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for many years, laser assisted in situ keratomileusis surgery (lasik) has been widely accepted as a procedure to correct visual errors. since the first femtosecond laser was introduced in 2002, all - laser lasik using a femtosecond laser has been gaining popularity. the clinical safety and benefits of femtosecond lasers for corneal flap creation have been reported, including ; predictable flap thicknesses, better astigmatic neutrality, and decreased epithelial injuries, when compared with mechanical microckeratomes.1 the use of femtosecond lasers has become more common with the recent release of five femtosecond laser systems : the intralase fs (abbot medical optics, inc, santa ana, ca, usa) ; femtec (20/10 perfectvision optische gerate gmbh, heidelberg, germany) ; visumax femtosecond laser system (carl zeiss meditec, jena, germany) ; femto ldv (ziemer ophthalmic systems ag, port, switzerland) ; and the wavelight fs200 femtosecond laser (alcon laboratories inc, fort worth, tx, usa). at our clinic, diffuse lamellar keratitis (dlk) is a sterile inflammation underneath the flap, known as one of the complications after lasik surgery.2 dlk is characterized by a diffuse, white, granular infiltrate that appears within a few days after lasik surgery.2 the etiology of dlk is still unknown ; however, it seems to have multiple causes, such as povidone - iodine solutions, hemorrhage, carboxymethylcellulose drops, bacterial endotoxins, epithelial defects caused by the surgery, debris from the microkeratome blade, atopy, marking pens, toxic chemicals, meibomian secretions, and traumatic flap dislocation or subsequent surgical repair.212 patients with dlk may claim other symptoms, such as irritation, blurred vision, pain, or sensitivity to light. however, some patients do not experience any symptoms in the early stages. when the symptoms of dlk are recognized, immediate treatment is necessary in order to prevent its progression. worsening cases may lead to scarring, stromal melt, haze, hyperopic shift, irregular astigmatism, or permanent visual loss.13 dlk is usually self - limiting or treated with topical medications, such as steroids, in its early stages. for severe cases, flap lifting and irrigation are needed.13 although there are numerous studies of dlk caused by mechanical microkeratomes, there are only a few reports of dlk incidence caused by femtosecond lasers. furthermore, we found no reports comparing the incidence of dlk between femtosecond lasers. in this study, we classified patients with dlk into five stages and compared two femtosecond lasers, the femto ldv and the intralase fs60, which are different in terms of their laser system specification, for the incidence of dlk. we also focused on examining the preoperative and postoperative visual acuity and evaluated whether dlk incidence would affect visual acuity after lasik surgery. in this prospective study, 818 consecutive eyes of 409 patients that had lasik using femto ldv or intralase fs60, at shinagawa lasik center, tokyo, japan in july 2010 were studied. during the period, 514 eyes of 257 patients (101 men and 156 women) with a mean age of 31.40 years 6.95 (range : 18 to 59 years) had bilateral lasik with the ziemer femto ldv (the femto ldv group), while 304 eyes of 152 patients (100 men and 52 women) with a mean age of 30.61 years 7.05 (range : 18 to 54 years) had bilateral lasik with intralase fs60 (the intralase fs60 group). all the surgical and pre / postoperative procedures were thoroughly explained to all participating patients and the patients provided written, informed consent before undergoing the procedure. all patients had a complete preoperative ophthalmologic examination that included uncorrected distance visual acuity (udva), corrected distance visual acuity (cdva), manifest refraction spherical equivalent (mrse), keratometry ; (ark-530a ; nidek co ltd, gamagori, japan), corneal topography (tms-4 ; tomey corp, nagoya, japan), specular microscopy ; (noncon robo fa-3509 ; konan medical inc, hyogo, japan), wavefront analysis (kr-9000pw ; topcon corp, tokyo, japan), measurement of the pupil before and after mydriasis, higher order aberration (opd - scan ii ; nidek co ltd), anterior eye segment (pentacam ; oculus optikgeriite gmbh, wetzlar, germany), corneal thickness (contact pachymeter sp-3000 ; tomey corp), and slitlamp biomicroscopy (carl zeiss meditec, jena, germany). when we noted irregular corneal topography during the pentacam and tms-4 examinations, an orbscan (bausch and lomb inc, rochester, ny, usa) image was obtained for additional evaluation. lasik was not performed for patients with keratoconus or an insufficient residual bed depth (of less than 300 m) after preoperative examination. the raster energy and side - cut energy were set at 100 nj for the femto ldv. for the intralase fs60, a bed energy level of 1.0 j, and a side - cut energy level of 0.80 j were set. after the flap was created with either laser, the flap was lifted and excimer laser ablation was performed using the wavefront optimized allegretto wave eye - q 400 hz (alcon laboratories inc, fort worth, tx, usa). follow - up visits were scheduled at 1 day, 1 week, and 3 months postsurgery. a slit - lamp biomicroscopic examination of the corneal flap and anterior segment of the patient s eyes was also carried out. on the day of surgery, a complete set of medications was given to all patients. patients used dexamethasone sodium m - sulfobenzoate 0.1% (dex ophthalmic solution 0.1% ; nitto medic co ltd, toyama, japan) once every hour, moxifloxacin hydrochloride 0.5% (vigamox ; alcon laboratories inc.) once every hour, then sodium hyaluronate 0.1% (hyalonsan ophthalmic solution 0.1% ; nitto medic co ltd) once every hour, and oxybuprocaine hydrochloride (benoxyl ; santen pharmaceutical co ltd, osaka, japan) for pain, if needed. the day after surgery, patients used dexamethasone sodium m - sulfobenzoate 0.1% five times a day, moxifloxacin hydrochloride 0.5% five times a day, and sodium hyaluronate 0.1% five times a day. the regimen of all eye drops was finished after 1 week. at their day-1 postoperative follow - up visit, patients were divided into four stages according to the widely known dlk classification system.13 the system was based on the extent of centripetal migration of any inflammatory cells. in the first stage of dlk, white granular cells appear at the laminar flap edge or hinge (stage 1). at stage 2, the visual axis is involved as the white granular cells develop at the center of the flap. at stage 3, the inflammation has progressed and spread over the flap, and there is an aggregation of denser white cells clumped over the visual axis. at stage 4, severe inflammation has resulted in collagenase release, stromal melt, and permanent scarring, associated with visual loss.13 in addition, we have adopted a method to divide stage 1 into two substages ; stage 1a and stage 1b, in order to define any postoperative dlk more precisely (figure 1). at stage 1a, dlk is barely seen on the peripheral part of the flap, whereas at stage 1b, any dlk is confined to the flap edge. when dlk was diagnosed in our clinic, immediate treatment was provided, and frequent follow - ups were scheduled to prevent further progression. the treatment procedure for dlk varied by stage. frequent use of a topical steroid drop (dexamethasone sodium m - sulfobenzoate 0.1%) was required five times a day for patients with stage 1, and every hour for patients with stage 2. however, if their symptoms were close to stage 3 or if the potential risk for developing stage 3 was expected, flap irrigation was considered in order to prevent the development of stage 3 and/or 4. for patients with stage 3 and/or 4 dlk, it was necessary to lift the flap and rinse the bed and the interface of the flap with a balanced salt solution (otsuka pharmaceutical co ltd, tokyo, japan) and a topical steroid. for patients with stage 2 or more, systemic medication (h2 blocker and prednisolone 30 mg per day) was prescribed for 3 days. in this study the data was analyzed with statistical software ; statview j 5.0 ; (abacus concepts, inc, piscataway, nj, usa). the mann whitney u - test was used to evaluate the statistical significance of the dlk incidence of each stage associated with these two femtosecond lasers, and also to evaluate the visual and refractive outcomes of patients with or without dlk. in this prospective study, 818 consecutive eyes of 409 patients that had lasik using femto ldv or intralase fs60, at shinagawa lasik center, tokyo, japan in july 2010 were studied. during the period, 514 eyes of 257 patients (101 men and 156 women) with a mean age of 31.40 years 6.95 (range : 18 to 59 years) had bilateral lasik with the ziemer femto ldv (the femto ldv group), while 304 eyes of 152 patients (100 men and 52 women) with a mean age of 30.61 years 7.05 (range : 18 to 54 years) had bilateral lasik with intralase fs60 (the intralase fs60 group). all the surgical and pre / postoperative procedures were thoroughly explained to all participating patients and the patients provided written, informed consent before undergoing the procedure. all patients had a complete preoperative ophthalmologic examination that included uncorrected distance visual acuity (udva), corrected distance visual acuity (cdva), manifest refraction spherical equivalent (mrse), keratometry ; (ark-530a ; nidek co ltd, gamagori, japan), corneal topography (tms-4 ; tomey corp, nagoya, japan), specular microscopy ; (noncon robo fa-3509 ; konan medical inc, hyogo, japan), wavefront analysis (kr-9000pw ; topcon corp, tokyo, japan), measurement of the pupil before and after mydriasis, higher order aberration (opd - scan ii ; nidek co ltd), anterior eye segment (pentacam ; oculus optikgeriite gmbh, wetzlar, germany), corneal thickness (contact pachymeter sp-3000 ; tomey corp), and slitlamp biomicroscopy (carl zeiss meditec, jena, germany). when we noted irregular corneal topography during the pentacam and tms-4 examinations, an orbscan (bausch and lomb inc, rochester, ny, usa) image was obtained for additional evaluation. lasik was not performed for patients with keratoconus or an insufficient residual bed depth (of less than 300 m) after preoperative examination. the raster energy and side - cut energy were set at 100 nj for the femto ldv. for the intralase fs60, a bed energy level of 1.0 j, and a side - cut energy level of 0.80 j were set. after the flap was created with either laser, the flap was lifted and excimer laser ablation was performed using the wavefront optimized allegretto wave eye - q 400 hz (alcon laboratories inc, fort worth, tx, usa). follow - up visits were scheduled at 1 day, 1 week, and 3 months postsurgery. a slit - lamp biomicroscopic examination of the corneal flap and anterior segment of the patient s eyes was also carried out. on the day of surgery, a complete set of medications was given to all patients. patients used dexamethasone sodium m - sulfobenzoate 0.1% (dex ophthalmic solution 0.1% ; nitto medic co ltd, toyama, japan) once every hour, moxifloxacin hydrochloride 0.5% (vigamox ; alcon laboratories inc.) once every hour, then sodium hyaluronate 0.1% (hyalonsan ophthalmic solution 0.1% ; nitto medic co ltd) once every hour, and oxybuprocaine hydrochloride (benoxyl ; santen pharmaceutical co ltd, osaka, japan) for pain, if needed. the day after surgery, patients used dexamethasone sodium m - sulfobenzoate 0.1% five times a day, moxifloxacin hydrochloride 0.5% five times a day, and sodium hyaluronate 0.1% five times a day. the regimen of all eye drops was finished after 1 week. at their day-1 postoperative follow - up visit, patients were divided into four stages according to the widely known dlk classification system.13 the system was based on the extent of centripetal migration of any inflammatory cells. in the first stage of dlk, white granular cells appear at the laminar flap edge or hinge (stage 1). at stage 2, the visual axis is involved as the white granular cells develop at the center of the flap. at stage 3, the inflammation has progressed and spread over the flap, and there is an aggregation of denser white cells clumped over the visual axis. at stage 4, severe inflammation has resulted in collagenase release, stromal melt, and permanent scarring, associated with visual loss.13 in addition, we have adopted a method to divide stage 1 into two substages ; stage 1a and stage 1b, in order to define any postoperative dlk more precisely (figure 1). at stage 1a, dlk is barely seen on the peripheral part of the flap, whereas at stage 1b, any dlk is confined to the flap edge. when dlk was diagnosed in our clinic, immediate treatment was provided, and frequent follow - ups were scheduled to prevent further progression. the treatment procedure for dlk varied by stage. frequent use of a topical steroid drop (dexamethasone sodium m - sulfobenzoate 0.1%) was required five times a day for patients with stage 1, and every hour for patients with stage 2. however, if their symptoms were close to stage 3 or if the potential risk for developing stage 3 was expected, flap irrigation was considered in order to prevent the development of stage 3 and/or 4. for patients with stage 3 and/or 4 dlk, it was necessary to lift the flap and rinse the bed and the interface of the flap with a balanced salt solution (otsuka pharmaceutical co ltd, tokyo, japan) and a topical steroid. for patients with stage 2 or more, systemic medication (h2 blocker and prednisolone 30 mg per day) was prescribed for 3 days. in this study, patients without any symptoms of dlk were classified as stage 0. the data was analyzed with statistical software ; statview j 5.0 ; (abacus concepts, inc, piscataway, nj, usa). the mann whitney u - test was used to evaluate the statistical significance of the dlk incidence of each stage associated with these two femtosecond lasers, and also to evaluate the visual and refractive outcomes of patients with or without dlk. all 409 patients (818 eyes) completed their scheduled day-1 postoperative follow - up visit, while 353 patients (706 eyes) returned for their week-1 postoperative examination, and 132 patients (264 eyes) returned for their 3-month postoperative examination. the patients who did nt return to our clinic had postoperative examinations at affiliated clinics. table 1 and figure 2 demonstrate the percentages of dlk incidence for both groups. at postoperative day 1, in the femto ldv group (totaling 514 eyes), dlk was found in 42 (8.17%) of the eyes, by slit - lamp examination ; of these, 35 eyes (6.81%) developed stage 1a and seven eyes (1.36%) developed stage 1b. the number of eyes with no dlk was 472 (91.83%) in the ldv group. in intralase fs60 group (totaling 304 eyes), 114 (37.5%) of the eyes had dlk ; of these, 96 eyes (31.58%) developed stage 1a and 18 eyes (5.92%) developed stage 1b. in this group as well, none of the eyes had dlk stage 2, 3, or 4. as a consequence, 190 eyes (62.5%) did not have dlk in intralase fs60 group. the dlk incidence rate was 8.17% in the femto ldv group and was 37.5% in the intralase fs60 group. there were significant differences observed between these two femtosecond lasers when the occurrence of dlk was compared at stage 1a and 1b, at postoperative day 1 (p < 0.0001 [mann whitney s u - test ]). dexamethasone sodium m - sulfobenzoate 0.1%, five times a day, was provided to the patients with dlk stage 1a and 1b. at 1 week the udva, cdva, and mrse were also examined, preoperatively and at 1 day, 1 week, and 3 months postop. table 2 demonstrates that the visual and refractive outcomes of this study were excellent in both groups. the safety index (mean postoperative cdva divided by mean preoperative cdva) at 1 day, 1 week, and 3 months postop was 0.97, 1.04, and 1.05, respectively in the femto ldv group and was 0.99, 1.06, and 1.08 (at 1 day, 1 week, and 3 months postop) in the intralase fs60 group. the efficacy index (mean postoperative udva divided by mean preoperative cdva) at 1 day, 1 week, and 3 months postop was 0.95, 1.01, and 1.02, respectively in the femto ldv group and was 0.95, 1.03, and 1.04, respectively in the intralase fs60 group. additionally, to evaluate whether the dlk incidence affected the visual outcomes, the udva, cdva, and mrse for patients with dlk stage 0 and stage 1 were also statistically analyzed (table 3) : patients who did nt have dlk symptoms were classified as stage 0 ; whereas patients with dlk stage 1a and/or 1b were classified as stage 1. for the femto ldv group, there were no significant differences between stages 0 and 1 at any preoperative and postoperative day. however, for the intralase group, significant differences were found between the two stages in regards to the cdva preoperatively, and udva and cdva at postoperative day 1 and week 1. when comparing the visual outcomes between the dlk and non - dlk groups for both femtosecond lasers, significant differences were observed only the for intralase group at certain postoperative days. however, as the visual and refractive results for both femtosecond laser groups were very good, these groups were not clinically different. moreover, the results of this analysis indicated that the occurrence of stage 1 dlk did not affect visual acuity. the safety and effectiveness of flap creation using a mechanical microkeratome and femtosecond lasers have been reported.14 microkeratomes used to be the primary means for flap creation in lasik surgery. however, the femtosecond laser is currently taking over that role and is becoming more common. in particular, one of the distinctive differences between the femtosecond laser and the microkeratome is the precision with which the flap is created.15 several studies have found that femtosecond lasers can create more predictable1,15 and smoother flaps than most mechanical microkeratomes.16 with the microkeratome, there is an inclination to have a disparity in the thickness when comparing the central and peripheral areas.15 of special note, one study comparing the microkeratome with three femtosecond lasers the intralase, visumax, and femto ldv reported that the femto ldv system was the most predictable with respect to flap thickness (intended versus measured).15 in regard to complications caused by the microkeratome and/or femtosecond laser, microkeratomes induced significantly more epithelial defects, such as buttonholes or decentered flaps, intraoperatively while femtosecond lasers had significantly higher incidences of gas breakthrough and postoperative dlk.17,18 the eyes with femtosecond - created flaps had a lower incidence of dry eye than the ones with microkeratome - created flaps.19 suction loss, which induces buttonholes and incomplete flaps, was reported when using a microkeratome.20 in the case of suction loss, immediate reattempts to cut the flap using femtosecond lasers after intraoperative suction loss did not affect the visual acuities or the refractive outcomes.21 regarding visual and refractive outcomes of lasik surgery, significantly better postoperative ucva and manifest refractive results were reported with femtosecond lasers.22 from previous studies, the femtosecond laser is generally considered to be superior to the microkeratome.15,16,19,22 the effect of the energy level used by femtosecond lasers on tissue has been investigated in previous studies. the effects on the corneal stromal cells by different femtosecond laser energy levels were analyzed, and researchers found that higher energy levels triggered greater cell death.23 moreover, greater inflammatory cell infiltration in the cornea has been observed with higher femtosecond laser energy levels.23 the possibility that lower - energy femtosecond lasers induce less tissue damage and inflammation was considered. other lasik complications, such as rainbow glare and transient light sensitivity, are also considered to be caused by femtosecond lasers.24,25 those studies indicate that lower pulse energy is one of the key parameters to reduce complication rates. dlk is defined as the formation of noninfectious corneal interlamellar inflammation.2 according to the literature, there are two major factors that induce inflammation during corneal flap creation using femtosecond lasers.23 one of the factors is that direct energy ablation by the femtosecond laser increases the number of necrotized cells in the stroma.23 femtosecond laser assisted flap creation is likely to contribute to greater inflammation, especially since higher energy levels result in greater stromal cell death.23 additionally, the other study reported that lower side - cut and angle energy eliminated the interface inflammation and also reported that use of higher laser energies during flap creation caused greater damage.26 applying this remarkable clinical experience to our study, the lower pulse energy level of the ldv system could have resulted in reduced stromal cell death and inflammation, and we hypothesize