result from congenital malformations, systemic inflammatory conditions, neoplasms, and spinal trauma. c1c2 stabilization can be challenging due to the complex anatomy of the c1 and c2 vertebrae. the proximity of neurovascular structures, the variability of vertebral artery anatomy, and the degree to which upper cervical pathology can distort normal anatomical structures calls for very precise placement of instrumentation to avoid surgical complications.1 2 3 4 various techniques have been developed to accomplish upper cervical fixation.5 6 7 posterior c1 lateral mass and c2 pedicle screw rod fixation, as described by harms and melcher, is a widely accepted, safe, and effective method to achieve fusion.8 9 10 11 traditionally, intraoperative fluoroscopy has been used to aid in instrumentation.12 13 however, just as surgical techniques have advanced through the years, imaging modalities have as well. three - dimensional intraoperative imaging with navigation has been implemented to help surmount some of the technical difficulties of screw placement in the upper cervical spine. these intraoperative tools increase the accuracy of screw placement in the spine and aid in the surgical decision making.14 15 16 17 studies have shown that o - arm usage allows for accurate screw placement in various levels of the cervical spine.14 18 19 20 when compared directly with conventional fluoroscopy, the o - arm has been shown to increase the accuracy of screw placement in the lower cervical spine.21 considering the unique challenges presented by surgically stabilizing the first two cervical vertebrae, we examined the surgical outcomes, radiation exposure, and accuracy of screw placement. consequently, the purpose of this study is to provide evidence that the use of intraoperative neuronavigation improves the safety and efficacy of surgical fixation in treating instability at the complex c1c2 region. seven patients (2 men, 5 women) with atlantoaxial instability underwent stabilization from february 2011 to january 2014. patients presented with a range of conditions leading to c1c2 instability including rheumatoid arthritis, odontoid fracture, c2 fracture, and ligamentous injury (fig. abbreviation : sd, standard deviation. a 43-year - old woman with rheumatoid arthritis, neck pain, and c1c2 instability. (a, b) postoperative computed tomography (ct) scans showing placement of c1 lateral mass screws. (d) lateral cervical x - ray demonstrating c1c2 fusion 12 months after surgery. from february 2011 to january 2014, all instances of atlantoaxial stabilization performed at our institution by a single surgeon were included in this study. the case series was approved by the institutional review board at our institution. in all seven cases, the radiologic evaluation was performed preoperatively and included plain x - rays in the lateral and anteroposterior planes as well as cervical computed tomographic (ct) scans and/or magnetic resonance imaging. operative data such as blood loss, complications, and operative time, defined as time from first incision to closure, was gathered from the anesthesia record. data is reported as averages standard deviation or standard error of measure. the patient 's head was placed in mayfield pins and aligned in a neutral position. lateral x - ray was obtained to assess the proper alignment and reduction. after the standard exposure was achieved, our institution uses the o - arm, a cone - beam ct scanner that, paired with the stealthstation surgical navigation system (medtronic, inc., minneapolis, minnesota, united states), allows for stereotactic intraoperative imaging and navigation in multiple planes. the images were transferred to the stealthstation navigation system, and the trajectories were planned in the c1 lateral mass and c2 pedicles. pilot holes were drilled bilaterally in c1 and c2, and o - arm navigation was used to maintain course. once the drilling was completed, a ball - tip probe was used to verify the integrity of the holes prior to final screw placement. the optimal screw length was calculated and the screws were placed under image guidance so that the appropriate positioning and good purchase were achieved. after screw placement, the screw trajectory was verified, and rods were placed into the screw heads and tightened. decortication was performed and the bone that was removed during the procedure was morselized and placed along the construct. the screw placement accuracy was determined from the postoperative ct scans that were analyzed by an independent neuroradiologist. screw placement was graded based on previously characterized grading systems for c1c3 lateral mass and c2 pedicle screws.3 22 briefly, screw position for lateral mass screws was defined as type i for ideal placement without cortical violation, type ii for acceptable placement with 50% of the diameter located within surrounding cortex and less than 1-mm protrusion from the anterior cortex, and type iii for unacceptable placement with clear violation of the transverse foramen or spinal canal.22 the medial and lateral