that the incidence of dlk may have been related to the energy level of the femtosecond laser. the femto ldv has different technical characteristics that distinguish it from conventional femtosecond lasers, such as the intralase fs60. the fundamental differences are the beam delivery and cutting process.27 a comparison of the technical features of these two femtosecond lasers is described in table 4. the intralase fs60 is characterized as having a high pulse energy and low pulse frequency, as it delivers pulse energies in the order of 1 j, with a repetition rate of 60 khz. on the other hand, the femto ldv creates a corneal flap with a 1,000 khz pulse rate and uses pulse energy levels in the nanojoule range. in this study, a 1 j mean raster energy and 0.8 j mean side - cut energy were used in the intralase system, and 100 nj energy was used by the femto ldv to create the stromal bed and side cut. these differences in system parameters affect the nature of the process.27 the low pulse energies of the femto ldv produce small and tightly overlapped dissection spots. this overlapped beam delivery makes a stromal flap cut without tissue bridges, and thus, the flap dissection is completed easily, with minimum damage to the corneal tissue.27 on the other hand, spots from intralase lasers do not overlap during the photodisruption process. thus two adjoining laser spots can be distant from each other,27 which leads to tissue bridges or uncut areas on the cornea, and this can create traction forces caused by dissection of the corneal lamellar, when using a blunt instrument to lift the flap. several articles have proved the superiority of the femto ldv and the intralase, so there are advantages to using these femtosecond lasers for lasik surgery. a study reported that flap dimensions and refractive results of lasik using the femto ldv were predictable, with an acceptable rate of complications and that no eyes had significant losses in cdva at 6 months postop.28 likewise, the predictability and accuracy of the intralase for flap creation, when compared to a microkeratome, were reported.29 in our previous studies, use of the femto ldv and intralase fs60 were evaluated in the lasik treatments of patients with corneal opacity.30 although these lasers do not have clinically significant differences in terms of visual outcomes, using the femto ldv, with its lower pulse energy, on areas of corneal opacity creates less gas, which reduces the chance of gas breakthrough in the subepithelial tissue.30 in our study analyzing the incidence of dlk following the use of these two lasers, we showed that the femto ldv induced significantly lower rates at postoperative day 1. however, no dlk of stage 2 or higher was found in either ldv or intralase groups at postoperative day 1. the clinical results of our experiment confirm that both the femto ldv and intralase fs60 were effective surgical tools for lasik surgery. consistent with previous studies,1, 15, 29 our findings indicate that both femtosecond lasers excel at lasik flap creation. in terms of dlk incidence, however, both lasers were excellent, and no clinical differences were found between them since both femtosecond lasers did not induce dlk stage 2 or higher, and any dlk presences cleared within 1 week. | purposeto compare the incidence of diffuse lamellar keratitis (dlk) after laser in situ keratomileusis (lasik) with flap creation using the femto ldv and intralase fs60 femtosecond lasers.0methodsa total of 818 consecutive myopic eyes had lasik performed using either femto ldv or intralase fs60 for flap creation. the same excimer laser, the allegretto wave eye - q laser, was used for correcting refractive errors for all patients. in the preoperative examination, uncorrected distance visual acuity, corrected distance visual acuity, and manifest refraction spherical equivalent were measured. at the postop examination, the same examinations were performed along with a slit - lamp biomicroscopic examination, and patients with dlk were classified into stages. for the statistical analysis of the dlk occurrence rate and the visual and refractive outcomes, the mann - whitney s u - test was used.resultsin the femto ldv group with 514 eyes, 42 (8.17%) had dlk. in the intralase fs60 group with 304 eyes, 114 (37.5%) had dlk. there was a statistically significant difference in the dlk incidence rate between these groups (p < 0.0001). both groups had excellent visual and refractive outcomes. although low levels of dlk were observed for both groups, they did not affect visual acuity.conclusionwhile there were significantly fewer incidences of low level dlk when using femto ldv, neither femtosecond laser induced high levels of dlk, and any postoperative dlk cleared up within 1 week. therefore, both lasers provide excellent results, with no clinical differences, and both excel at flap creation for lasik. |
the changes in oncologic therapies are achieving longer survivals of patients with many tumour types. this increase in survival together with the better sensitivity of imaging techniques can be associated with an increase in the incidence of metastasis on the longterm, what can render diagnostic and therapeutic challenges in the near future. in this sense it is interesting to note the recent increase in the number of reported cases of metastasis to unusual sites, like the pancreas or the spleen with most series published in the last 5 years. the objective of the present report is to review the cases of pancreatic and splenic metastasis diagnosed at a single center between 1998 and 2010 and to comment on diagnostic and therapeutic aspects in these patients. the review of the electronic database files of the surgical pathology department of the hospital fundacin jimnez daz, a tertiary hospital attending over 400.000 people in an urban area of madrid (spain), has rendered 156 pancreatectomy specimens (both whipple procedure and partial resections) and 345 splenectomy ones in the last 12 years. among these cases we have found 3 cases of pancreatic metastasis (1.9% of the specimens) and 5 cases of splenic metastasis (1.5% of the specimens). table 1summary of the caseslocationsex / ageprimarytherapy of the primary tumourtime since diagnosis of the primarypresenting symptomspreoperative diagnosis of the metastasistherapyoutcomehead of the pancreasfemale 58malignant melanoma, clark level 4 of the legsurgery : resection and lymphadenectomy (0/18 lymph nodes affected by tumour)8 yearsjaundice and painpancreatic adenocarcinomacephalic duodeno - pancreatectomy (whipple procedure)good evolution surgery ; dead of widespread disease 8 months after surgeryhead and body of pancreasfemale 71renal clear cell carcinoma (left side) pt1a n0 m0surgery : radical nephrectomy15 monthsincidental finding during surveillanceneuroendocrine pancreatic tumorssubtotal pancreatectomygood evolution after surgery ; alive and disease free 8 months after surgerytail of the pancreasfemale 53renal clear cell carcinoma pt1b n0 m0surgery : radical nephrectomy9 yearsincidental finding during surveillanceneuroendocrine pancreatic tumorresection of the tail of the pancreas and splenectomygood evolution after surgery ; alive and disease free 12 months after surgeryspleenfemale 75ovarian serous poorly differentiated carcinomasurgery : omentectomy, splenectomy, histerectomy and bilateral oophorectomysynchronous with the primaryinvolvement found during surgery of the primarynonesplenectomy and chemotherapydead of disease 9 months after surgeryspleenfemale 74ovarian high grade carcinomasurgery : omentectomy, splenectomy, histerectomy and bilateral oophorectomysynchronous with the primaryinvolvement found during surgery of the primarynonesplenectomy and chemotherapydead of disease 15 months after surgeryspleenfemale 77high grade papillary serous carcinoma of probable endometrial originsurgery : splenectomy, histerectomy and bilateral oophorectomysynchronous with the primaryinvolvement found during surgery of the primarynonesplenectomy chemotherapydead of disease 42 months after surgeryspleenmale 72squamous cell carcinoma of the lungchemotherapy with good response14 monthsincidental finding during surveillancemetastasis vs. lymphomasplenectomydead of disease 12 months after surgeryspleenmale 63enteroid well - differentiated adenocarcinoma of the large intestine t3n1m0surgery and chemotherapy6 yearsimaging findingmetastasissplenectomy with resection of isolated hepatic metastasisdead of disease 8 months after splenectomy all the cases of pancreatic metastasis in our series were metachronous with the primary tumour and the time elapsed between therapy of the primary tumour and recurrence was fairly long (8 and 9 years in two patients). it is worth noting that in the three cases the pancreas was the only location of the tumour recurrence and imaging studies revealed no lesions elsewhere in any of the cases. two patients were asymptomatic, while the third had jaundice and pain that led to a preoperative diagnosis of pancreatic adenocarcinoma. in neither case there was any clinical suspicion of tumour recurrence and the other two cases were diagnosed of neuroendocrine primary pancreatic tumours, despite the biochemical assays failed to show any abnormal hormone secretion. all the patients were surgically treated with a radical or partial pancreatectomy depending on the location of the mass. it is interesting to note that in one patient the lesions were multiple (2 in the head of the pancreas and 1 in the body), while the other was a solitary nodule in the pancreatic tail (figure 1). two of the patients (both with metastasis from a clear cell renal carcinoma ; figure 2) are doing well and remain well and disease free, almost one year after diagnosis. the third one, who had metastasis from a malignant melanoma, operated 8 years before and who had no previous recurrence of the tumour, died of widespread disease 8 months after whipple procedure (figure 3). however, at the time of pancreatectomy both imaging neither studies nor clinical examination revealed metastases elsewhere and they developed later in the course of the disease and eventually killed the patient. figure 1computed tomography scan showing a nodule in the tail of the pancreas. figure 2a) hematoxylin - eosin stained medium power image of the pancreatic metastasis from a clear cell carcinoma. note the presence of pancreatic endocrine normal cells at the periphery of the metastatic nodule (h e, 200 ; immunohistochemistry for cd10). a) hematoxylin - eosin stained medium power image of the pancreatic metastasis from a clear cell carcinoma. note the presence of pancreatic endocrine normal cells at the periphery of the metastatic nodule (h e, 200 ; immunohistochemistry for cd10). figure 3a) hematoxylin - eosin stained medium power image of the pancreatic metastasis from malignant melanoma. a) hematoxylin - eosin stained medium power image of the pancreatic metastasis from malignant melanoma. three of our cases were synchronous with the primary tumours, located two in the ovaries and one in the endometrium. all these women had widespread disease in the abdominal cavity at the time of the primary surgery and died of disease on the short - medium term, despite aggressive resection and postoperative chemotherapy. only two of the splenic metastases in our series were metachronous with the primary tumour. one was a metastasis from an squamous cell carcinoma of the lung, treated with chemotherapy 14 months earlier, that recurred as an isolated splenic mass (figure 4). the second was a m. jesscolonic adenocarcinoma that recurred 6 years after colectomy as hepatic and splenic metastasis (figure 5). in both cases surgery was deemed necessary both to confirm diagnosis and to improve the patients outcome. however, despite surgical therapy and postoperative chemotherapy both patients died within one year. figure 4hematoxylin - eosin stained medium power image of the splenic metastasis from squamous cell carcinoma. note normal splenic tissue in the left bottom of the image (he, 200). hematoxylin - eosin stained medium power image of the splenic metastasis from squamous cell carcinoma. note normal splenic tissue in the left bottom of the image (he, 200). figure 5gross photograph showing the large splenic nodule with a smaller satellite one, corresponding histologically to metastases from an enteroid adenocarcinoma of the large intestine. gross photograph showing the large splenic nodule with a smaller satellite one, corresponding histologically to metastases from an enteroid adenocarcinoma of the large intestine. metastasis to unusual sites are being increasingly reported, a fact that can be in part explained by : i) the longer survival of oncologic patients related to improved therapies ; ii) the better standardized oncological follow - up after primary therapy ; iii) the improved sensitivity of the imaging techniques ; iv) and a more aggressive surgical and oncological attitude in cases with metastasis. in this report we have reviewed the metastasis to the pancreas and the spleen in a tertiary hospital in madrid (spain). our experience seems to fit the most recently reported reviews on this topic. in a recent editorial by zerbi. the authors emphasize the dramatic increase in the incidence of pancreatic metastasis since 2008. according to their review pancreatic metastasis represent 2% of all pancreatic tumours (1.9% in our series). prognosis after resection can be related to the tumour type and the therapeutic options after it. the most frequent type is renal clear cell carcinoma (rcc), as already described by other authors. it is followed by colorectal cancer, melanoma and sarcoma, but in the literature there are reports of metastasis from almost any tumour type, including lobular breast carcinoma and even as the first symptom of an occult primary. one peculiar feature of rcc is the long latency between diagnosis of the primary tumour and appearance of the pancreatic metastasis. in one of our patients 9 years elapsed between radical nephrectomy and pancreatic metastasis. in this time the patient remained disease - free with no further therapy. although metastasis from rcc have been described as hypervascular with imaging techniques and both our cases showed this imaging feature, preoperative diagnosis in our patients was neuroendocrine pancreatic tumour, a much more frequent occurrence. in this sense, we should take into account the possibility of metastatic spread from a primary tumour, independently of the time elapsed since the first malignant lesion. there has been much controversy in the literature regarding resection for pancreatic metastasis. in a recent systematic review of over 400 patients with pancreatic metastasis of rcc, a 5-year survival over 70% was found after resection ; this figure seems to justify metastasectomy in patients able to tolerate pancreatic resection, for it equates the results obtained after hepatic or lung metastasectomy, a therapeutic approach which is now widely accepted in the oncologic literature. in this sense it would be useless to perform fine needle aspiration cytology of pancreatic masses suspicious of metastasis, as therapy would be surgical, mainly when there is no metastatic spread elsewhere. most authors recommend standard radical pancreatic resection, as the experience of some authors employing atypical more conservative resections describes a 29% rate of local recurrences. however it is worth noting that, to date, there are no reports comparing surgery to the modern biological therapies that are increasingly used in renal carcinomas. if diagnosis of metastasis could be confirmed, the chance to employ these new drugs could avoid surgery in responding cases and reduce the important morbidity and mortality related to pancreatic surgery. surgical results seem not to be changed by multiplicity of the metastatic nodules within the pancreas, a fact which was considered rare, but reaches 39% of the cases in some recent reviews. one of our patients showed this pattern of involvement with three metastatic nodules that obliged to perform a subtotal pancreatectomy from which she recovered uneventfully. metastatic malignant melanoma behaves very poorly according to most reports and prognosis is bad, with a median survival time of 14 months, even when metastasis were limited to the pancreas. however, the lack of alternative therapeutic options for this tumour and the possible symptoms associated with it can justify surgery as a valid alternative, with palliative or curative intention, as happened in our case. the experience with other tumours is very scarce, but recent reviews indicate surgery is justified for isolated pancreatic metastasis from colonic tumours and sarcomas of the gastrointestinal stromal tumour (gist) type, but not for lung tumours. however, in these cases long survival is only achieved when postoperative chemotherapy or molecular targeted therapy are employed. the incidence of splenic involvement in autopsy series from oncologic patients has ranged from 27%. however, less than 150 cases of isolated splenic metastasis undergoing splenectomy have been reported to date. a recent review by gatenby. as it is also the case in pancreatic metastasis, metastasis to the spleen can appear long after diagnosis and therapy for the primary tumour. in our series we have found two cases originating in the ovaries and one in the endometrium, both metachronous with the primary tumour, and two metachronous ones from the lung and the colon. the lung seems an infrequent origin for splenic metastasis and less than 10 cases have been published in the world literature ; the interval between diagnosis and metastastic involvement has widely varied from 0 to 8 years. the literature indicates prognosis is better for metachronous metastasis over synchronous ones and also for metastasis originating in the colon and the ovary. splenectomy seems to be clearly indicated for solitary lesions, even when suspicious of metastasis, due to the low incidence of complications of this type of surgery and also to the need to confirm diagnosis to decide further therapy. we should not forget that malignant lymphoma can also arise as a solitary splenic nodule and imaging does not allow differential diagnosis of the histology of the lesions. in our series prognosis of the patients with splenic metastasis has been uniformly poor. nevertheless, it seems an improved outcome may be seen in patients for whom there were effective adjuvant chemotherapeutic options, low probability of other metastatic disease and less aggressive tumour biology. in this report we review the cases of isolated pancreatic and splenic metastases in our tertiary hospital. our experience seems to fit to the reported literature regarding tumour types and outcome of the patients. according to the present literature metastasectomy | we have reviewed the electronic biopsies database files of the department of surgical pathology, fundacin jimnez daz in madrid (spain). in this time period (19982010) we have found 3 pancreatic metastasis and 5 splenic metastasis. two of the pancreatic metastases were originated in clear cell renal cell carcinomas. the last pancreatic metastasis was from a malignant cutaneous melanoma diagnosed and treated 8 years before. as for splenic metastasis, three of them were diagnosed during the abdominal surgery for primary therapy of the tumour (2 ovaries and one endometrium), while the remaining 2 corresponded to metastasis from a lung primary diagnosed 1 year before and a colonic primary diagnosed 6 years before. the patients with splenic metastasis died on the short term with progression of the disease despite resection of the splenic lesions, while the patients with pancreatic metastasis have survived longer. |
survey 1 sampled 1 natural village from each of 5 different towns in hengxian county. (towns in china have several administrative villages, the basic level of administration, which often comprise > 1 natural villages) each sampled natural village had 500600 residents, all of whom were eligible for survey participation. the same procedures were followed in survey 2, but only 3 towns were sampled. dozens of households were surveyed from each administrative village, and all household members > 5 years of age were eligible for participation ; 200 villagers were selected in each administrative village. in each survey, a single fecal sample from each participant was used to prepare a kato - katz thick smear to determine the presence of helminths and count c. sinensis eggs. participants were categorized as 70 years of age. we defined infection intensity as the geometric mean number of eggs per gram of feces (epg) in the egg - positive survey participants ; lg(epg + 1) transformation was used to calculate infection intensity in all survey participants. we used student t test and analysis of variance to compare infection intensities between sexes and among surveys, respectively ; fisher least significant difference test was used to compare between individual surveys. logarithmic correlation was used to explore the relationship between prevalence and infection intensity in age groups. survey 1 had 2,623 participants ; surveys 2 and 3 had 1,748 and 3,437, respectively (table 1). during surveys 13, the standardized prevalences were 19.7%, 30.5%, and 46.5%, respectively, for clonorchiasis and 86.3%, 25.1%, and 7.0%, respectively, for sths. the corresponding standardized prevalences were 69.8%, 13.8%, and 0.5% for roundworm infections ; 55.7%, 11.2%, and 1.4% for whipworm infections ; and 24.3%, 6.3%, and 5.3% for hookworm infections. # 48 children 1 type of helminth. during surveys 13, the standardized prevalences of clonorchiasis were 10.4%, 20.8%, and 29.2%, respectively, among females and 28.5%, 39.6%, and 62.9%, respectively, among males. the corresponding standardized prevalences of sths were 86.2%, 29.5%, and 9.4% among females and 86.3%, 20.9%, and 4.7% among males. the corresponding standardized prevalences of roundworm, whipworm, and hookworm infections among males and females showed a decreasing trend similar to that for the total sths. in surveys 13, clonorchiasis prevalence increased fairly steadily by age group to middle age, after which, prevalence continued to increase with age in survey 1 and decreased with age in surveys 2 and 3 (figure 1, panel a). sths prevalence changed irregularly by age group in surveys 1 and 2 but increased gradually by age group in survey 3 (figure 1, panel b). prevalences of roundworm and whipworm infections usually peaked among children 514 years of age ; hookworm infections peaked in middle - aged age groups in surveys 1 and 2 and in older age groups in survey 3. prevalence of clonorchiasis (a) and soil - transmitted helminthiases (sths) (b) among sex and age groups during 3 parasitic disease surveys, hengxian county, china, 19892011. green indicates the first survey (1989) ; purple indicates the second survey (2002) ; red indicates the third survey (2011). in c. sinensis egg positive participants, the geometric mean numbers of epg were 367.3 epg, 833.3 epg, and 942.9 epg in surveys 13, respectively (f = 36.2, p<0.001) (table 2). differences were significant between survey 1 and surveys 2 and 3 (p<0.001) but not between survey 2 and survey 3 (p = 0.210). in surveys 13, geometric mean numbers of epg were 241.7 epg, 364.9 epg, and 405.1 epg, respectively, among females (f = 3.3, p<0.05) and 419.8 epg, 1,199.7 epg, and 1,363.2 epg, respectively, among males (f = 43.6, p<0.001). among all participants, the geometric mean numbers of epg were 1.6 epg, 6.3 epg, and 38.2 epg during surveys 13, respectively (f = 522.9, p<0.001) ; differences between surveys were all significant (p<0.001). in surveys 13, geometric mean numbers of epg were 0.6 epg, 2.2 epg, and 6.8 epg, respectively, among females (f = 137.6, p<0.001) and 3.1 epg, 13.5 epg, and 171.5 epg, respectively, among males (f = 474.4, p<0.001). among egg - positive participants and all participants, infection intensities were all significantly lower among females than males, and infection intensities increased as ages increased, reaching a peak in middle - aged persons and then decreasing in older persons (figure 2). infection intensity of clonorchiasis among persons in different age groups during 3 parasitic disease surveys, hengxian county, china, 19892011. green indicates the first survey (1989) ; purple indicates the second survey (2002) ; red indicates the third survey (2011). in surveys 13, clonorchiasis prevalence in different age groups was significantly correlated with the corresponding infection intensity in terms of the geometric mean number of epg after logarithmic transformation (p<0.01). determination coefficients (r) were 0.709, 0.891, and 0.748 in surveys 13, respectively, when only egg - positive participants were included in the analysis ; corresponding r were 0.901, 0.980, and 0.997, respectively, for all participants. analysis of data from 3 parasitic disease surveys conducted in hengxian county over the last 22 years showed substantial decreases in the trend of sths prevalence and substantial increases in the patterns of clonorchiasis prevalence and infection intensity. these findings indicate a transitioning pattern of the most prominent parasitic disease changing from sths to clonorchiasis. distribution patterns of prevalence and infection intensity among sex and age groups showed that men have been most affected by increases in c. sinensis transmission and infection. thus, control measures and health education programs are urgently needed, especially among men, to reduce c. sinensis disease and transmission. this trend may partly be explained by massive health education programs and chemotherapy for sths, which were implemented in schools in china after the first national parasitic disease survey (8). in addition, over the last 30 years, rapid economic development has occurred in china, resulting in increased availability of safe water and establishment of sanitary lavatories for improved hygiene (9). furthermore, the increased use of chemical fertilizer has reduced the use of human feces as fertilizer (a source of helminth transmission) (10). however, economic development has also promoted development of aquaculture and made it economically possible for more residents to include freshwater fish in their diets, a source of c. sinensis metacercariae when consumed raw (11). clonorchiasis is a neglected parasitic disease in china : no nationwide intervention or control programs have been implemented to reduce the caseload (12). a third national survey is expected to be conducted beginning in 2014, which will provide updated data regarding the national status of clonorchiasis and an impetus for public health control / intervention and education programs. a national clonorchiasis surveillance system should also be established to gather up - to - date information and inform public health policymakers. | during 19892011, three parasitic disease surveys were conducted in hengxian county, china, where soil - transmitted helminthiases and foodborne clonorchiasis are endemic. we compared the data and found that the prevalence of helminthiases decreased and the prevalence and intensity of clonorchiasis increased over time, especially among men. clonorchiasis control / intervention measures are urgently needed in this area. |
isolated coracoid process fracture is a rare injury.1 furthermore, acute subscapularis tear is infrequent condition and often related with trauma rather than degenerative process.2 in our case, fracture of the coracoid process was associated with subscapularis tear without any shoulder dislocation. there was only one case report of isolated coracoid process fracture with acute subscapularis tear which was described as an unstable shoulder.1 a 61 year old male driver presented with right shoulder pain due to trauma which occurred 2 days ago. the patient fell from 2 meter height (stair case) with the shoulder in abduction and then hit the ground directly impacting the anterior shoulder. he had no history of shoulder trauma previously or any other shoulder disease. at initial radiologic evaluation, a displaced coracoid process fracture was identified on the axillary lateral view [figure 1 ]. it was displaced about 2.5 cm inferiorly without any other shoulder girdle fracture or dislocation [figure 2 ]. it was a midprocess level fracture, a type ii injury as per the classification of eyres. and ogawa.34 from the coracoid fracture classification point of view, conservative treatment could be sufficient. however, at physical examination, the patient revealed a positive belly press test and internal rotation lag sign, although there was mild pain that limited complete examination. for further evaluation, magnetic resonance arthrography (mra) of the shoulder was performed and complete and retracted tear of the subscapular tendon was identified [figure 3 ]. no evidence of shoulder dislocation such as an anteroinferior labral tear or a hill - sachs lesion was seen. x - ray (r) shoulder joint axillary view showing coracoid process fracture fragment (white arrow) three - dimensional computed tomography reconstruction image showing of displaced coracoid process fracture preoperative magnetic resonance arthrography axial image showing complete subscapularis tear (white arrow) during surgery, ruptured full thickness subscapularis tendon was easily identified and reducible to the lesser tuberosity [figure 4 ]. however, as the coracoid process fracture fragment was too small and fragile to fix, we excised the coracoid process bone fragment and reattached the remaining conjoined tendon (short head of bicep brachii, coracobrachialis) and pectoralis minor tendon to the pectoralis major. intraoperative clinical photograph showing ruptured subscapularis tendon and easily reducible to footprint postoperatively, the patient wore internal rotation brace for 6 weeks, after which, the stretching exercises were started. at 3 months after injury, 6 month after the surgery, the patient showed a negative belly press test and a negative internal rotation lag sign. he recovered to show normal range of motion and no other symptoms affecting activities of daily living with a constant score of 89. followup magnetic resonance imaging (mri) at 6 months showed healed subscapularis tendon without any atrophic changes [figure 5 ]. coracoid process fracture is an uncommon injury, occurring in about 2 - 5% of all scapular fractures. it is often associated with other shoulder suspensory complex injuries such as acromioclavicular injuries, clavicular fractures, shoulder dislocations, etc.3 it is recommended that a coracoid process fracture at the base of the process may need surgical intervention because it disturbs the coracoclavicular ligament and results in unstable connection between the scapula and the clavicle. on the other hand, coracoid process fractures distal to the coracoclavicular ligament can be treated conservatively.34 in our case, isolated coracoid process fracture distal to the coracoclavicular ligament was combined with acute subscapularis tear without any dislocation. there has been only one similar previous report but it was described as an unstable shoulder.1 the mechanism of this type of injury has been described as acute shoulder external rotation and extension trauma which results in shoulder dislocation. however, there was no dislocation or instability in our case contrary to the previous report. we hypothesize that there could be impact between the coracoid process and the lesser tuberosity of the humerus, which leads to subscapularis tear but without shoulder dislocation. surgical interventions are decided based on the site of the fracure.34 in our case, coracoid process fracture itself could be treated conservatively since the fracture involved midportion of the coracoid process and did not disturb the coracoclavicular ligaments. however, our clinical evaluation suggested weakness of subscapularis. therefore, the patient was taken up for surgery for repairing the isolated full thickness subscapularis tear which seemed to be acute. it is known that acute rotator cuff tears should be treated within 3 weeks and if the treatment is delayed, it is hard to have functional recovery.5 during surgery, coracoid process fractured fragment was fragile and hence excised and reattachment of the conjoined tendon and pectoralis minor to the pectoralis major was done, which resulted in a good functional recovery without any deficits. it is important to see for associated injuries not only within the bony structure but also amongst the surrounding soft tissues such as the rotator cuff and the labrum. if proper diagnosis and appropriate treatment is not promptly initiated then full recovery of shoulder function becomes difficult. mri is beneficial so that we should not miss associated soft tissue injuries along with coracoid fracture. hence we recommend using mri or mra without hesitation for coracoid process fractures with internal rotation weakness and hypothesize new collision type injury between lesser tuberosity and coracoid process in high energy fall down trauma. | coracoid process fracture is an uncommon injury and can be easily missed. an associated acute subscapularis tear is still rare. herein, we describe a 61 year old male who fell from a 2 meter height (stair case) and presented with isolated coracoid process fracture with acute subscapularis tear without dislocation of (r) shoulder joint. the plain x - rays, ct scan and mr arthrography comprised the diagnosis. he was operated upon with reattachment of subscapularis to lesser tuberosity and conjoint tendons to pectoralis major. at 6 mo followup he had good range of motion and his mri revealed complete healing. |
it is well known that the incidence of gastric carcinoma is higher for men than for women. although the incidence of gastric carcinoma varies geographically, men are more commonly affected.1 several lines of evidence support this lower incidence of gastric carcinoma in women than in men. some investigators have indicated that there are no differences in survival by sex, whereas others found better survival for women.2 - 4 few studies are available concerning the relationship between sex and gastric carcinoma survival. also, little is known about the clinicopathological features of female gastric carcinoma (fgc) patients. in the present study, we aimed to clarify the magnitude of the gender difference in gastric carcinoma survival and the prognostic factors in women with gastric carcinoma who underwent curative resection. from 1995 to 2005, 2701 patients with gastric carcinoma were treated curatively by the department of surgery, chonnam national university hospital. of these, information on the patient 's age, gender, operative type, tumor size, tumor location, macroscopic appearance, depth of invasion, histologic type, presence of lymph node invasion, and stage at initial diagnosis was obtained from the hospital records. the 6 american joint committee on cancer tnm staging system was used for pathologic staging.5 histological evaluation was performed according to the japanese general rules for gastric cancer study in surgery and pathology.6 the survival rates of the patients were calculated by using the kaplan - meier method and the relative prognostic importance of the parameters was investigated by using the cox proportional hazards model. the chi - square test was used to evaluate the statistical significance of differences, and p values less than 0.05 were considered significant. from 1995 to 2005, 2701 patients with gastric carcinoma were treated curatively by the department of surgery, chonnam national university hospital. of these, information on the patient 's age, gender, operative type, tumor size, tumor location, macroscopic appearance, depth of invasion, histologic type, presence of lymph node invasion, and stage at initial diagnosis was obtained from the hospital records. the 6 american joint committee on cancer tnm staging system was used for pathologic staging.5 histological evaluation was performed according to the japanese general rules for gastric cancer study in surgery and pathology.6 the survival rates of the patients were calculated by using the kaplan - meier method and the relative prognostic importance of the parameters was investigated by using the cox proportional hazards model. the chi - square test was used to evaluate the statistical significance of differences, and p values less than 0.05 were considered significant. table 1 summarizes the clinicopathologic findings of the 940 (34.8%) fgc patients and 1,761 (65.2%) male gastric carcinoma (mgc) patients with curative resection. there was a significant difference in mean age between the fgc patients (55.8 years) and the mgc group (57.4 years) (p0.05). the lower third of the stomach was the most common site of gastric carcinoma in both groups. the most common type of advanced gastric carcinoma in the fgc patients was the ulcerating infiltrative type (386/603, 64.1%). there was no significant difference in macroscopic appearance in early and advanced gastric carcinoma between the two groups. using the ptnm system, 348 fgc patients were classified as pt1, 154 as pt2, 410 as pt3, and 28 as pt4. according to the grade of anaplasia, 133 tumors were well - differentiated, 184 were moderately differentiated, 441 were poorly differentiated, 48 were mucinous, and 102 were signet ring cell carcinomas. significantly more fgc patients had a poorly differentiated histology and more mgc patients had a well - differentiated histology (p<0.001). a total of 525 fgc patients were pn0 and 415 had lymph node metastasis. by disease stage, 444, 194, 255, and 47 fgc patients were stage i, ii, iii, and iv, respectively. the fgc and mgc patients had similar distributions with respect to depth of invasion, nodal involvement, and tumor stage at the initial diagnosis. of the fgc patients, 638 (67.9%) were classified as either stage i or ii at initial diagnosis. tumor size, depth of invasion, tumor location, operative type, tumor stage at the initial diagnosis, and the presence of lymph node invasion were significant prognostic factors in the univariate analysis (table 2). the multivariate analysis showed that three factors were independent, statistically significant parameters associated with survival : serosal invasion (risk ratio, 2.485 ; 95% confidence interval [ci ], 1.554 - 3.792 ; p<0.001), lymph node metastasis (risk ratio, 2.837 ; 95% ci, 1.871 - 4.302 ; p<0.001), and operative type (risk ratio, 0.554 ; 95% ci, 0.853 - 8.915 ; p<0.01) (table 3). the overall 5-year survival rate of the fgc patients was significantly higher than that for the mgc patients (53.4% vs. 47.6% ; p=0.010) (fig. the overall 5-year survival rate of the early fgc patients was significantly higher than that of the mgc patients (94.3% vs. 86.1% ; p=0.046) (fig. the overall 5-year survival rates of advanced fgc and mgc patients with curative resection did not differ significantly (41.63% vs. 37.4% ; p=0.077) (fig. although many studies of the prognostic factors in patients with gastric carcinoma have been reported, only a few have examined the prognosis of fgc patients. therefore, we analyzed the clinicopathologic features of fgc patients undergoing curative resection and their surgical outcomes. the incidence of gastric carcinoma is known to be higher for men than for women.1,7 - 9 chandanos and lagergren reported that the incidence of gastric carcinoma shows a male dominance, with a male - to - female ratio of about 2:1. they suggested that the male dominance in the incidence of gastric carcinoma can at least be partly explained by a protective effect of estrogen in women.10 in agreement with their result, the male - to - female ratio in the present study was 1.9:1. poorly differentiated histology was more common in fgc patients than in mgc patients, as in yu and zhao's11 report. however, the use of histologic differentiation as a prognostic factor is another controversial subject, although several studies have examined the prognostic relevance of histologic grade in patients with gastric carcinoma. furthermore, our study showed that histologic differentiation had no statistical significance as a prognostic factor. traditionally, the depth of invasion and the presence or absence of lymph node metastasis are the most important clinicopathologic factors influencing the prognosis of patients with gastric carcinoma.12 - 14 in the present study, multivariate analysis showed that three factors were independent, statistically significant parameters associated with survival : serosal invasion, lymph node metastasis, and operative type. lymph node metastasis is thought to be an important prognostic factor in carcinoma of the stomach. in this study, bonezziti.2 found that female patients with negative nodes at pt1 or pt2 had better survival. bando.15 also reported that sex should be taken into account as well as clinicopathological variables related to lymph node metastases when determining appropriate therapy for early gastric carcinoma. schafmayer.16 reported that the extent of lymphadenectomy influenced long - time survival in female patients with gastric carcinoma. in gastric carcinoma, the depth of wall invasion is another important prognostic factor in addition to lymph node metastasis. moriguchi.12 and adachi.14 demonstrated that the depth of wall invasion provides useful prognostic information in patients with gastric carcinoma. some studies have suggested that the extent of gastric resection was a significant independent predictor of survival in patients with node - positive gastric carcinoma. kim.17 found that patients who had a distal gastrectomy showed a significantly better long - term prognosis than did patients who underwent total gastrectomy. they interpreted their results in terms of indications for total gastrectomy and the relative risks for more distant and extensive lymph node metastasis. several studies have reported a better prognosis for women than for men.2,3,7 similar to these results, we observed a positive significant effect of female gender on survival outcome in our study. our results showed that the overall 5-year survival rate of the fgc patients was significantly higher than that for mgc patients (53.4% vs. 47.6% ; p=0.010). when fgc patients were divided into those with early and those with advanced carcinoma, the overall 5-year survival rate of the early fgc patients was significantly higher than that of the mgc patients (94.3% vs. 86.1% ; p=0.0462). however, the overall 5-year survival rates of advanced fgc and mgc patients with curative resection did not differ statistically (41.63% vs. 37.4% ; p=0.0766) in this study. this suggests that early detection is important to achieve better survival rates. with regard to long - term survival, schafmayer.16 reported that no significant difference could be shown between men and women. however, splenectomy had a significant effect on long - term survival between the two groups. when the spleen was preserved, women showed a significantly improved survival rate compared with men with preserved spleens. therefore, those authors proposed that gender differences should be taken into account when analyzing the long - term data of oncological patients.16 kim.8 proposed that sex hormones such as estrogens contribute to the survival differences between men and women with gastric carcinoma. in conclusion, serosal invasion, lymph node metastasis, and type of operation were statistically significant parameters associated with survival. also, we found that the overall 5-year survival rate of fgc patients with curative resection (53.4%) was higher than that of mgc patients. in advanced cases, however, no significant difference was observed in the overall 5-year rate between the fgc and mgc patients. therefore, early detection is more important for improving the prognosis of female patients with gastric cancer than for male patients. | little is known about the clinicopathological features of female gastric carcinoma (fgc) patients. we compared the clinicopathologic features and outcomes of fgc patients with curative resection with those of male gastric carcinoma (mgc) patients. we reviewed the hospital records of 940 fgc patients between 1986 and 2005 at chonnam national university hospital. multivariate analysis showed that presence of serosal invasion, lymph node metastasis, and operative type were significant prognostic factors for survival of fgc patients with curative resection. furthermore, the overall 5-year survival rate of fgc patients with curative resection (53.4%) was higher than that of mgc patients (47.6%, p0.05). therefore, serosal invasion, lymph node metastasis, and type of operation were statistically significant parameters associated with survival. early detection is more important for improving the prognosis of female patients with gastric cancer than for male patients. |