displacement of the c2 pedicle screws was defined as previously described3 : grade 0, no deviation ; grade 1, deviation less than 2 mm ; grade 2, deviation more than 2 mm and less than 4 mm ; grade 3, deviation more than 4 mm. patients were followed clinically at increasing intervals after surgery with imaging to evaluate the fusion progression. no complications occurred during surgery, such as vertebral artery, nerve root, or spinal cord injury. the initial intraoperative ct was used to determine the proper cervical alignment of the patient ; no patients required repositioning prior to the surgery. the mean operative time was 188.7 minutes (range 155 to 213), and the mean blood loss was 271.4 ml (range 50 to 900). the patients received on average 39.0 mgy of radiation with use of the o - arm. a total of 30 screws were placed in the cervical spine : 14 screws (46.7%) in c1, 8 screws (26.7%) in c2, and 8 screws (26.7%) in c3. in three instances, the c2 anatomy was determined to be not conducive to screw placement, so c3 was used as the site of instrumentation instead. in one instance, it was deemed necessary to place the screws in each of the first three cervical vertebrae. a total of two pedicle screws (25%) were classified as grade 0 (table 3). two screws (25%), 4 screws (50%), and 0 screws (0%) were graded as 1, 2, and 3, respectively (table 3). the average deviation for all c2 pedicle screws was 1.5 mm, and no screws had cortical violation. the lateral mass screws placed in the first and/or third cervical vertebra had an average deviation of 2.1 mm. all but two were classified as type i or ideal placement (table 4). no screws required repositioning. note : grading of c1c3 lateral mass screws22 : type i, ideal placement without cortical violation ; type ii, acceptable placement with 50% of diameter located within surrounding cortex and 2 mm and 4 mm. the mean follow - up was 12 months for patients who underwent atlantoaxial fusion. at follow - up, only one patient showed motion on flexion extension films, but subsequent ct imaging demonstrated intact instrumentation and solid fusion. intraoperative ct combined with navigation is a useful technology that offers significant advantage in the operating room. it enables the surgeon to precisely track the trajectory of surgical instruments and can improve hardware placement. intraoperative imaging provides the advantage of capturing the patient 's anatomy in the surgical position, which is particularly helpful in cases with complex anatomy or where pathology distorts the normal anatomic structure, as can occur with c1c2 pathology. in fact, a mounting body of evidence has shown that intraoperative navigation can improve the accuracy of screw placement during spine surgery and the intraoperative anatomical localization and can reduce morbidity.23 24 although both the o - arm and conventional fluoroscopy are used intraoperatively, the o - arm has an advantage in that it produces a three - dimensional image. as such, it allows the surgical team to ensure that the patient 's vertebrae are aligned in the axial plane in addition to the lateral and oblique planes (as provided by fluoroscopy). this multidimensional visualization correlates with the intraoperative findings, which is particularly important because the simple act of turning the patient prone can produce a discrepancy between the preoperative ct imaging and the intraoperative surgical anatomy and alignment.25 one of the main concerns with screw placement in the superior cervical vertebrae is direct vertebral artery injury. lateral displacement of the screw can compromise the medial border of the transverse foramen. with the use of intraoperative navigation, the trajectory of each screw can be easily visualized to ensure the vertebra can safely accept a screw, the bony course is able to be maintained, and the surrounding structures including vertebral artery, nerve root, or spinal cord are not injured. the combination of the o - arm imaging and stealthstation navigation suite allows the surgeon to plan the trajectories in the complicated spinal anatomy. this tool can help navigate difficult cases, as mentioned by acosta, who reported that 24% of their screws could not have been safely placed without intraoperative imaging.26 in our experience, three patients who underwent treatment for atlantoaxial subluxation had screws placed in c3 instead of c2, because the c2 anatomy was deemed inappropriate for screw placement. this decision about the surgical approach would otherwise not have been made without intraoperative imaging and trajectory planning. as mentioned earlier, placing screws into the c1 and c2 vertebrae can be difficult, especially when inserting screws into the c2 pedicles. despite these difficulties, the pedicles are desired targets for screws in the c2 vertebrae because they offer much better biomechanical stability.27 28 29 30 relying only on conventional means, meta - analysis has shown a misplacement rate of c2 pedicle screws from 0.9 to 44.7%.31 32 33 34 35 misplacement in the c1 and c2 vertebrae can be particularly dangerous due to the potential for damage to the surrounding neurovasculature structures.3 navigation has been used to mitigate the difficulties of pedicle screw placement with positive results, showing a misplacement rate from 1 to 11.1%.15 17 19 20 35 36 37 van de kelft placed 1,922 pedicle screws in 353 patients at the thoracic, lumbar, and sacral levels with a 97.5% accuracy using the o - arm and stealthstation navigation.17 also using intraoperative neuronavigation, larson reported a 98.2% accuracy rate in 1,511 pedicle screws placed in adults.36 at the cervical spine, ishikawa noted that of 108 pedicle screws, 88.9% had no perforation, 8.3% had less than 2 mm of perforation, and 2.7% were between 2 and 4 mm outside of the bone.19 ling reported that 98% of their screws were placed in ideal positions in 21 patients treated with a harm 's construct or occipital cervical fusion using the o - arm, though 2 of the 20 placed in the c2 pedicle deviated by 1 mm.20 in our case series, 90% had less than 4 mm of deviation from the ideal trajectory. in our study, no screws required removal and replacement. the ability to correct screw placement if necessary in the operating room is advantageous and may prevent reoperation for anatomical misalignment and/or poorly placed screws. despite the benefits of utilizing o - arm technology, some potential drawbacks do exist. as with any form of ct, although the operating staff are able to leave the room and avoid virtually any radiation exposure,38 published numbers show that o - arm patients undergo a higher level of radiation (highest tissue dose = 40 mgy with two o - arm scans) than they would with conventional fluoroscopy (highest tissue dose = 6 mgy).39 tabaraee investigated the radiation dose to cadavers following exposure by either the c - arm or o - arm. the study found cadavers received higher doses of radiation with the use of the o - arm, even though the cadavers exposed to c - arm radiation also had a postoperative conventional ct scan.40 the risks of increased radiation exposure must be weighed with the benefits of utilizing the o - arm technology. one reported limitation of intraoperative neuronavigation includes the increased operative time involved to acquire intraoperative ct images and navigation registration. the mean operative times for atlantoaxial fusion vary greatly in the literature.25 41 42 yang compared c1 lateral mass and c2 pedicle screws placed with conventional fluoroscopy to iso - c three - dimensional navigation and found no significant difference in operative times.42 in our center, we can acquire data in 18 to 20 minutes from the time the o - arm is positioned and draped and the intraoperative ct is completed. this time is comparable to the time it takes us to set up and access images with biplanar fluoroscopy. further comparisons between intraoperative navigation and conventional methods for screw placement are necessary to determine if any benefit or detriment exists in operative times with newer technology. another problem that can arise from o - arm usage is the potential navigation errors. the navigation reference frame has to be attached to a fixed point on the patient, and any movement that dislodges the reference frame will result in a navigational error. oftentimes the reference frame is attached to the spinous process of c3 or another vertebra, which has the potential for slight movement during procedures. the operating team must take steps to ensure that the reference frame is fixed throughout the entire procedure. even with the reference frame in the right position, the o - arm is reported to have an inherent amount of error. oertel found a 2.8 1.9-degree difference between the angulation of the actual and virtual pedicle screws.43 when compared with actual dissection of human cadavers, santos discovered that the overall accuracy of the o - arm images in depicting pedicle screw placement in the thoracolumbar spine was only 73%.44 errors related to navigation may lead to inaccuracies during screw placement. our experience with c1c2 fusion shows that o - arm imaging and navigation can offer adequate screw placement accuracy without complications and with good surgical outcomes. this robust technology can assist in the placement of instrumentation during difficult procedures like atlantoaxial fixation. further investigation may include minimally invasive surgery using intraoperative navigation and performing a cost benefit analysis comparing the o - arm with other imaging modalities and navigation. | study design case series of seven patients. objective c2 stabilization can be challenging due to the complex anatomy of the upper cervical vertebrae. we describe seven cases of c1c2 fusion using intraoperative navigation to aid in the screw placement at the atlantoaxial (c1c2) junction. methods between 2011 and 2014, seven patients underwent posterior atlantoaxial fusion using intraoperative frameless stereotactic o - arm surgical imaging and stealthstation surgical navigation system (medtronic, inc., minneapolis, minnesota, united states). outcome measures included screw accuracy, neurologic status, radiation dosing, and surgical complications. results four patients had fusion at c1c2 only, and in the remaining three, fixation extended down to c3 due to anatomical considerations for screw placement recognized on intraoperative imaging. out of 30 screws placed, all demonstrated minimal divergence from desired placement in either c1 lateral mass, c2 pedicle, or c3 lateral mass. no neurovascular compromise was seen following the use of intraoperative guided screw placement. the average radiation dosing due to intraoperative imaging was 39.0 mgy. all patients were followed for a minimum of 12 months. all patients went on to solid fusion. conclusion c1c2 fusion using computed tomography - guided navigation is a safe and effective way to treat atlantoaxial instability. intraoperative neuronavigation allows for high accuracy of screw placement, limits complications by sparing injury to the critical structures in the upper cervical spine, and can help surgeons make intraoperative decisions regarding complex pathology. |
conservative therapy ranges from parenteral nutrition, gastric drainage, and antibiotics to interventional techniques such as implantation of covered stents and closure with clips. esophageal stenting or clipping of esophageal leaks often has to be combined with simultaneous external drainage 1 2. endoscopic vacuum therapy (evt) has been reported as a novel treatment option for esophageal leakage 3 4. most retrospective studies of the technology summarize heterogeneous cases of anastomotic insufficiency and spontaneous and iatrogenic perforation 5 6 7 8 9 10 11. we gained experience with evt since 2005 and the aim of this paper is to present our results in treatment of iatrogenic perforation of the esophagus in a series of 10 patients. between 2007 and 2014, 10 patients from our clinic were found to have iatrogenic perforation caused by endoscopic procedures (7 male/3 females), aged 28 to 82 years. since 2007, open pore drainage consisting of a gastric tube and open pore polyurethane foam (v.a.c., san antonio, usa ; endo - sponge, b. braun melsungen ag, melsungen, germany ; suprasorbcnp wundschaum, lohmann & rauscher gmbh & co. kg, neuwied, germany) was used for therapy. the foam fixed at the tip of the tube was placed solely by endoscopic means with a grasper (fig. 1 a, fig. 2 a). the open pore foam adheres to the tissue when connected with an electronic vacuum device (kci v.a.c. freedome, kci usa inc., san antonio, texas, usa, setting : 125 mmhg, continuous, intensity 10). intraluminal (il) and intracavitary (ic) variants of therapy have been described previously in detail 12. open pore polyurethane foam (pu) fixed at tip of drainage tube (t), endoscope (e), grasper (g), b open pore drainage. left : short polyurethane foam (pu) for intracavitary evt ; right : long pu for intraluminal evt ; endoscope (e) ; drainage tube (t). esophageal placement of open pore drainage (polyurethane foam [pu ], suture [s ], drainage tube [t ], grasper [g ]. b after 2 days of intracavitary evt (wound edges [we ] of perforation defect). c after 3 days of intraluminal evt (wound edges [we ] stick together). d 8 days after a 5-day treatment with evt (erosion pattern has disappeared and a tiny scar [sc ] is the residuum of the perforation defect [esophageal lumen { l } ]). otherwise, the open pore foam was placed directly intraluminally onto the epithelium of the esophagus covering the perforation area. contamination of the wound with gastric fluids and saliva was eliminated. wound secretion and edema b after application of vacuum suction, the esophageal lumen collapses around and with the open pore foam. for intraluminal evt, we used foam that measured of 1.5 to 2 cm in diameter and up to 12 cm in length. for intracavitary placement, the tube exited nasally and, if necessary, a nasolabial suture was placed to prevent dislocation. drainage was removed orally in the first 2 to 5 days simply by withdrawing the tube to allow endoscopic inspection of the inner wound. if the area of perforation was free of inflammation and granulation closed or covered the inner wound, the therapy was discontinued. when endoscopic control showed regular intracorporeal wound healing, a patient was started on a soft diet. after completion of vacuum therapy, follow - up endoscopies were performed to monitor the healing process. all perforations were diagnosed endoscopically in median time on the same day or within 24 hours after a perforation event. esophageal perforations were located from the cricopharyngeus (4 out of 10) to the esophagogastric junction (2 out of 10). eight patients were treated with intraluminal evt (il), one with intracavitary evt (ic) and one patient with both types of treatment (conversion from intracavitary to intraluminal therapy)(video 1). after removal of the foam, epithelial tissue that had come into contact with it during evt showed a typical pimpled erosion pattern (fig. 2 d). in some patients, we observed open pores