rehabilitation therapy includes various treatments (eg, physical therapy, occupational therapy, speech pathology) aimed to increase patient functional independence, prevent further loss of function, and improve quality of life for individuals with disabilities. restorative nursing care is a program available in nursing homes that helps maintain patient progress made during rehabilitation therapy and enhances functional capacity. according to the us department of veterans affairs / department of defense (va / dod) clinical practice guideline for the management of adult stroke rehabilitation care, effective multidisciplinary rehabilitation therapy and restorative nursing care improve stroke survivors functional recovery.1 in addition to its 134 community living centers (clcs), the va also places veteran enrollees needing post - acute institutional care in private community nursing homes (cnhs) under a per diem contractual program. nursing home contracts outside the va health care system meet veterans increasing needs in post - acute institutional care and were implemented in response to the millennium act of 1999 requirement.2 under the per diem contract program, veteran cnh placement is decided based upon several conditions such as va clc bed availability, patient long - term care needs defined by patient hospital discharge assessment, patient priority for va health care (eg, service connected status, level of disability, and income), and patient preferences (eg, proximity to veterans home or family).3 veterans length of stay (los) and rehabilitation therapy needs at the cnh are defined in the individual contract between local va facility and cnhs. however, the contract may be renewed and the veteran s los may be extended if needed. va handbook 1143.2, va community nursing home oversight procedures, outlines the per diem program and specific requirements for a cnh qualifying for a contract.4 each year, va contracts with > 2,500 cnhs, and these contracts are usually initiated at local va medical centers.4,5 the us general accountability office criticized the cnh per diem program in their report dated 2001 and 2004 for lacking a system - wide quality monitoring and oversight mechanism and lacking national data to track health services and care quality for veteran residents.4,6 in an early study, johnson reported that va - contracted cnhs differed from their counterparts without a va contract ; they were more likely to be larger and for - profit and less likely to be hospital - based and to meet centers for medicare and medicaid services (cms) recommended minimum certified nursing assistant and registered nurses staffing. additionally, the va - contracted cnhs had more residents enterally tube fed, newly catheterized, mobility restrained, and who had developed new pressure ulcers.7 in a recent study comparing post - stroke rehabilitation and restorative care utilization by veterans in clcs and cnhs, we found that before and after risk adjustment, cnh veterans had significantly more average rehabilitation therapy days, but clc veterans had significantly more average days for restorative nursing care.8 in that study, we were limited by imperfect matching of available data for cnhs and clcs. for example, the resident / nurse ratio and facility quality deficiency scores were not available for clcs.7 furthermore, we excluded 594 clc cnh dual users in our analysis to enhance the comparability between the two types of facilities. the earlier limitations motivated further assessment of the rehabilitation therapy and restorative nursing care received by veterans with stroke at cnhs with va contract. the objectives of this retrospective observational study were, 1) to determine rehabilitation therapy and restorative nursing care utilization by veterans with stroke in va - contracted cnhs, and 2) to assess risk - adjusted regional variations in the utilization of rehabilitation therapy and restorative nursing care. such information is important as post - acute rehabilitation enhances stroke survivors functional recovery and daily living activities.911 cnhs are major non - va health care recovery resources for veterans with stroke, and yet our understanding of this topic is limited. our research questions were as follows : how many veterans with stroke have received rehabilitation therapy and restorative nursing care during their stay at the cnhs ? are there any geographic variations in the utilization of rehabilitation therapy and restorative nursing care across the five va regions ? we hypothesized that there are no significant regional variations in the utilization of rehabilitation therapy and restorative nursing care after adjusting for the potential risk factors. the study sample included all veterans diagnosed with stroke and admitted to cnhs with a va contract from january 1, 2006, through december 31, 2009. in the minimum dataset (mds) used for the study, the variable, ilt_stroke, indicates the primary medical diagnosis of the veteran residents at the time of their admission to the cnhs. all eligible veterans were followed up for a maximum of 12 months post - admission date. this study was approved by the institutional review board (irb) at the university of florida and the research and development committee (r&dc) at the north florida / south georgia veterans health system, gainesville, fl, usa. this study was of minimal risk with a retrospective design and used existing administrative data from multiple data sources. as such, the study was reviewed and approved by our irb and r&dc as an expedited protocol with a waiver of informed consent. first, the cms mds (mds 2.0) provided the veterans baseline sociodemographics and service utilization at the cnhs. the mds is a standardized clinical assessment tool of health status of all residents in medicare- and/or medicaid - certified nursing homes. the mds contains items on residents health care utilization and functional capabilities, and it helps nursing home staff identify residents health problems.12 rehabilitation therapy and restorative nursing care utilization data were collected as a part of the mds assessment information. federal government mandates these facilities to conduct an assessment for all residents at admission, after a significant change in health status, quarterly, annually, and at discharge, regardless of payment source.13 second, we used va national inpatient and outpatient data files obtaining the veterans relevant va inpatient / outpatient clinical information 12 months prior to their nursing home admission.14 finally, the va national fee basis inpatient and outpatient treatment files provided veterans relevant sociodemographic and clinical information on covered care provided outside of va facilities. we used a unique patient identifier (scrambled social security number [ssn ]) linking the multiple - source data as follows : 1) to identify eligible veterans in the 20062009 cms mds data and create two identifier files (scrambled ssn for veterans and facility identifier file for the cnhs) ; 2) to apply the veteran identifier in the va and fee basis inpatient and patient data sets obtaining and confirming the sociodemographic and clinical information ; and 3) to match the facilities in the medicare nursing home compare file to obtain facility characteristics. the mds recorded the number of days for each specific type of the rehabilitation therapy and restorative nursing care provided to a resident for 15 minutes per day in the last 7 days prior to an assessment date. for rehabilitation therapy, we first calculated the average number of days for each type of rehabilitation therapy (i.e., physical, occupational, speech, and/or respiratory therapy), then we derived a total score summing across all types of rehabilitation therapies by geographic region. similarly, we applied the steps mentioned earlier, obtaining the regional totals and average days for restorative nursing care (i.e., active range of motion, walking, passive range of motion, bed transfer, dressing and/or grooming, eating and/or swallowing, bed mobility, and communication). these restorative techniques denoted the nursing interventions in assisting, promoting, or maximizing a resident s ability, independent living, and functional potential. myva regions include five geographic regions : the north atlantic, southeast, midwest, continental, and pacific regions. a map of the districts can be found at http://www.blogs.va.gov/vantage/16786/.15 the regional assignment was based on cnh s state location. if multiple facilities were used by a veteran during the follow - up time period, we chose the facility with the longest days of stay and the most post - admission assessments. the facility characteristics are as follows : 1) rural / urban facility was designated by linking cnh zip codes with the zip codes in the va rural / urban definition. with this va definition, urban refers to population density 1,000 civilians per square mile and rural refers to any non - urban rural or highly rural area.16,17 2) beds number was the number of facility medicare - certified beds during the study time frame. 3) resident / nurse ratio was the number of residents divided by the total number of full - time registered nurses per facility. 4) resident / bed ratio was the number of residents divided by the total number of facility - certified beds. 5) ownership referred to non - profit, for - profit, or local government owned. 6) hospital - based status indicated whether a facility was affiliated with a hospital during the study. 7) weighted deficiency sum score (numerical) : scope and severity is a national system used by all - state survey agencies to rate cnh deficiencies. to be certified for medicare and medicaid programs, the weighted deficiency sum score was calculated by multiplying cms deficiency weights by the number of scope and severity identified for each facility. the weighted sum score indicated that a facility s deficiency rating did not meet the federal regulatory requirements for the standard quality of care : the higher the score, the worse the quality of care.18 the veteran characteristics are as follows : 1) age was veteran s age at nursing home admission using the formula : age = (admission date this variable was divided into five ranges (i.e., 64, 6569, 7074, 7579, and 80 years). this categorical age variable allows us to make a distinction between different age groups and the utilization outcomes. for example, veterans 65 years and older are more likely to be dually eligible for va and medicare health care programs, and dually eligible veterans are likely to receive more services than those veterans who only have va health care coverage. 3) education was divided into five categories : < high school, high school, some college, bachelor degree, and graduate degree. 4) race / ethnicity was categorized into three groups (white, african - american, and all others). 5) marital status was coded as never married, married, widowed, or separated / divorced. 6) va health care priority was derived from two variables existing in the va inpatient databases : percent service - connected, a number between 0 and 100, is determined based on the major diagnosis responsible for the hospital stay, and means test indicator is used to determine veteran eligibility to receive care within the va health care system in addition to the percent service - connected. for this study, va health care priority was coded as high if veterans percent service - connected and means test category indicated that they were all compensable service - connected (10%) or non - service - connected (eg, prisoner of war, world war i, and low - income veterans). all others were coded as low.19,20 7) admission source indicated veteran s location before nursing home admission. we coded this variable as acute care hospital, non - acute hospital, nursing home, and all others. 8) baseline activities of daily living (adl) score was composed of the seven mds functional measures sum (i.e., bed mobility, transfer, locomotion, dressing, eating, toilet use, and personal hygiene) at admission. in the mds 2.0, each measure was entered as a value of 0 through 4, with 0 representing independent status and 4 representing a totally dependent status. the adl functioning total score at baseline or admission ranged from 0 to 28, with lower values representing higher functional status.21,22 9) comorbidity was measured using the modified charlson s comorbidity index by dhoore.23 the index assigned weights for major comorbid diseases. the total assigned weights represented a measure of comorbid disease burden : the higher the score, the more severe the comorbid disease burden. the va national inpatient and outpatient files and va fee basis inpatient and outpatient files were used to obtain the diagnoses for the comorbid sum score calculation. sas version 9.4 (sas institute, cary, nc, usa) was used for all data analyses. first, descriptive statistics were obtained on all the variables. statistical inference (chi - squared test or fisher s exact test on discrete variables and analysis of variance [anova ] test on continuous variables) was performed to compare the facility and veteran characteristics and utilization between the five regions. second, a two - part regression model with backward elimination method was fitted, 1) to estimate the risk - adjusted probability of receiving rehabilitation therapy and restorative care using logistic regression, and 2) to evaluate the risk - adjusted expected weekly days for the utilization among the users based on general linear regression. consequently, we removed the patient rural / urban resident variable from our final models due to high collinearity with facility rural / urban characteristics. the study sample included all veterans diagnosed with stroke and admitted to cnhs with a va contract from january 1, 2006, through december 31, 2009. in the minimum dataset (mds) used for the study, the variable, ilt_stroke, indicates the primary medical diagnosis of the veteran residents at the time of their admission to the cnhs. all eligible veterans were followed up for a maximum of 12 months post - admission date. this study was approved by the institutional review board (irb) at the university of florida and the research and development committee (r&dc) at the north florida / south georgia veterans health system, gainesville, fl, usa. this study was of minimal risk with a retrospective design and used existing administrative data from multiple data sources. as such, the study was reviewed and approved by our irb and r&dc as an expedited protocol with a waiver of informed consent. first, the cms mds (mds 2.0) provided the veterans baseline sociodemographics and service utilization at the cnhs. the mds is a standardized clinical assessment tool of health status of all residents in medicare- and/or medicaid - certified nursing homes. the mds contains items on residents health care utilization and functional capabilities, and it helps nursing home staff identify residents health problems.12 rehabilitation therapy and restorative nursing care utilization data were collected as a part of the mds assessment information. federal government mandates these facilities to conduct an assessment for all residents at admission, after a significant change in health status, quarterly, annually, and at discharge, regardless of payment source.13 second, we used va national inpatient and outpatient data files obtaining the veterans relevant va inpatient / outpatient clinical information 12 months prior to their nursing home admission.14 finally, the va national fee basis inpatient and outpatient treatment files provided veterans relevant sociodemographic and clinical information on covered care provided outside of va facilities. we used a unique patient identifier (scrambled social security number [ssn ]) linking the multiple - source data as follows : 1) to identify eligible veterans in the 20062009 cms mds data and create two identifier files (scrambled ssn for veterans and facility identifier file for the cnhs) ; 2) to apply the veteran identifier in the va and fee basis inpatient and patient data sets obtaining and confirming the sociodemographic and clinical information ; and 3) to match the facilities in the medicare nursing home compare file to obtain facility characteristics. the mds recorded the number of days for each specific type of the rehabilitation therapy and restorative nursing care provided to a resident for 15 minutes per day in the last 7 days prior to an assessment date. for rehabilitation therapy, we first calculated the average number of days for each type of rehabilitation therapy (i.e., physical, occupational, speech, and/or respiratory therapy), then we derived a total score summing across all types of rehabilitation therapies by geographic region. similarly, we applied the steps mentioned earlier, obtaining the regional totals and average days for restorative nursing care (i.e., active range of motion, walking, passive range of motion, bed transfer, dressing and/or grooming, eating and/or swallowing, bed mobility, and communication). these restorative techniques denoted the nursing interventions in assisting, promoting, or maximizing a resident s ability, independent living, and functional potential. myva regions include five geographic regions : the north atlantic, southeast, midwest, continental, and pacific regions. a map of the districts can be found at http://www.blogs.va.gov/vantage/16786/.15 the regional assignment was based on cnh s state location. if multiple facilities were used by a veteran during the follow - up time period, we chose the facility with the longest days of stay and the most post - admission assessments. the facility characteristics are as follows : 1) rural / urban facility was designated by linking cnh zip codes with the zip codes in the va rural / urban definition. with this va definition, urban refers to population density 1,000 civilians per square mile and rural refers to any non - urban rural or highly rural area.16,17 2) beds number was the number of facility medicare - certified beds during the study time frame. 