obstructed partly by viscous saliva or slime. six of the 10 patients were ventilated during the treatment period, four of them only during the placement procedures. systemic antibiotics were given in eight cases but as shown in the table, there were no ventilation - associated morbidities. no dilation of defects no ventilation - associated problems surgical therapy of ulcus ventriculi bleeding clinically asymptomatic patient did not consent to follow - up endoscopy. in all patients treated with intraluminal evt, the wound edges were found to be closed on the first control endoscopy (fig. one asymptomatic patient did not consent to follow - up endoscopy. in all 10 patients (100 %), the perforation leakages were healed in a median of 5 (3 7) days after administration of evt. no extra endoscopic intervention (clip, stent) or other surgical procedures were necessary. the healing process was closely monitored by control endoscopy ; a second cycle of evt was not necessary in any of the patients. in contrast to anastomotic insufficiency, sudden transmural injury results in a localized inflammation in cases of early iatrogenic perforation. but if saliva, gastric, and choline secretions or infective fluids are brought into continuous contact with extraluminal tissue through the perforation due to the physiologic intrathoracic negative pressure, development of mediastinitis can be promoted. in our limited series, we were able to start evt immediately after diagnosis within 24 hours after a perforation event. recent studies with evt show a success rate of 85 % to 95 % for use of the approach to treat esophageal anastomotic insufficiencies and perforations in more than 120 patients 7 8 9 10 11 12. endoscopic stenting for sealing esophageal leaks is an established method with over 20 years of clinical experience. however, in six of our patients, perforations were located either ciropharyngeal or near the esophagogastric junction. in those locations, limitations of stent therapy have been described, due to dislocation, migration, persisting leakage and foreign body sensation 13. we suppose that stenting would not have been an optimal treatment in these cases. to date, two studies comparing stenting with evt both have suggested that evt has an advantage over use of stents in terms of outcomes for sealing esophageal leaks 9 10. few published reports exist about experience with closure of early - detected esophageal perforation use the over - the - scope - clip method. the procedure seems to be successful but also appears to be associated with considerable risks 14. we suspect that our short treatment duration (median 5 days) and success rate of 100 % also depends on the parameters of the negative pressure we use. two technical prerequisites exist for evt : open pore drainage and negative pressure. based on our experience with the technology, we believe that it is important to ensure the best suction effect and adherence of the foam within tissue -specific parameters. permanent suction works with multiple open pores, even if some of them may be obstructed. since we began performing evt on the esophagus in 2006, we have continued to successfully use the same electronic device. we assume that these modern devices may be not suitable for evt in the esophagus. in 2008 intraluminal vacuum application resulted in occlusion of the esophagus lumen and contraction of adjacent tissue. the procedure was easy to perform and feasible in nine of 10 patients with iatrogenic perforation. we have observed that swallowing viscous saliva can result in blockage of open pores in the foam. our observations suggest, however, that the blockage was not complete and that drainage was still effective. however, blockage of the foam may increase risk that the vacuum will be ineffective. therefore, we recommend routine endoscopic examination of patients undergoing evt 3 days after the procedure or in case of any irregular symptoms. surgical monitoring following evt is the same as after any other superficial surgical procedure : inspection of the wound is necessary to supervise wound healing. six of the patients in our series were ventilated during the treatment period to avoid swallowing of saliva and to ensure effective suction of tissue. this might be one of the disadvantages of evt, yet in our series, we did not observe any comorbidities caused by the short periods of ventilation. because the duration of treatment with evt was short, all of the patients in our series received parenteral nutrition. in cases of anastomotic insufficiency, because the treatment duration was significantly longer, we use adapted feeding tubes 5. because none of the patients required external thoracic drainage, it appears that by combining evacuation and a sealing effect, evt resulted in sufficient drainage in all cases. the advantages of evt are its easy application throughout the whole length of the esophagus from the cricopharyngeus to the esophagogastric junction and the ability to simultaneously close and promote internal drainage in one endoscopic step. the treatment period is short and no complications have been observed. our study suggests that evt will play an important role in endoscopic management of all types of esophageal perforation. | background and study aims : endoscopic vacuum therapy (evt) has been reported as a novel treatment option for esophageal leakage. we present our results in the treatment of iatrogenic perforation with evt in a case series of 10 patients. patients and methods : an open pore polyurethane drainage was placed either intracavitary through the perforation defect or intraluminal covering the defect zone. application of vacuum suction with an electronic device (continuous negative pressure, 125 mmhg) resulted in defect closure and internal drainage. results : esophageal perforations were located from the cricopharyngeus (4/10) to the esophagogastric junction (2/10). evt was feasible in all patients. eight patients were treated with intraluminal evt, one with intracavitary evt, and one with both types of treatments. all perforations (100 %) were healed in within a median of (3 7) days. no stenosis occurred, no complications were observed, and no additional operative treatment was necessary. conclusions : our study suggests that intraluminal evt will play an important role in endoscopic management of esophageal perforation. |
enteric fever is endemic in developing countries such as india where sanitary conditions and potable water is not accessible to all. patients typically present at the end of the first week after the onset of symptoms with fever, influenza - like symptoms with a dull frontal headache, malaise, etc., but with few physical signs. a coated tongue, tender abdomen, hepatomegaly, and splenomegaly are common. blanching erythematous maculopapular lesions commonly called rose spots are reported in 530% of cases. if untreated or where the implicated organism is resistant to the treatment being given, there may be seeding of salmonellae in various organs of the body., such as liver, splenic, and anterior abdominal wall, but bilateral breast abscesses are a rare presentation. we present here a rare case of a young nonlactating, immunocompetent female who presented with bilateral breast abscesses due to salmonella typhi. a 29-year - old female presented to the surgical outpatient department (opd) at our hospital with the complaint of a lump in both the breasts for 7 days associated with pain. the lumps were felt around the nipple, tender to touch with the overlying skin warm. fever was high grade with a peak of 39c and touched baseline only with the intake of antipyretics. two weeks prior to the development of breast pain and lump, the patient had visited the medical outpatient department with the complaint of fever. she was empirically started on amoxicillin plus clavulanic acid 625 mg bid for 7 days, but did not respond and continued to have fever. although the blood culture had grown salmonella typhi and the isolate was resistant to ampicillin, she did not visit the hospital for follow - up and so the treatment could not be modified accordingly. she was a mother of two children, but at presentation she was neither pregnant nor lactating. on examination, there was a centrally located lump in the left breast measuring around 8 8 cm and another lump in her right breast just below the areola measuring around 3 3 cm. a palpable axillary lymph node around 2 cm in size was present on the left side. pus was drained from both the sides, which on culture grew non - lactose - fermenting colonies. the organism was identified as salmonella typhi by biochemical reactions and agglutination test with specific antisera. the isolate was sensitive to chloramphenicol, cefotaxime, ciprofloxacin, and co - trimoxazole but resistant to ampicillin and nalidixic acid. the patient had a report of a positive blood culture for salmonella typhi from our laboratory 2 weeks prior to the presentation, which had a similar sensitivity pattern. the widal test showed a negative result with the titer of to=160 and th and ah=40. her hemogram revealed normal values [haemoglobin (hb) : 10.4 g / dl, total leukocyte count (tlc) : 10,000/mm, differential leukocyte count (dlc) ; neutrophils : 80% ; lymphocytes : 18% ; eosinophils : 2% ]. biochemistry investigations were also within normal limits except that her serum glutamic oxaloacetic transaminase (sgot) and serum glutamic pyruvic transaminase (sgpt) were raised with values of 96.59 and 113.78 u / l, respectively. the patient was started on oral ciprofloxacin 500 mg bid for 2 weeks to which she responded favorably with regression of the lesion. the stool and urine cultures were negative for salmonella typhi during presentation and subsequent visits. it is capable of forming abscesses in various organs such as liver, subcutaneous tissue, muscles, and skin. the possible causes may be infective bile from carriers, hematogenous spread from distant site, and lymphatic spread from gastrointestinal tract. bilateral breast abscesses due to salmonella typhi the occurrence of breast abscesses in patients with typhoid has been