3) resident / nurse ratio was the number of residents divided by the total number of full - time registered nurses per facility. 4) resident / bed ratio was the number of residents divided by the total number of facility - certified beds. 5) ownership referred to non - profit, for - profit, or local government owned. 6) hospital - based status indicated whether a facility was affiliated with a hospital during the study. 7) weighted deficiency sum score (numerical) : scope and severity is a national system used by all - state survey agencies to rate cnh deficiencies. to be certified for medicare and medicaid programs, the weighted deficiency sum score was calculated by multiplying cms deficiency weights by the number of scope and severity identified for each facility. the weighted sum score indicated that a facility s deficiency rating did not meet the federal regulatory requirements for the standard quality of care : the higher the score, the worse the quality of care.18 the veteran characteristics are as follows : 1) age was veteran s age at nursing home admission using the formula : age = (admission date date of birth)/365.25 days. this variable was divided into five ranges (i.e., 64, 6569, 7074, 7579, and 80 years). this categorical age variable allows us to make a distinction between different age groups and the utilization outcomes. for example, veterans 65 years and older are more likely to be dually eligible for va and medicare health care programs, and dually eligible veterans are likely to receive more services than those veterans who only have va health care coverage. 3) education was divided into five categories : < high school, high school, some college, bachelor degree, and graduate degree. 4) race / ethnicity was categorized into three groups (white, african - american, and all others). 5) marital status was coded as never married, married, widowed, or separated / divorced. 6) va health care priority was derived from two variables existing in the va inpatient databases : percent service - connected, a number between 0 and 100, is determined based on the major diagnosis responsible for the hospital stay, and means test indicator is used to determine veteran eligibility to receive care within the va health care system in addition to the percent service - connected. for this study, va health care priority was coded as high if veterans percent service - connected and means test category indicated that they were all compensable service - connected (10%) or non - service - connected (eg, prisoner of war, world war i, and low - income veterans). all others were coded as low.19,20 7) admission source indicated veteran s location before nursing home admission. we coded this variable as acute care hospital, non - acute hospital, nursing home, and all others. 8) baseline activities of daily living (adl) score was composed of the seven mds functional measures sum (i.e., bed mobility, transfer, locomotion, dressing, eating, toilet use, and personal hygiene) at admission. in the mds 2.0, each measure was entered as a value of 0 through 4, with 0 representing independent status and 4 representing a totally dependent status. the adl functioning total score at baseline or admission ranged from 0 to 28, with lower values representing higher functional status.21,22 9) comorbidity was measured using the modified charlson s comorbidity index by dhoore.23 the index assigned weights for major comorbid diseases. the total assigned weights represented a measure of comorbid disease burden : the higher the score, the more severe the comorbid disease burden. the va national inpatient and outpatient files and va fee basis inpatient and outpatient files were used to obtain the diagnoses for the comorbid sum score calculation. sas version 9.4 (sas institute, cary, nc, usa) was used for all data analyses. first, descriptive statistics were obtained on all the variables. statistical inference (chi - squared test or fisher s exact test on discrete variables and analysis of variance [anova ] test on continuous variables) was performed to compare the facility and veteran characteristics and utilization between the five regions. second, a two - part regression model with backward elimination method was fitted, 1) to estimate the risk - adjusted probability of receiving rehabilitation therapy and restorative care using logistic regression, and 2) to evaluate the risk - adjusted expected weekly days for the utilization among the users based on general linear regression. consequently, we removed the patient rural / urban resident variable from our final models due to high collinearity with facility rural / urban characteristics. this study comprised 6,206 veterans diagnosed with stroke at 2,511 va - contracted cnhs between 2006 and 2009. north atlantic region facilities were significantly different from other regions in many ways : larger in average certified beds, higher in resident / beds ratio, lower in resident / full - time nurse ratio, and more likely to be non - profit homes and run by local governments with lower weighted deficiency sum score. continental region facilities were more likely to be in rural areas and have higher resident / full - time nurse ratio, but their resident / bed ratio was lower than other regions. pacific region facilities were more likely to be hospital - based, for - profit, and in urban areas. continental and pacific region facilities had the worst deficiency score, although continental region facilities have twice as many residents per nurse as pacific region facilities. north atlantic cnh veterans were significantly older with lower education level and more likely to be married. southeast cnh veterans were more likely to be african - american and admitted from acute care hospital with high va health care priority. midwest cnh veterans were more likely to be younger, white, and single with lower average adl score at baseline. pacific cnh veterans were more likely to be non - white and non - african - american with higher education level and a heavier burden of comorbid conditions. table 2 provides the regional comparisons of veterans average weekly days for rehabilitation therapy and restorative nursing care during their cnh stays. among the 6,206 veterans, the proportion of rehabilitation therapy and restorative nursing care users was 75.7% and 30.1%, respectively, and the observed weekly rehabilitation therapy and restorative nursing care utilization averaged 6.44.4 and 5.96.2 days, respectively. north atlantic region had the highest rates of rehabilitation therapy and restorative nursing care, but southeast region reported more average days in rehabilitation therapy and midwest region reported more days in restorative nursing care. the most commonly used rehabilitation therapies included physical and occupational therapy, and the commonly used restorative nursing care activities were active range of motion, walking, and passive range of motion. the estimated probability of receiving rehabilitation therapy and restorative nursing care was 76% and 30%, respectively, with significant regional variations (north atlantic : 88%/34% ; southeast : 83%/23% ; midwest : 66%/28% ; continental : 66%/30% ; and pacific : 70%/34%) (table 4). significant covariates in the rehabilitation therapy model were rural facility, less certified beds, lower resident / nurse ratio, for - profit facilities, hospital - based facilities, lower deficiency sum score, race / ethnicity, admission from acute care facilities, low va health care priority, and poor baseline adl. significant covariates in the restorative nursing care model include less certified beds, lower resident / nurse ratio, nonprofit facilities, non - hospital - based facilities, admission from non - acute care facilities, and poor baseline adl (table 3). part 2 model findings are shown in table 5. the expected weekly days of rehabilitation therapy and restorative nursing care were 6.4 and 5.9 days among users, respectively, with significant regional variations (north atlantic : 6.6/6.1 ; southeast : 7.2/5.1 ; midwest : 5.9/7.3 ; continental : 5.8/5.6 ; and pacific : 6.2/5.5 days) (table 4). significant covariates in the rehabilitation therapy model were urban facilities, less certified beds, for - profit facilities, hospital - based facilities, married, admitted from acute care facilities, low va health care priority, less comorbid burden, and higher adl. significant covariates in the restorative model included non - profit and hospital - based facilities (table 5). the first objective of this study was to determine rehabilitation therapy and restorative nursing care utilization by veterans with stroke in va - contracted cnhs. our results showed that 75.7% and 30.1% of the veterans in our study have received rehabilitation therapy and restorative nursing care, respectively, during their cnh stays. the number of pre - adjusted rehabilitation therapy and restorative nursing care days averaged 6.4 and 5.9 days per week, respectively (table 2). we found no relevant literature to benchmark our findings. in an early study on physical rehabilitation among elderly residents at skilled nursing facilities in michigan and ohio, the rehabilitation therapy prevalence was 79.9% for the residents.24 however, this study differed from ours in that the study residents were older than our veteran cohort and the reported rehabilitation prevalence was for all medical diagnoses, whereas our study focused solely on stroke diagnoses. regardless, we found that rehabilitation therapy prevalence was high and the users received almost one therapy daily. the high prevalence of rehabilitation therapy may be associated with two factors : 1) it may demonstrate the actual therapies delivered to veterans by the providers following the treatment plan specified in their per diem contract with va ; 2) alternatively, the high utilization may represent upcodes in therapy minutes by the providers. a recent study reported that nursing homes may upcode their residents rehabilitation therapy minutes to increase revenue.25 according to the report, upcoding is visualized by plotting the frequency of therapy minutes and noting a clustering of high frequencies just above the amounts necessary to place reimbursement in the next higher category. furthermore, providers have economic incentives to upcode the therapy as rehabilitation minutes documented in the mds determine the resource utilization group score calculation, which in turn determines the providers amount of reimbursement from medicare. to test this hypothesis of reported rehabilitation minutes upcoding, we examined rehabilitation minutes frequency of our data and observed a similar pattern in minutes upcoding with a previous research report;25 however, our finding in minutes upcoding was less pronounced. if upcoding is present, its blunted nature may be due to a removal of incentive when care is paid for by per diem contract. our results also showed that the most commonly used rehabilitation therapies were physical therapy and occupational therapy, and the most commonly used restorative nursing techniques included active range of motion, walking, and passive range of motion (table 2). these specific therapy types and restorative techniques were consistent with clinical guideline - recommended long - term rehabilitation goals of care for stroke survivors : to regain ability in carrying out adl, reduce the effect of remaining deficits, and live as independently as feasible.1 with rehabilitation therapy, physical therapists help stroke patients regain the use of their stroke - impaired limbs and establish physical training programs to retain the newly learned skills. similarly, occupational therapists help stroke patients acquire skills needed for performing self - directed activities (eg, self - grooming, preparing meals, and housecleaning). with training, registered and licensed practical nurses and/or nursing assistants help patients maintain the functioning achieved in rehabilitation therapy using different restorative nursing techniques such as actively help (passive) or help without physical assistance (active) to move the impaired limbs, perform exercises, or facilitate walking. contrary to our expectations, we found that less than one - third of the veterans received restorative nursing care during their cnh stays. this step down from rehabilitation therapy to restorative nursing care may be because providers lacked financial incentive to provide and/or document the care in the mds assessment form since restorative nursing care is not as prominent in determining facility reimbursement and quality of care assessment. it may also indicate a mismatch between the amount of staffing needed and the amount of staffing a facility can afford. the second objective of the study was to assess the regional variations in rehabilitation therapy and restorative nursing care utilization. contrary to our hypothesis, we observed a significant variation in rehabilitation therapy and restorative nursing care utilization across the five myva regions. consistently, our results demonstrated that veterans in the north atlantic and southeast cnhs were significantly more likely to receive rehabilitation therapies than their counterparts from other regions in terms of observed, adjusted probability and expected utilization. however, veterans in the southeast facilities were significantly less likely to receive restorative nursing care compared with veterans in other regions before or after risk adjustment. to understand the potential contributing factors for the variation, we found that veterans in north atlantic and southeast facilities were significantly more likely to be admitted directly from acute care settings and have poorer average adl score compared with veterans from other regions. our study and findings are important on several fronts : first, the study fills in the gaps by providing rehabilitation therapy and restorative nursing care utilization by all veterans with stroke at the va - contracted cnhs. second, our findings will help va policymakers at different levels to understand the types and intensity (number of weekly days) of rehabilitation therapy and restorative nursing care received by the veterans at the va - contracted cnhs, as well as the geographic variation of these health services utilizations. third, our study results will lay the ground work for developing future research in the field, such as quality of care for the veterans and the impact of veterans rehabilitation and restorative nursing care utilization on the changes of their functional outcomes (i.e., adl, cognitive function, and depression status). our study results were based upon all veterans diagnosed with stroke residing in va - contracted cnhs over a period of 4 years (20062009) ; nonetheless, facility variation in va s contract and policy implications should be considered with caution. since this study is part of a larger study intended to compare the rehabilitation utilization and functional outcomes between va clcs and cnhs, we were limited in including some of the variables commonly used in cnh studies (eg, the resource utilization group score). we included a number of facility and veteran characteristic variables by linking the mds data with the va and non - va inpatient and outpatient utilization databases. the majority of veterans with stroke received rehabilitation therapy, and about one - third had restorative nursing care during their cnh stays. we found significant variations in the observed probability and estimated rehabilitation utilization across the five myva regions. our findings provide needed information about veterans post - stroke rehabilitation therapy at cnhs and insights regarding the care variations across the five newly defined myva regions. | introductioneffective post - acute multidisciplinary rehabilitation therapy improves stroke survivors functional recovery and daily living activities. the us department of veterans affairs (va) places veterans needing post - acute institutional care in private community nursing homes (cnhs). these placements are made under the same rules and regulations across the va health care system and through individual per diem contracts between local va facilities and cnhs. however, there is limited information about utilization of these veterans health services as well as the geographic variation of the service utilization.aimthe aims of this study were to determine rehabilitation therapy and restorative nursing care utilization by veterans with stroke in va - contracted cnhs and to assess risk - adjusted regional variations in the utilization of rehabilitation therapy and restorative nursing care.methodsthis retrospective study included all veterans diagnosed with stroke residing in va - contracted cnhs between 2006 and 2009. minimum dataset (a health status assessment tool for cnh residents) for the study cnhs was linked with veterans inpatient and outpatient data within the va health care system. cnhs were grouped into five va - defined geographic regions : the north atlantic, southeast, midwest, continental, and pacific regions. a two - part model was applied estimating risk - adjusted utilization probability and average weekly utilization days. two dependent variables were rehabilitation therapy and restorative nursing care utilization by veterans during their cnh stays.resultsthe study comprised 6,206 veterans at 2,511 cnhs. rates for utilization of rehabilitation therapy and restorative nursing care were 75.7% and 30.1%, respectively. veterans in north atlantic and southeast cnhs were significantly (p<0.001) more likely to receive rehabilitation therapies than veterans from other regions. however, veterans in southeast cnhs were significantly (p<0.001) less likely to receive restorative nursing care compared with veterans in all other regions, before and after risk adjustment.conclusionthe majority of veterans with stroke received rehabilitation therapy, and about one - third had restorative nursing care during their stay at va - contracted cnhs. significant regional variations in weekly days for rehabilitation therapy and restorative nursing care utilization were observed even after adjusting for potential risk factors. |