shown to be around 0.3% by klose and sebening (1930) and 0.5% by pezinski (1937) in a study of 1 196 cases of typhoid over a period of 2 years. in females, other authors have also reported similar cases of unilateral breast abscess due to salmonella typhi. razeq and edelstein had isolated salmonella landweisser and salmonella serogroup b in breast abscess, respectively. in a recently published study from kuwait, a very rare serotype, salmonella enterica serotype poona, was isolated from a case of breast abscess that was associated with erythema nodosum. on analyzing the available literature on breast abscesses due to salmonella spp., we found that most of the patients were immunocompetent females between the ages of 23 and 45 years. however, no common predisposing factors could be elucidated. in the present case, besides fever, which could also be a feature of breast abscess, there was no other clinical feature of typhoid. the patient had been initially started on amoxicillin in combination with clavulanic acid, but as the strain was resistant to it she did not show any response. the patient did not turn up for the scheduled follow - up after the blood culture and sensitivity report, leading to the progression of the disease and localization in the breast tissue. it was only when she visited the surgical opd 2 weeks later with the complaint of bilateral breast abscesses, the pus could be drained and was sent for culture and sensitivity testing. the patient responded to oral ciprofloxacin with reduced swelling of the abscesses and was afebrile thereafter. any breast abscess in a nonlactating female with a history of typhoid fever in the recent past and no other predisposing factors must be evaluated, keeping the possibility of a salmonella breast abscess in mind. also, a combination of medical and surgical management helps in such a case when supported by a microbiological culture and sensitivity report. | focal infection is an uncommon complication of salmonella septicemia, particularly in immunocompetent patients. the localization of salmonella infection to breast tissue is regarded as a rare event. we report a case of bilateral breast abscesses due to salmonella enterica serotype typhi in a nonlactating female and highlight the fact that salmonella spp. should be included in differential diagnosis of abscesses in individuals coming from endemic areas with the history of recent typhoid fever and should be treated accordingly. |
patients with cancer have several risk factors for wernicke encephalopathy, including the use of chemotherapeutic agents, nutritional imbalance, performance of bypass surgery, and disease progression due to fast - growing tumor cells. the classical symptoms of wernicke encephalopathy are global confusion, ataxia, and ophthalmoplegia, but only one third of patients present with all of these classical symptoms. korsakoff syndrome, which is characterized by a chronic, striking loss of working memory, often follows or accompanies wernicke encephalopathy. although wernicke encephalopathy can be resolved by rapid repletion of thiamine, korsakoff syndrome can not be resolved. this is the first reported case of wernicke - korsakoff syndrome in a patient with primary peritoneal cancer. a 76-year - old woman with primary peritoneal cancer and multiple lymph node metastases received 6 cycles of paclitaxel and carboplatin. six months after her last chemotherapy cycle, she was admitted to the hospital because of sustained nausea and general weakness, which she had experienced for several months. chest and abdominal - pelvic computed tomography scans showed that peritoneal metastasis and lymph node metastasis had progressed. four days after admission, she developed disorientation and dizziness, which were followed by ataxia and diplopia. her laboratory findings were as follows : white blood cell count 6.59 10/l, hemoglobin 12.0 g / dl, hematocrit 35.8%, blood platelet count 230 10/l, serum total protein 6.1 g / dl, albumin 3.3 g / dl, glucose 111 mg / dl, aspartate aminotransferase 21 iu, alanine aminotransferase 14 iu, blood urea nitrogen 9.0 mg / dl, creatinine 0.15 mg / dl, na 139 mm, k 4.6 mm, cl 107 mm, and ca 0.98 mg / dl. magnetic resonance imaging (mri) of the brain showed hyperintensity in the mammillary body, medial aspects of both halves of the thalamus, cerebral aqueduct, tectal plate, and dorsal medulla on t2 fluid - attenuated inversion recovery (flair) images (fig. the patient 's serum thiamine level was 2.0 g / dl (normal range 2.07.2 g / dl). thiamine (100 mg) was urgently administered intravenously, and ataxia and diplopia improved 2 days after thiamine injection. fifteen days after the thiamine injection, she had recovered from her symptoms but displayed anterograde amnesia and disorientation with respect to time. a follow - up mri scan of the brain showed improvement in the abnormal lesion (fig. the classic clinical triad of wernicke encephalopathy comprises nystagmus and ophthalmoplegia, changes in mental status, and ataxia. predisposing factors for wernicke encephalopathy include a staple diet of polished rice, chronic alcohol abuse, chronic malnutrition, gastrointestinal surgical procedures, recurrent vomiting, chronic diarrhea, cancer, use of chemotherapeutic agents, systemic disease, magnesium depletion, the use of chemical compounds (including drugs), and unbalanced nutrition. thiamine is an important coenzyme in intermediated carbohydrate metabolism, lipid metabolism, the production of amino acids, and the production of glucose - derived neurotransmitters. thiamine deficiency mainly results in changes to the macroscopic and microscopic findings of brain lesions. notably, 100% of patients with wernicke encephalopathy show typical histopathological changes in the medial dorsal thalamic nucleus. additionally, one third of patients show histopathological changes in the superior vermis of the cerebellum. several reports have shown that some cases of thiamine deficiency are related to cancer [3, 4, 5, 6, 7 ]. causes of thiamine deficiency in cancer patients include the use of thiamine by fast - growing neoplastic cells, poor dietary intake (which is related to appetite and nausea), significant malabsorption, and the use of specific types of chemotherapeutic agents. in the present case, thiamine deficiency was probably related to fast - growing neoplastic cells and poor dietary intake. the patient recovered completely from ophthalmoplegia after a few hours, from ataxia after a few days, and from mental changes after 23 weeks of thiamine replacement therapy. however, irreversible brain damage or death occurs in approximately 20% of cases of wernicke encephalopathy, and the chronic irreversible form of the encephalopathy (korsakoff syndrome) is associated with a survival rate of approximately 85%. in the present case, however, she developed new symptoms of anterograde amnesia and disorientation with respect to time. many reports have described cases of wernicke encephalopathy in patients with cancer, but korsakoff syndrome has not been reported in such patients. indeed, this is the first reported case of wernicke - korsakoff syndrome in a patient with primary peritoneal cancer. in addition, it is difficult to diagnose wernicke encephalopathy if the patient has developed neurologic symptoms related to his or her cancer, because neurologic symptoms in patients with cancer can be related to many factors, such as electrolyte imbalance, cerebral metastasis, chemotherapeutic treatments, infection, and opioid overdose. to diagnose wernicke encephalopathy, wernicke encephalopathy is confirmed by measuring blood thiamine concentration or red blood cell transketolase activity. in our case, the blood thiamine level was 2.0 g / dl, which is the lower limit of normal. an isocratic high - performance liquid chromatography method for thiamine, thiamine phosphate, and thiamine diphosphate in human erythrocytes is suitable for diagnosing wernicke encephalopathy, but this test was not available. mri is a valuable tool for confirming a diagnosis of wernicke encephalopathy ; it has a high specificity of 93% and a sensitivity of 53%. in the present case, a follow - up mri scan showed improvement in the previously abnormal findings, but korsakoff syndrome remained. primary peritoneal cancer is a rare cancer with a behavior that is very similar to epithelial ovarian cancer. the development of wernicke encephalopathy has been reported in patients with gastrointestinal tract cancer, head and neck cancer, lung cancer, breast cancer, lymphoma, and leukemia ; however, wernicke - korsakoff syndrome has not been reported previously in patients with primary cancers [3, 4, 5, 7, 8, 9 ]. the case reported here involved wernicke - korsakoff syndrome in a patient with primary peritoneal cancer. therefore, clinicians should consider wernicke encephalopathy in cancer patients with risk factors for this disease, such as nutritional imbalance due to long - term parenteral nutritional supply, use of chemotherapeutic agents, and fast - growing tumor cells. clinicians should remember that wernicke encephalopathy occurs in patients with unbalanced nutrition, as well as in those with subacute or chronic disease that increases metabolism. | wernicke encephalopathy is a disease that constitutes a medical emergency, but one that can be reversed with thiamine repletion if it is recognized early. patients with cancer have a high risk of wernicke encephalopathy because of malnutrition, the use of chemotherapeutic agents, and disease progression. korsakoff syndrome can follow or accompany wernicke encephalopathy. although patients can recover from wernicke encephalopathy via rapid repletion of thiamine, few patients recover from korsakoff syndrome. here, the case of a 76-year - old female patient who had primary peritoneal cancer and developed wernicke - korsakoff syndrome as a result of prolonged nutritional imbalance and fast - growing tumor cells is reported. the patient 's neurologic symptoms improved, but she did not recover from the psychiatric effects of the disease. |
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