most patients are adults and are infected by eating raw or undercooked freshwater snails or other paratenic hosts such as freshwater shrimps, frogs, or monitor lizards. the diagnosis is mostly made clinically by the evidence of eosinophils in cerebrospinal fluid (csf) constituting more than 10% of total csf white blood cells. the diagnosis may be missed because meningism signs including fever and neck stiffness are found infrequently. however, clinical manifestations in children may be different from those of adult patients. here, we report a series of eom cases in children in terms of clinical manifestations and outcomes in the endemic area of eom associated with a. cantonensis. twenty patients below the age of 15 were diagnosed as eom associated with a. cantonensis at srinagarind hospital, khon kaen university, thailand. the diagnosis of eom was made if csf wbc count was > 10 cells / mm and more than 10% of these were eosinophils. additionally, negative results should be yielded from the csf by gram, acid - fast and india ink staining, and cultures. we excluded patients with other possible causes of eosinophilic pleocytosis in csf such as neurognathostomiasis, tuberculosis, or malignancy. clinical manifestations and outcomes were recorded including baseline characteristics, symptoms, physical signs, laboratory results, treatment, and outcomes. data included age, gender, season of presenting symptoms defined by thailand meteorology classification (winter, summer, or rainy), incubation period (number of days after the last exposure to snails or paratenic hosts to the first day of developing symptoms), duration of headache, history of paresthesia, history of nausea or vomiting, fever (oral temperature more than 37.8), general physical signs, cranial nerve abnormalities, cerebellar signs, papilledema, and neck stiffness. treatment and clinical outcomes such as duration of headache, number of lumbar punctures were also reviewed. all data were presented using descriptive statistics and compared with the data of adult eom patients. most patients were males (78.9%), had fever (78.9%), nausea or vomiting (63.2%), a history of ingestion of raw freshwater snails (68.4%), neck stiffness (68.4%), and about half of them were infected during winter season (52.6%). data were summarized and compared with those from adult patients as shown in tables 1 and 2. the median duration of headache after treatment was 4 days (range 1 - 21 days) as shown in table 3. the median numbers of repeated lumbar puncture was 2.5 times (range 1 - 6 times). although our sample number is small, children with eom revealed more systemic responses, as were apparent from the high proportion of patients with fever (78.9% vs 10%) and nausea / vomiting (63.2% vs 38.8%). two children exhibited hepatomegaly, which was never seen in adult patients (10.5% vs 0% : tables 1 and 2). in addition, cerebellar abnormality and thrombocytosis have never been reported in adult patients according to the literature [4,6 - 9 ]. compared to adult patients, a higher proportion of children showed cranial nerve abnormalities (both cranial nerve vi and vii), neck stiffness (68.4% vs 47.5%), and papilledema (31.6% vs 2.5%). clinical signs of meningism (fever, headache, and neck stiffness) were much more frequent in child patients compared with adults (68.4% vs 9.0%). in contrast, hyperesthesia, the specific sign for angiostrongyliasis in adults, was not found in children. regarding laboratory results (table 2), all variables were quite comparable between children and adults except for thrombocytosis and high csf opening pressure. these clinical features recall a report from taiwan that showed high proportion of fever (91.5%) in children with eom. the explanation may be due to systemic responses and high intracranial pressure, evidenced by higher csf opening pressures in children than in adults (400 vs 280 mmh2o) as shown in table 2. most clinical variables in this study and the study from taiwan were quite comparable except numbers of patients with meningoencephalitis and worm recovery in the csf (table 4). in thailand, the rate of meningoencephalitis was low particularly in adults (< 5%). in this study the definition of meningoencephalitis may be different between taiwan and thailand studies. in thailand, physicians defined meningoencephalitis only if the patients turned to permanent unresponsive or comatose condition. however, in taiwan studies, patients were defined as meningoencphalitis if the patients had alteration in mental or consciousness level at presentation. the worm recovery in the csf was generally low, although it was reported to be 41.5% in a taiwan study. the treatment outcomes shown in table 3 may not be realistic because data shown for adult patients included those who were treated with placebo, whereas children in the present study were treated with analgesics, corticosteroids, and anthelmintics. the duration of headache in children seemed to be shorter but a higher proportion required repeated lumbar puncture. this may imply that children with eom may resolve more quickly than adults despite having more severe systemic responses as discussed earlier. children with eom, however, needed more frequent reduction of intracranial pressure by repeated lumbar puncture compared with adult patients (52.6% vs 40%, table 3). the limitations of this study were the small number of patients and incomplete data as a consequence of the retrospective nature of the study. having a history of eating raw freshwater snails is an important risk factor for angiostrongyliasis, yet was found in only 68.4% of child patients compared to almost 100% in adult patients [4,6 - 9 ]. other than eating, direct contact with snails and slugs may be another risk factor for eom in children. a report from taiwan showed that having snails as a pet was another route of infection. in conclusion, clinical features and outcomes of eom caused by a. cantonensis in children were different from those in adult patients. compared with previous data of adult patients, high proportions of fever, nausea, vomiting, hepatomegaly, neck stiffness, and cranial | eosinophilic meningitis, caused by the nematode angiostrongylus cantonensis, is prevalent in northeastern thailand, most commonly in adults. data regarding clinical manifestations of this condition in children is limited and may be different those in adults. a chart review was done on 19 eosinophilic meningitis patients aged less than 15 years in srinagarind hospital, faculty of medicine, khon kaen university, thailand. clinical manifestations and outcomes were reported using descriptive statistics. all patients had presented with severe headache. most patients were males, had fever, nausea or vomiting, stiffness of the neck, and a history of snail ingestion. six patients had papilledema or cranial nerve palsies. it was shown that the clinical manifestations of eosinophilic meningitis due to a. cantonensis in children are different from those in adult patients. fever, nausea, vomiting, hepatomegaly, neck stiffness, and cranial nerve palsies were all more common in children than in adults. |
a 49-year - old man was admitted to pusan national university hospital with complaints of squeezing chest pain and drowsy mentality. his systolic blood pressure was 70 mmhg, and his heart rate was 120 bpm. computed tomography revealed acute type a aortic dissection (ataad) that involved the aortic arch and the descending thoracic aorta (fig. 1). an emergency operation was performed. regional cerebral oxygen saturation (rso2) was monitored upon arrival at the operation room, and it was maintained during surgery using an invos system (somanetics, troy, mi, usa) (fig. the arterial perfusion line was placed on the right subclavian artery that appeared to be visually normal. after initiating cardiopulmonary bypass (cpb), after aortic cross clamping, the ascending aorta and the aortic valve were observed through aortotomy. an intimal tear was located in the ascending aorta, and the coaptation of the aortic valve was good. ascending and hemiarch replacement was planned because the visible arch and the descending aorta were intact. the proximal innominate artery was clamped, and perfusion catheters were inserted into the left carotid and left subclavian arteries. then, the right rso2 decreased abruptly, and it did not recover as compared to the left one. despite declamping and rechecking the inside of the innominate artery, we found no luminal problem. as dynamic carotid artery obstruction was suspected, the operation was continued because such obstructions usually resolve after operation. although the distal aortic anastomosis was completed and an adequate amount of blood flow to the branch of artificial graft was restarted, the right rso2 did not increase. another intimal tear on the proximal right common carotid artery, which was obstructing the blood flow, was identified using angiography. a 1280 mm smart control nitinol stent system (cordis co., bridgewater, nj, usa) was inserted into the right common carotid artery (fig. the total bypass time, aortic cross clamp time, and selective antegrade cerebral perfusion time were 227, 191, and 85 minutes, respectively. the patient was extubated on postoperative day 2 and discharged on postoperative day 13 without neurologic deficit. despite advances in surgical techniques, the mortality rates of ataad have remained high in patients with brain malperfusion. occlusion of the aortic arch vessels, which is propagated from the dissected aorta, commonly leads to neurological deficit. the main factor causing malperfusion is the dynamic obstruction of the false lumen or hypotension itself. in most cases, this can be relieved by restoring sufficient true luminal blood flow via surgery. in the present case the patient was treated via stent placement at the time of the surgery, using a hybrid technique. first, whenever the patient 's condition is acceptable, evaluations of the aortic arch vessels are essential for selecting the arterial approach for cardiopulmonary bypass in ataad involving the aortic arch. a previous study elucidated when the malperfusion occurs : 1) pre - surgery malperfusion, 2) malperfusion at the institution of cardiopulmonary bypass, 3) malperfusion occurring after aortic cross clamping, and 4) malperfusion postdistal aortic anastomotic construction. clamping the innominate artery for sacp may have caused injuries such as intimal tear and dissection, resulting in a reduction of carotid blood flow. this speculation is consistent with the fact that rso2 dramatically decreased when sacp was initiated. malperfusion, at the beginning of cpb, can be generated not only by retrograde perfusion via the femoral artery but also by antegrade perfusion via the subclavian artery. if rso2 drops at the time of cpb initiation, surgeons should promptly change the arterial cannulation site. in addition, it is important to initiate monitoring before anesthetic induction to detect problems by observing serial saturation changes. near - infrared spectroscopy (nirs) provides continuous rso2 monitoring and is a simple noninvasive method for monitoring cerebral perfusion. nirs - guided sacp allows a safe approach to aortic arch surgery, and a unilateral progressive discrepancy in rso2 indicates the occurrence of brain malperfusion. it is recommended that additional perfusion should be addressed immediately, when major hemispheric discrepancies appear. in our case third, if rso2 drops without any apparent anatomic or procedural reason, diagnostic angiography, as a hybrid technique, is strongly recommended. as for the discrepancy in rso2 in our case, even though aortic replacement was completed, we ascertained the luminal integrity of the innominate artery and suspected insufficient brain perfusion due to dissection or re - dissection of the carotid artery. angiography is needed to confirm the status of the carotid artery lumen. in our case, we found a tear on the right common carotid artery and achieved rso2 recovery through prompt carotid stent placement. after an open arterial stenosis, sufficient brain perfusion was restored, and no postoperative neurological symptoms developed. there are several published experiences with acute carotid revascularization in ataad cases involving the aortic arch vessels. in these cases, carotid stent placement was performed to recanalize the artery or seal an intimal flap, and a high success rate was achieved. in summary, in ataad patients with decreased mentality, preoperative evaluation of the aortic arch vessels, continuous monitoring of rso2, and the use of a hybrid technique that involves confirmatory angiography for detected problems will reduce neurologic complications from brain malperfusion. in addition, if a hybrid operation room is available, surgeons can save additional time in finding and fixing vascular complications. | a 49-year - old man with drowsy mentality was diagnosed with acute type a aortic dissection ; he underwent an emergency operation. when selective antegrade cerebral perfusion was initiated, the right regional cerebral oxygen saturation (rso2) decreased as compared to the left one. adequate blood flow was perfused through the branch of the artificial graft, after distal anastomosis, but the right rso2 did not recover. angiography revealed another intimal tear on the right common carotid artery. a stent was then inserted. the right rso2 promptly increased to the same level as that of the left one. the patient was discharged without any neurologic complications. |
during december 2006march 2007, a total of 23 human sylvatic rabies cases occurred in puno (n = 6) and madre de dios (n = 17), peru. the affected population consisted mainly of gold miners and their families who relocate to the area during the rainy season for small - scale mining before returning to their original towns for agricultural activities during the rest of the year. a vampire bat (d. rotundus) rabies virus variant was identified from clinical samples of deceased patients (2). after the rainy season ended, a team from the us naval medical research center detachment, the ministerio de salud (peruvian ministry of health), the servicio nacional de sanidad agraria (agricultural health service), and the museo de historia natural (natural history museum) from the universidad nacional mayor de san marcos (san marcos university) traveled to the area to conduct bat collections. the objective of the survey was to identify the prevalence of rabies infection among hematophagous (vampire) and nonhematophagous bats and to assess the distribution of bat genera within the outbreak area. the first collection was conducted in the region of madre de dios (location a, 13 753.53s, 702428.27w) from may 2 through may 4, 2007. the second site was located in the region of puno (location b, 131529.57s, 701939.14w) and sampled from may 6 through may 10, 2007 (figure). these nets were situated along the river banks, in secondary forests, and in manioc and banana plantations. in addition to the above habitats, bats in location b were collected in cattle grazing areas and in 2 caves. mist nets were set out every afternoon before dusk, checked every 1.5 hours, and closed at midnight, after 7 hours. each animal was placed inside a canvas bag, transported to a processing location, and kept until the following morning when they were processed as previously described (3). brain tissues and blood samples collected on necropsy were kept in a liquid nitrogen container for storage and shipping to the centers for disease control and prevention (cdc) in atlanta, ga, usa. carcasses were tagged and stored in formalin for shipment to the museo de historia natural in lima for positive species identification. blood samples processed at cdc were tested for rabies virus specific antibodies by using a rapid fluorescent focus inhibition test (rffit). brain stems were tested by direct fluorescence antibody (dfa) for evidence of active disease. antibody prevalence was calculated by using the binomial exact method ; antibody prevalence rates between bat species, location, and genera were compared by using the and fisher exact tests. all analyses were performed with stata 10.0 (statacorp, college station, tx, usa). sufficient quantity of serum for rffit was available from only 165 (85%) of the sampled animals, which were included in this study. the bats that were collected represented 6 genera, including 10 species ; 103 (62%) were females ; 62 (38%), males ; 25 (15%), juveniles ; 140 (85%, adults ; and 125 (76%) were carollia spp. one hundred thirty - seven animals (83%) were collected from community b. all vampire bats (n = 7) were collected in this location as well as other non - carollia insectivorous and frugivorous bats (p = 0.001). bats from the genus carollia were collected more frequently from natural, non - disturbed refuges (e.g., creeks, caves), while other insectivorous, frugivorous, and vampire bats were found in more visibly disturbed or modified foraging areas (e.g., plantations, cattle farms) (p<0.001). seventeen bats were antibody positive to rabies virus (cut - off value 0.5 iu), for an antibody prevalence of 10.3% (95% confidence interval 6.116.0). antibody prevalence was similar (p = 1.000) among vampire bats (1/7, 14%), carollia spp. (12/125, 10%), and other nonvampire bat genera (uroderma, sturnira, platyrhinus, and artibeus) (4/33, 12%) (table 2). no statistical differences were found between antibody prevalence and sex (p = 0.111), age (p = 0.078), habitat (p = 1.000), or collection site (p = 1.000) using fisher exact test. information on refuges and foraging areas is missing for 9 individuals, including 2 positive carollia perspicillata bats. in recent years, cases of rabies among humans in urban areas (transmitted by domestic animals) have declined considerably in the americas. this is likely the result of an aggressive initiative by the member states of the pan american health organization (paho) to eliminate urban rabies in the americas (4). consequently, rabies infections acquired by humans from wild animals, or sylvatic rabies, now represents the primary source for human infection in the region (1), with a similar trend for peru (5). haematophagous bats, including d. rotundus, are usually the species associated with sylvatic bat rabies outbreaks in south america, but little is known about the role of nonhematophagous bats. an investigation in chile found that nonvampire bats may in fact serve as adequate vectors of sylvatic rabies and even confirmed a single human infection of nonvampire - bat variant rabies linked to a nonhematophagous bat (6,7). likewise, paho reported 3 cases of sylvatic rabies transmitted by nonhematophagous bats in 2004 (4). this may suggest that insectivorous and frugivorous bats have a more specific role in the transmission of rabies virus in south america. the antibody prevalence of 10.3% found in our study is concordant with the 37% antibody rates found in nonhematophagous bats in colima, mexico (8) and 7.6% and 12.8% antibody prevalence among vampire and nonvampire bats in grenada and trinidad, respectively (9). although the transmission of rabies virus seems to occur very early in life (10,11), our study did not demonstrate evidence of antibodies to the virus among juvenile bats. likewise, the distribution of gender suggested higher antibody prevalence among females, although the finding was not significant, perhaps due to small sample size. the different distribution of bat species may be related to food availability, which would explain why d. rotundus bats were found near cattle farms and the more ubiquitous distribution of carollia perspicillata bats, which feed primarily on fruit in addition to pollen and insects. the bat species collected in our study have been previously found in areas at similar altitudes in peru ; therefore, their distribution in this area follows a regular pattern (12,13). additionally, the lack of surveillance for bat populations in the area prevents further inference about a possible source of infection among these bat populations. although none of the bats tested in our investigation had active rabies infections, both vampire and nonvampire bats had evidence of antibodies to rabies virus, which perhaps suggests ongoing cross - species transmission (spillover) among multiple bat species. peru and other south american countries should enforce the comprehensive, more aggressive preventive measures suggested at the xi reunion de directores de los programas nacionales de control de rabia en america latina (meeting of the directors of national rabies control programs in latin america) for certain prioritized areas. these activities include preexposure prophylaxis, surveillance, closer coordination with local animal health authorities, and community education. | after a human rabies outbreak in southeastern peru, we collected bats to estimate the prevalence of rabies in various species. among 165 bats from 6 genera and 10 species, 10.3% were antibody positive ; antibody prevalence was similar in vampire and nonvampire bats. thus, nonvampire bats may also be a source for human rabies in peru. |
dna (genetic material) in the nucleus and mitochondria of cells, recognized is the most critical target of ionizing radiation (2). ionizing radiation generates reactive oxygen species (ros) in the radiation - exposed cells, these free radicals such as hydroxyl (oh) and (h) can induce damage to critical macromolecules such as dna (3). dna damage may cause mutations and cancer (3). since radiation - induced cellular damage is attributed primarily to the harmful effects of free radicals, molecules with radical scavenging properties are particularly promising as a radioprotector (4). selenium and vitamin - e are two famous radioprotectors that much research has been done on them (5,6). selenium is a trace element that is naturally present in many foods and available as a dietary supplements (5,7). selenium has been shown to be a direct free radical scavenger and an indirect antioxidant via its stimulatory actions on antioxidant enzymes activity and inhibitory actions on pro - oxidative enzymes activity (7). many studies indicated selenium antioxidant properties can effects on dna repair, apoptosis, and the endocrine and immune systems, moreover selenium might play a role in the prevention of cancer (5,8 - 10). vitamin - e is the collective name for a group of fat - soluble compounds with distinctive antioxidant activities. it is found naturally in some foods and available as dietary supplements (11). antioxidant nutrients like vitamin - e protect cell constituents from the damaging effects of free radicals that if unchecked might contribute to cancer development (6). for assessing chromosome damage in this study, a micronucleus is a test that is used in toxicological screening for potential genotoxic compounds. this assay is now recognized as one of the most successful and reliable assays for genotoxic carcinogens, i.e., carcinogens that act by causing genetic damage (12). in this analyze, the cytochinesis is blocked and any numerical or structural damages to chromosomes would be viewed as minute nuclei in binucleate cells called micronucleus (mnbi). micronuclei (mn) may originate from either acentric chromosomal fragments or whole chromosome delayed in anaphase. this assay has being used widely to investigate the effects of different probable radioprotectors (12,13). although many studies showed that treatment of human peripheral blood lymphocytes in vitro with selenium and vitamin - e reduce micronuclei induced by gamma irradiation, the efficiency of these substances has been not evaluated in vivo by oral administration. here, we evaluate the efficacy of selenium and vitamin - e separately and synergistically in human volunteer 's blood lymphocytes with a method combining in vivo exposure of the volunteers to the selenium and vitamin - e and in vitro testing of the radioprotective effect against 6mv x - rays this study was performed after obtaining permission from the medical ethics committee of the tehran university of medical science. healthy, non - smoking male volunteers (meansd age of 263yrs) with an average weight of 69 kg, whom had no medicines or drugs taken for at least two months prior to sampling and not suffering from any known serious acute or chronic illness, were selected and informed consent was obtained from them. these volunteers divided into three groups include a, b and c. after of overnight fasting, five volunteers (a group) were given one gelatin capsules containing 800 iu selenium, five volunteers (b group) were given capsule containing 100 mg of vitamin - e and five volunteers were given vitamin - e plus selenium gelatin capsule(containing 400 iu vitamin e and 50 mg selenium) at 8 am. all vitamin - e and selenium gelatin capsules contain standard dose (fda approve) that were purchased from american natural company. then blood samples were collected in heparinized tubes 10 min before (0 hr) and 1, 2, and 3 hr after selenium (a group), vitamin - e (b group) and selenium plus vitamin - e (c group) ingestion. at each sampling time, for each volunteer, 1ml aliquots of heparinized whole blood was divided into two 25 ml culture flasks. one flask was the non - irradiated control sample, and another was irradiated at 37c (temperature) and 87.9 kpa (pressure) with 6mv x - rays accelerator (oncor, germany siemens) with a dose of 200 cgy at a 100 cm ssd and 10field size. in order to provide uniformity of dose distribution throughout the whole sample, two opposite x - rays beams were directed perpendicular to the longer axis of the flask containing the blood sampels. in each of these two fractions, half of the planned dose was delivered (14). subsequently, 0.5ml of each samples (non - irradiated control and irradiated) was added to 4.5ml of rpmi 1640 culture medium (sigma, u.s), which contained a mixture of 20% fetal calf serum, 100l / ml phytohaemagglutinin (sigma, u.s), 100l / ml penicillin, 250g / ml streptomycin and 2 mm glutamine (sigma, u.s) at final concentration. all cultures were incubated at 371c in a humidified atmosphere of 5% co2 and 95% o2. cytochalasin b (sigma - aldrich, final concentration : 6g / ml) was added after 44 hr of culture (13). after 72 hr of incubation, the cells were collected by centrifugation for 5 min at 800r.p.m, resuspended in 0.075 m cold potassium chloride for 6 min at 800r.p.m and immediately fixed in a fixative solution)methanol : acetic acid,6:1) three times. the fixed cells were dropped onto clean microscopic slides, air - dried and stained with giemsa solution. all slides were evaluated at 1000 magnification to determine the frequency of micronuclei in cytokinesis - blocked binucleate cells with the well preserved cytoplasm. small nuclei were scored as micronuclei if their diameters were between 1/16 to 1/3 of main nuclei and were non - refractile, not linked to main nuclei and not overlapping the main nuclei (13). at each blood collection time and for each volunteer, 1000cells were examined from the irradiated and control cultures in duplicate to record the frequency of micronuclei. the t - test was used to compare the incidence of micronuclei in each time point (1, 2, 3 hr) with the 0 hr time irradiated control points. this study was performed after obtaining permission from the medical ethics committee of the tehran university of medical science. healthy, non - smoking male volunteers (meansd age of 263yrs) with an average weight of 69 kg, whom had no medicines or drugs taken for at least two months prior to sampling and not suffering from any known serious acute or chronic illness, were selected and informed consent was obtained from them. these volunteers divided into three groups include a, b and c. after of overnight fasting, five volunteers (a group) were given one gelatin capsules containing 800 iu selenium, five volunteers (b group) were given capsule containing 100 mg of vitamin - e and five volunteers were given vitamin - e plus selenium gelatin capsule(containing 400 iu vitamin e and 50 mg selenium) at 8 am. all vitamin - e and selenium gelatin capsules contain standard dose (fda approve) that were purchased from american natural company. then blood samples were collected in heparinized tubes 10 min before (0 hr) and 1, 2, and 3 hr after selenium (a group), vitamin - e (b group) and selenium plus vitamin - e (c group) ingestion. at each sampling time, for each volunteer, 1ml aliquots of heparinized whole blood was divided into two 25 ml culture flasks. one flask was the non - irradiated control sample, and another was irradiated at 37c (temperature) and 87.9 kpa (pressure) with 6mv x - rays accelerator (oncor, germany siemens) with a dose of 200 cgy at a 100 cm ssd and 10field size. in order to provide uniformity of dose distribution throughout the whole sample, two opposite x - rays beams were directed perpendicular to the longer axis of the flask containing the blood sampels. in each of these two fractions, half of the planned dose was delivered (14). subsequently, 0.5ml of each samples (non - irradiated control and irradiated) was added to 4.5ml of rpmi 1640 culture medium (sigma, u.s), which contained a mixture of 20% fetal calf serum, 100l / ml phytohaemagglutinin (sigma, u.s), 100l / ml penicillin, 250g / ml streptomycin and 2 mm glutamine (sigma, u.s) at final concentration. all cultures were incubated at 371c in a humidified atmosphere of 5% co2 and 95% o2. cytochalasin b (sigma - aldrich, final concentration : 6g / ml) was added after 44 hr of culture (13). after 72 hr of incubation, the cells were collected by centrifugation for 5 min at 800r.p.m, resuspended in 0.075 m cold potassium chloride for 6 min at 800r.p.m and immediately fixed in a fixative solution)methanol : acetic acid,6:1) three times. the fixed cells were dropped onto clean microscopic slides, air - dried and stained with giemsa solution. all slides were evaluated at 1000 magnification to determine the frequency of micronuclei in cytokinesis - blocked binucleate cells with the well preserved cytoplasm. small nuclei were scored as micronuclei if their diameters were between 1/16 to 1/3 of main nuclei and were non - refractile, not linked to main nuclei and not overlapping the main nuclei (13). at each blood collection time and for each volunteer, 1000cells were examined from the irradiated and control cultures in duplicate to record the frequency of micronuclei. the t - test was used to compare the incidence of micronuclei in each time point (1, 2, 3 hr) with the 0 hr time irradiated control points. there were no side effects observed after the single oral dose ingestion of selenium and vitamin e in all five volunteers at 0 hr (before selenium and vitamin e administration). the obtained data (table 1, 2 and 3) showed a significant increase in the incidence of micronuclei in x - rays irradiated lymphocytes in compare to the control cells without irradiation. typical micronucleated binucleate the percentage of micronuclei induced by irradiation in lymphocytes of all volunteers at 1, 2 and 3 hr after eating of selenium alone (a group) was 6.080.22, 7.740.34 and 9.320.42, respectively. the percentage of micronuclei found in the selenium - treated cell groups were significantly much lower than the control point with radiation alone at 0 hr. lymphocytes in the blood samples collected at 1 hr after the oral eating of materials (exposed in vitro to 2 gy of 6 mv radiation) exhibited significant decrease in the incidence of micronuclei as compared with similarly irradiated lymphocytes in the blood collected at 0 hr (p<0.05). the total micronuclei values were 45% fold less after one hr from the oral administration of selenium. total micronuclei usually were lower at one hr as compared with those at 2 and 3 hr after the oral administration of selenium alone (table 1). the percentage of micronuclei induced by irradiation in lymphocytes of all volunteers at 1, 2 and 3 hr after eating of vitamin - e alone(b group) was 6.560.44, 8.20.4, and 9.740.26, respectively. the percentage of micronuclei found in the vitamin - e treated cell groups were significantly much lower than the control point with radiation alone at 0 hr. lymphocytes in the blood samples collected at 1 hr after the oral eating of vitamin - e (exposed in vitro to 2 gy of 6 mv radiation) exhibited significant decrease in the incidence of micronuclei as compared with similarly irradiated lymphocytes in the blood collected at 0 hr (p<0.05). the total micronuclei values were 41% fold less after 1 hr from the oral administration of vitamin e. total micronuclei usually were lower at 1 hr as compared with those at 2 and 3 hr after the oral administration of vitamin e alone (table 2). the percentage of micronuclei induced by irradiation in lymphocytes of all volunteers at 1, 2 and 3 hr after eating of selenium plus vitamin - e (c group) was 5.40.6, 6.40.3 and 6.80.3, respectively. the percentage of micronuclei found in the selenium and vitamin - e treated cell groups were significantly much lower than the control point with radiation alone at 0 hr. lymphocytes in the blood samples collected at 1 hr after the oral eating of selenium and vitamin - e (exposed in vitro to 2 gy of 6 mv radiation) exhibited significant decrease in the incidence of micronuclei as compared with similarly irradiated lymphocytes in the blood collected at 0 hr (p<0.001). the total micronuclei values were 50% fold less after 1 hr from the oral administration of selenium and vitamin e. total micronuclei usually were lower at 1hr as compared with those at 2 and 3 hr after the oral administration of selenium and vitamin e synergistically (table 3). differences between the total number of micronuclei recorded after treatments with selenium and vitamin - e in irradiated samples and not treatment irradiated samples were statistically significant (p<0.05). radioprotectors with high performance can be used to protect people in cases like occupational exposures, nuclear accidents, radiotherapy and cosmic rays exposures (15 - 17). two - thirds of chromosomal damage induced by ionizing radiation are due to free radicals ; it is regarded as the indirect effects of radiation (3). compounds include antioxidant element reduce the effects of ionizing radiations in living systems such chromosomal damage due to scavenging free radicals (4). many studies have demonstrated selenium and vitamin - e ability to scavenge free radicals (5,6,8,10,11). the data from noaman. suggests the synergistic use of vitamin - e and selenium which cause a greater reduction in dangerous enzymes such : glutathione (gsh), glutathione peroxidase (gsh - px), and superoxide dismutase (sod) in a blood sample of irradiated rats in compare separately use of vitamin e and selenium (19). moreover many studies have shown the radioprotective effect of selenium and vitamin - e separately, since both of them are natural and inexpensive compounds, however, few studies have been carried out on these two, according to human volunteers (6,7,9,18). this study confirmed the ability of x - rays -radiation to increase the incidence of mn in exposed human lymphocytes, as reported previously in many other investigations(2,3,18,20). the result of our study did not show any side effect from eating selenium and vitamin - e in human volunteers. high reduction about 50% in chromosomal abnormalities by exposure to 2gy of 6-mv - radiation in blood lymphocytes seen after administration of selenium and vitamin e synergistically. in vitro, similar studies on human blood lymphocytes also show a significant decrease in chromosomal damage in the presence of selenium and vitamin - e after irradiation of gamma rays (6,9,18). in this study 2 gy of 6mv - radiation was selected because it is a standard dose fraction that is applied daily to cancer patients in fractionation regimes in radiotherapy and high sensitivity of human dna is in 2 gy radiation (21). the results of this study showed the greatest effect of radioprotection on blood lymphocytes (in all three groups) was one hour after administration of selenium and vitamin - e, and after that, this effect was reduced. the results of this study also showed that concomitant use of selenium and vitamin e have a more radioprotective effect than using these separately. first, the data group was small ; further investigation using a larger data set is needed. second, various parameters of the immune system can effect the action of radioprotector agents, therefore, it seems these parameters must be evaluated separately. this study showed selenium and vitamin - e can decrease dna damage inflicted by x - rays radiation in volunteers ' human peripheral blood lymphocytes if it is given before exposure. selenium and vitamin - e are natural compounds and no side effects found in this study. this study showed that selenium and vitamin - e are effective radioprotectors because these compounds reduced dna damage in blood lymphocytes. this study has produced a new data set contribute to the growing body of evidence about radiation protection efficiency of selenium and vitamin - e (separately and synergistically) and confirms that synergistically use these compound can be more effective than separate use of them. the results also suggest that simultaneous ingestion of these radioprotective agents could increase the radiation protection efficiency of each one. however, before they are approved for clinical use, further experimental studies by using different cytogenetic must be done. moreover, conjugation of this compound with monoclonal antibody can increase the effect of these compounds in cancer radiotherapy. | background : critical macromolecules of cells such as dna are in exposure to damage of free radicals that induced from the interaction of ionizing radiation with biological systems. selenium and vitamin - e are natural compounds that have been shown to be a direct free radical scavenger. the aim of this study was to investigate the radioprotective effect of selenium and vitamin - e separately and synergistically against genotoxicity induced by 6mv x - rays irradiation in blood lymphocytes. methods : fifteen volunteers were divided into three groups include a, b and c. these groups were given selenium (800iu), vitamin - e (100 mg) and selenium (400iu) + vitamin - e (50 mg), respectively. peripheral blood samples were collected from each group before (0hr) and 1, 2 and 3hr after selenium and vitamin - e administration (separately and synergistically). then the blood samples were irradiated to 200cgy of 6mv x - rays. after that lymphocyte samples were cultured with mitogenic stimulation to determine the chromosomal aberrations with micronucleus assay in cytokinesis - blocked binucleated cells. results : the lymphocytes in the blood samples collected at one hr after ingestion selenium and vitamin - e, exposed in vitro to x - rays exhibited a significant decrease in the incidence of micronuclei, compared with control group at 0hr. the maximum protection and decrease in frequency of micronuclei (50%) were observed at one hr after administration of selenium and vitamin - e synergistically. conclusion : the data suggest that ingestion of selenium and vitamin - e as a radioprotector substance before exposures may reduce genetic damage caused by x - rays irradiation. |
in this work we investigate the image set of integer - valued polynomials over z. the set of these polynomials is a ring usually denoted by : int(z){fq[x]|f(z)z}. since an integer - valued polynomial f(x) maps the integers in a subset of the integers, it is natural to consider the subset of the integers formed by the values of f(x) over the integers and the ideal generated by this subset. the fixed divisor of f(x) is the ideal of z generated by the values of f(n), as n ranges in z : d(f)=d(f, z)=(f(n)|nz). the fixed divisor of f(x) is the ideal of z generated by the values of f(n), as n ranges in z : d(f)=d(f, z)=(f(n)|nz). it is well - known that for every integer n1 we haved(x(x1)(x(n1)))=n ! so that the so - called binomial polynomials bn(x)x(x1)(x(n1))/n ! are integer - valued (indeed, they form a free basis of int(z) as a z - module ; see). notice that, given two integer - valued polynomials f and g, we have d(fg)d(f)d(g) and we may not have an equality. for instance, consider f(x)=x and g(x)=x1 ; then we have d(f)=d(g)=z and d(fg)=2z. if fint(z) and nz, then directly from the definition we have d(nf)=nd(f). if cont(f) denotes the content of a polynomial fz[x ], that is, the greatest common divisor of the coefficients of f, we have f(x)=cont(f)g(x), where gz[x ] is a primitive polynomial (that is, cont(g)=1). in particular, the fixed divisor is contained in the ideal generated by the content. hence, given a polynomial with integer coefficients, we can assume it to be primitive. in the same way, if we have an integer - valued polynomial f(x)=f(x)/n, with fz[x ] and nn, we can assume that (cont(f),n)=1 and f(x) to be primitive. the next lemma gives a well - known characterization of a generator of the above ideal (see [1, lemma 2.7 ]). lemma 1.1let fint(z) be of degree d and set1)d1=sup{nz|f(x)nint(z)},2)d2=gcd{f(n)|nz},3)d3=gcd{f(0),,f(d) }, then d1=d2=d3. let fint(z) be of degree d and set1)d1=sup{nz|f(x)nint(z)},2)d2=gcd{f(n)|nz},3)d3=gcd{f(0),,f(d) }, then d1=d2=d3. we remark that the value d1 of lemma 1.1 is plainly equal to : d1=sup{nz|fnint(z)}. moreover, given an integer n, we have this equivalence that we will use throughout the paper, a sort of ideal - theoretic characterization of the arithmetical property that all the values attained by f(x) are divisible by n : f(z)nzfnint(z) (nint(z) is the principal ideal of int(z) generated by n). from 1) of lemma 1.1 we see immediately that if f(x)=f(x)/n is an integer - valued polynomial, where fz[x ] and nn coprime with the content of f(x), then d(f)=d(f)/n, so we can just focus our attention on the fixed divisor of a primitive polynomial in z[x ]. we want to give another interpretation of the fixed divisor of a polynomial fz[x ] by considering the maximal ideals of int(z) containing f(x) and looking at their contraction to z[x ]. that is, an ideal i of int(z) is unitary if it contains a non - zero integer, or, equivalently, iq[x]=q[x ] (where iq[x ] denotes the extension ideal in q[x ]). the next results are probably well - known, but for the ease of the reader we report them. the first lemma says that a principal unitary ideal i is generated by a non - zero integer, which generates the contraction of i to z. in particular, this lemma establishes a bijective correspondence between the nonzero ideals of z and the set of principal unitary ideals of int(z). if iz = nz with n0 then i = nint(z). in particular, nint(z)z = nz. moreover, n1int(z)=n2int(z) with n1,n2z if and only if n1=n2. let iint(z) be a principal unitary ideal. if iz = nz with n0 then i = nint(z). in particular, nint(z)z = nz. moreover, proofif i=(f) for some fint(z) then deg(f)=0 since a non - zero integer n is in i. since f(x) is integer - valued it must be equal to an integer and so it is contained in iz = nz. hence we get the first statement of the lemma. if n1int(z)=n2int(z) then n1=n2f with fint(z) ; this forces f to be a non - zero integer, so that n1 divides n2. if i=(f) for some fint(z) then deg(f)=0 since a non - zero integer n is in i. since f(x) is integer - valued it must be equal to an integer and so it is contained in iz = nz. if n1int(z)=n2int(z) then n1=n2f with fint(z) ; this forces f to be a non - zero integer, so that n1 divides n2. proofsuppose ii = niint(z), where niz, niz = iiz, for i=1,2. we have n1zn2z = nz, where n = lcm{n1,n2}. the ideal i1i2 is unitary since ni1i2. in particular, we have i1i2nint(z). suppose ii = niint(z), where niz, niz = iiz, for i=1,2. we have n1zn2z = nz, where n = lcm{n1,n2}. the ideal i1i2 is unitary since ni1i2. in particular, we have i1i2nint(z). the previous lemma implies the following decomposition for a principal unitary ideal generated by an integer n, with prime factorization n=ipiai. we havenint(z)=ipiaiint(z)=ipiaiint(z) where the last equality holds because the ideals piaiz are coprime in z, hence they are coprime in int(z). the extended fixed divisor of f(x) is the minimal ideal of the set { nint(z)|nz, fnint(z)}. we denote this ideal by d(f). the extended fixed divisor of f(x) is the minimal ideal of the set { nint(z)|nz, fnint(z)}. we denote this ideal by d(f). equivalently, in the above definition, we require that nint(z) contains the principal ideal in int(z) generated by the polynomial f(x). lemma 1.2, lemma 1.3 show that the minimal ideal in the above definition does exist : it is equal to the intersection of all the principal unitary ideals containing f(x). notice that the extended fixed divisor is an ideal of int(z), while the fixed divisor is an ideal of z. the polynomial f(x) is image primitive if and only if its extended fixed divisor is the whole ring int(z). in the next sections we will study the extended fixed divisor by considering the p - part of it, namely the principal unitary ideals of the form pnint(z), pz being prime and n a positive integer. the following proposition gives a link between the fixed divisor and the extended fixed divisor : the latter is the extension of the former and conversely. prooflet d, dz be such that d(f)=dz and d(f)=dint(z). since d(f)int(z)=dint(z) is a principal unitary ideal containing f(x), from the definition of extended fixed divisor, we have d(f)dint(z). we also have f(x)/dint(z) and so dd, by characterization 1) of lemma 1.1. let d, dz be such that d(f)=dz and d(f)=dint(z). since d(f)int(z)=dint(z) is a principal unitary ideal containing f(x), from the definition of extended fixed divisor, we have d(f)dint(z). we also have f(x)/dint(z) and so dd, by characterization 1) of lemma 1.1. as already remarked in, the rings z and int(z) share the same units, namely { 1}. then [5, proposition 2.1 ] can be restated as follows. then f(x) is irreducible in int(z) if and only if f(x) is not contained in any proper principal unitary ideal of int(z). then f(x) is irreducible in int(z) if and only if f(x) is not contained in any proper principal unitary ideal of int(z). the next lemma has been given in and is analogous to the gauss lemma for polynomials in z[x ] which are irreducible in int(z). then f(x) is irreducible in int(z) if and only if it is irreducible in z[x ] and image primitive. then f(x) is irreducible in int(z) if and only if it is irreducible in z[x ] and image primitive. for example, the polynomial f(x)=x2+x+2 is irreducible in q[x ] and also in z[x ] since it is primitive (because of gauss lemma). but it is reducible in int(z) since its extended fixed divisor is not trivial, namely it is the ideal 2int(z). so in int(z) we have the following factorization : f(x)=2x2+x+22 and indeed this is a factorization into irreducibles in int(z), since the latter polynomial is image primitive and irreducible in q[x ], and by [5, lemma 1.1 ], the irreducible elements in z remain irreducible in int(z). so the study of the extended fixed divisor of the elements in int(z) is a first step toward studying the factorization of the elements in this ring (which is not a unique factorization domain). here is an overview of the content of the paper. at the beginning of the next section we recall the structure of the prime spectrum of int(z). then, for a fixed prime p, we describe the contractions to z[x ] of the maximal unitary ideals of int(z) containing p (lemma 2.1). in theorem 2.1 we describe the ideal ip of z[x ] of those polynomials whose fixed divisor is divisible by p, namely the contraction to z[x ] of the principal unitary ideal pint(z), which is the ideal of integer - valued polynomials whose extended fixed divisor is contained in pint(z). it turns out that ip is the intersection of the aforementioned contractions. in the third section we generalize the result of the second section to prime powers, by means of a structure theorem of loper regarding unitary ideals of int(z). we consider the contractions to z[x ] of the powers of the prime unitary ideals of int(z) (lemma 3.1). in remark 2 we give a description of the structure of the set of these contractions ; that allows us to give the primary decomposition of the ideal ipn = pnint(z)z[x ], made up of those polynomials whose fixed divisor is divisible by a prime power pn. we shall see that we have to distinguish two cases : pn and p > n (see also the examples in remark 3). this result was already known in a slightly different context by dickson (see [7, p. 22, theorem 27 ]), but our different proof uses the primary decomposition of ipn and that gives an insight to generalize the result to the second case. in proposition 3.2 we give a set of generators for the primary components of ipn, in the case p > n. finally in the last section, as an application, we explicitly compute the ideal ipp+1. we recall that the prime ideals of int(z) are divided into two different categories, unitary and non - unitary. if it is unitary then its intersection with the ring of integers is a principal ideal generated by a prime p. non - unitary prime ideals : pz={0}. in this case p is a prime (non - maximal) ideal and it is of the formbq = qq[x]int(z) for some qq[x ] irreducible. by gauss lemma is maximal and is of the formmp,={fint(z)|f()pzp } for some p prime in z and some zp, the ring of p - adic integers. so if we fix the prime p, the elements of zp are in bijection with the unitary prime ideals of int(z) above the prime p. moreover, mp, is height 1 if and only if is transcendental over q. if is algebraic over q and q(x) is its minimal polynomial then mp,bq every prime ideal of int(z) is not finitely generated. for a detailed study of spec(int(z)) see. if we denote by d(f, zp) the fixed divisor of fint(z) viewed as a polynomial over the ring of p - adic integers zp (that is, d(f, zp) is the ideal (f()|zp)), gunji and mcquillan in observed thatd(f)=pd(f, zp) where the intersection is taken over the set of primes in z. moreover, d(f, zp)=d(f)zpzp. remember that given an ideal iz and a prime p we have izp = zp if and only if i(p), so that in the previous equation we have a finite intersection. since zp is a dvr we have d(f, zp)=pnzp, for some integer n (which of course depends on p), so that the exact power of p which divides f(z) is the same as the power of p dividing f(zp). without loss of generality, we can restrict our attention to the p - part of the fixed divisor of a polynomial fz[x ]. we begin our research by finding those polynomials in z[x ] whose fixed divisor is divisible by a fixed prime p, namely the ideal pint(z)z[x ]. then mp,z[x]=(p, xa), where az is such that a(modp). moreover, if zp is another p - adic integer, we have mp,z[x]=mp,z[x ] if and only if (modp). then mp,z[x]=(p, xa), where az is such that a(modp). moreover, if zp is another p - adic integer, we have mp,z[x]=mp,z[x ] if and only if (modp). prooflet a be an integer as in the statement of the lemma ; it exists since z is dense in zp for the p - adic topology. we immediately see that p and xa are in mp,. then the conclusion follows since (p, xa) is a maximal ideal of z[x ] and mp,z[x ] is not equal to the whole ring z[x ]. the second statement follows from the fact that (p, xa)=(p, xb) if and only if ab(modp). let a be an integer as in the statement of the lemma ; it exists since z is dense in zp for the p - adic topology. we immediately see that p and xa are in mp,. then the conclusion follows since (p, xa) is a maximal ideal of z[x ] and mp,z[x ] is not equal to the whole ring z[x ]. the second statement follows from the fact that (p, xa)=(p, xb) if and only if ab(modp). we have just seen that the contraction of mp, to z[x ] depends only on the residue class modulo p of. so, if p is a fixed prime, the contractions of mp, to z[x ] as ranges through zp are made up of p distinct maximal ideals, namely{mp,z[x]|zp}={(p, xj)|j{0,,p1}}. conversely, the set of prime ideals of int(z) above a fixed maximal ideal of the form (p, xj) is { mp,|zp,j(modp) }, since bq are non - unitary ideals and p is the only prime integer in mp,. for a prime p and an integer j{0,,p1 }, we set : mp, j = mj(p, xj). whenever the notation mp, j is used, it will be implicit that j{0,,p1}. the next lemma computes the intersection of the ideals mp, j, for a fixed prime p, by finding an ideal whose primary decomposition is given by this intersection (and its primary components are precisely the p ideals mp, j). from now on we will omit the index p. lemma 2.2let pz be a prime. if p is a prime minimal over j, then we see immediately that p = mj for some j{0,,p1 }, since mj is a maximal ideal.. then the minimal primary decomposition of j is of the formj=j=0,,p1qj where qj is an mj - primary ideal. since ximj for all i{0,,p1}{j }, we have (xj)qj, so indeed qj=(p, xj) for each j=0,,p1. if p is a prime minimal over j, then we see immediately that p = mj for some j{0,,p1 }, since mj is a maximal ideal. then the minimal primary decomposition of j is of the formj=j=0,,p1qj where qj is an mj - primary ideal. since ximj for all i{0,,p1}{j }, we have (xj)qj, so indeed qj=(p, xj) for each j=0,,p1. the next proposition characterizes the principal unitary ideals in int(z) generated by a prime p. proposition 2.1let pz be a prime. then the principal unitary ideal pint(z) is equal topint(z)=zpmp,. let pz be a prime. then the principal unitary ideal pint(z) is equal topint(z)=zpmp,. proofwe trivially have that pint(z) is contained in the above intersection, since p is in every ideal of the form mp,. on the other hand, this intersection is equal to { fint(z)|f(zp)pzp}. if f(x) is in this intersection, since f(x) is integer - valued and pzpz = pz, we have f(z)pz. this is equivalent to saying that f(x)/pint(z), that is, fpint(z). we trivially have that pint(z) is contained in the above intersection, since p is in every ideal of the form mp,. on the other hand, this intersection is equal to { fint(z)|f(zp)pzp}. if f(x) is in this intersection, since f(x) is integer - valued and pzpz = pz, we have f(z)pz. this is equivalent to saying that f(x)/pint(z), that is, fpint(z). in particular, the previous proposition implies that int(z) does not have the finite character property (we recall that a ring has this property if every non - zero element is contained in a finite number of maximal ideals). from the above results we get the following theorem, which characterizes the ideal of polynomials with integer coefficients whose fixed divisor is divisible by a prime p, that is, the ideal pint(z)z[x ]. theorem 2.1let p,p1(xj)). notice that lemma 2.2 gives the primary decomposition of pint(z)z[x ], so mj for j=0,,p1 are exactly the prime ideals belonging to it. as a consequence of this theorem we get the following well - known result : if fz[x ] is primitive and p is a prime such that d(f)p then pdeg(f).,p1(xj) is p. we remark that by fermats little theorem the ideal on the right - hand side of the statement of theorem 2.1 is equal to (p, xpx). this amounts to saying that the two polynomials x(x(p1)) and xpx induce the same polynomial function on z / pz. we remark that proposition 2.1 also follows from a general result contained in : every unitary ideal in int(z) is an intersection of powers of unitary prime ideals (namely the maximal ideals mp,). in particular, every mp,-primary ideal is a power of mp, itself, since mp, is maximal. from the same result we also have the following characterization of the powers of mp,, for any positive integer n : mp,n={fint(z)|f()pnzp}. this fact implies the following expression for the principal unitary ideal generated by pn:(1)pnint(z)=zpmp,n. we remark again that the previous ideal is made up of those integer - valued polynomials whose extended fixed divisor is contained in pnint(z). similarly to the previous case n=1 (see theorem 2.1) we want to find the contraction of this ideal to z[x ], in order to find the polynomials in z[x ] whose fixed divisor is divisible by pn. like before, we begin by finding the contraction to z[x ] of mp,n, for each zp. the next lemma is a generalization of lemma 2.1. then mp,nz[x]=(pn, xa), where az is such that a(modpn). the ideal mp,nz[x ] is mp, j - primary, where j(modp). moreover, if zp is another p - adic integer, we have mp,nz[x]=mp,nz[x ] if and only if (modpn). then mp,nz[x]=(pn, xa), where az is such that a(modpn). the ideal mp,nz[x ] is mp, j - primary, where j(modp). moreover, if zp is another p - adic integer, we have mp,nz[x]=mp,nz[x ] if and only if (modpn). let az be such that a(modpn) (again, such an integer exists since z is dense in zp for the p - adic topology). we have (pn, xa)mp,nz[x ] (notice that if n>1 then (pn, xa) is not a prime ideal). to prove the other inclusion the leading coefficient of xa is a unit) we havef(x)=q(x)(xa)+f(a). since f()pnzp and pn|a we have pn|f(a). since mp,n is an mp,-primary ideal in int(z) and the contraction of a primary ideal is a primary ideal, by lemma 2.1 we get the second statement. finally, like in the proof of lemma 2.1, we immediately see that (pn, xa)=(pn, xb) if and only if ab(modpn), which gives the last statement of the lemma. the case n=1 has been done in lemma 2.1. for the general case, let az be such that a(modpn) (again, such an integer exists since z is dense in zp for the p - adic topology). we have (pn, xa)mp,nz[x ] (notice that if n>1 then (pn, xa) is not a prime ideal). to prove the other inclusion the leading coefficient of xa is a unit) we havef(x)=q(x)(xa)+f(a). since f()pnzp and pn|a we have pn|f(a). since mp,n is an mp,-primary ideal in int(z) and the contraction of a primary ideal is a primary ideal, by lemma 2.1 we get the second statement. finally, like in the proof of lemma 2.1, we immediately see that (pn, xa)=(pn, xb) if and only if ab(modpn), which gives the last statement of the lemma. remark 1it is worth to write down the fact that we used in the above proof : given a polynomial fz[x ], we have(3)f(pn, xa)f(a)0(modpn). it is worth to write down the fact that we used in the above proof : given a polynomial fz[x ], we have(3)f(pn, xa)f(a)0(modpn). remark 2if p is a fixed prime and n is a positive integer, lemma 3.1 impliesip, n{mp,nz[x]|zp}={(pn, xi)|i=0,,pn1}. let us consider an ideal i = mp,nz[x]=(pn, xi) in ip, n, with iz, i(modpn). it is quite easy to see that i contains (mp,z[x])n = mp, jn=(p, xj)n, where j{0,,p1 }, j(modp) (notice that ji(modp)). if n>1 this containment is strict, since xi(p, xj)n. we can group the ideals of ip, n according to their radical : there are p radicals of these pn ideals, namely the maximal ideals mp, j, j=0,,p1. this amounts to making a partition of the residue classes modulo pn into p different sets of elements congruent to j modulo p, for j=0,,p1 ; each of these sets has cardinality pn1. every ideal in ip, n, j is mp, j - primary and it contains the n - th power of its radical, namely mp, jn.now we want to compute the intersection of the ideals in ip, n, which is equal to the ideal ipn in z[x ] (see (1) and (2)). we can express this intersection as an intersection of mp, j - primary ideals as we have said above, in the following way (in the first equality we make use of eq.,p1qp, n, j whereqp, n, jij(modp)(pn, xi) (notice that the intersection is taken over the set { i{0,,pn1}|ij(modp) }). the ideal qp, n, j is an mp, j - primary ideal, for j=0,,p1, since the intersection of m - primary ideals is an m - primary ideal. we will omit the index p in qp, n, j and in mp, j if that will be clear from the context. the mp, j - primary ideal qn, j is just the intersection of the ideals in ip, n, j, according to the partition we made. it is equal to the set of polynomials in z[x ] which modulo pn are zero at the residue classes congruent to j modulo p (see (3) of remark 1). notice that there are no embedded components in this primary decomposition, since the prime ideals belonging to it (the minimal primes containing ipn) are { mj|j=0,,p1 }, which are maximal ideals.we recall that if i and j are two coprime ideals in a ring r, that is i+j = r, then ij = ij (in general only the inclusion ijij holds). the condition for two ideals i and j to be coprime amounts to saying that i and j are not contained in a same maximal ideal m, that is, i+j is not contained in any maximal ideal m. if m1 and m2 are two distinct maximal ideals then they are coprime, and the same holds for any of their respective powers. if r is noetherian, then every primary ideal q contains a power of its radical and moreover if the radical of q is maximal then also the converse holds (see). so if qi is an mi - primary ideal for i=1,2 and m1,m2 are distinct maximal ideals, then q1 and q2 are coprime.since { mj}j=0,,p1 are p distinct maximal ideals, for what we have just said above we havej=0, if p is a fixed prime and n is a positive integer, lemma 3.1 impliesip, n{mp,nz[x]|zp}={(pn, xi)|i=0,,pn1}. let us consider an ideal i = mp,nz[x]=(pn, xi) in ip, n, with iz, i(modpn). it is quite easy to see that i contains (mp,z[x])n = mp, jn=(p, xj)n, where j{0,,p1 }, j(modp) (notice that ji(modp)). if n>1 this containment is strict, since xi(p, xj)n. we can group the ideals of ip, n according to their radical : there are p radicals of these pn ideals, namely the maximal ideals mp, j, j=0,,p1. this amounts to making a partition of the residue classes modulo pn into p different sets of elements congruent to j modulo p, for j=0,,p1 ; each of these sets has cardinality pn1. every ideal in ip, n, j is mp, j - primary and it contains the n - th power of its radical, namely mp, jn. now we want to compute the intersection of the ideals in ip, n, which is equal to the ideal ipn in z[x ] (see (1) and (2)). we can express this intersection as an intersection of mp, j - primary ideals as we have said above, in the following way (in the first equality we make use of eq.,p1qp, n, j whereqp, n, jij(modp)(pn, xi) (notice that the intersection is taken over the set { i{0,,pn1}|ij(modp) }). the ideal qp, n, j is an mp, j - primary ideal, for j=0,,p1, since the intersection of m - primary ideals is an m - primary ideal. we will omit the index p in qp, n, j and in mp, j if that will be clear from the context. the mp, j - primary ideal qn, j is just the intersection of the ideals in ip, n, j, according to the partition we made. it is equal to the set of polynomials in z[x ] which modulo pn are zero at the residue classes congruent to j modulo p (see (3) of remark 1). notice that there are no embedded components in this primary decomposition, since the prime ideals belonging to it (the minimal primes containing ipn) are { mj|j=0,,p1 }, which are maximal ideals. we recall that if i and j are two coprime ideals in a ring r, that is i+j = r, then ij = ij (in general only the inclusion ijij holds). the condition for two ideals i and j to be coprime amounts to saying that i and j are not contained in a same maximal ideal m, that is, i+j is not contained in any maximal ideal m. if m1 and m2 are two distinct maximal ideals then they are coprime, and the same holds for any of their respective powers. if r is noetherian, then every primary ideal q contains a power of its radical and moreover if the radical of q is maximal then also the converse holds (see). so if qi is an mi - primary ideal for i=1,2 and m1,m2 are distinct maximal ideals, then q1 and q2 are coprime. since { mj}j=0,,p1 are p distinct maximal ideals, for what we have just said above we havej=0, now we want to describe the mj - primary ideals qn, j, for j=0,,p1. the next lemma gives a relation of containment between these ideals and the n - th powers of their radicals. for each j=0,,p1, we haveqn, jmjn. let p be a fixed prime and n a positive integer. for each j=0,,p1, we haveqn, jmjn. as a consequence of this lemma, we get the following result : corollary 3.1let p be a fixed prime and n a positive integer. then we have : ipn(p,j=0,,p1(xj))n. proofby (4) and lemma 3.2 we haveipn=j=0,,p1qn, jj=0,,p1mjn where the last containment follows from lemma 3.2. finally, by lemma 2.2, the product of the ideals mjn is equal toj=0,,p1mjn=(p,j=0,,p1(xj))n. notice that the product of the mjs is actually equal to their intersection, since they are maximal coprime ideals. lemma 3.2 we haveipn=j=0,,p1qn, jj=0,,p1mjn where the last containment follows from lemma 3.2. finally, by lemma 2.2, the product of the ideals mjn is equal toj=0,,p1mjn=(p,j=0,,p1(xj))n. notice that the product of the mjs is actually equal to their intersection, since they are maximal coprime ideals. the last formula of the previous proof gives the primary decomposition of the ideal (p,j=0,,p1(xj))n.,p1 }, the reverse containment of lemma 3.2 does not hold, that is, the n - th power of mj can be strictly contained in the mj - primary ideal qn, j. for example (again, we use (3) to prove the containment):x(x2)(k=0,,3(23,x2k))(2,x)3. because of that, in general we do not have an equality in corollary 3.1.,pn1(xi)). see for example : p=2, n=2 : 2x(x1)i=0,,3(4,xi)(4,i=0, the reverse containment of lemma 3.2 does not hold, that is, the n - th power of mj can be strictly contained in the mj - primary ideal qn, j. for example (again, we use (3) to prove the containment):x(x2)(k=0,,3(23,x2k))(2,x)3. because of that, in general we do not have an equality in corollary 3.1.,pn1(xi)). see for example : p=2, n=2 : 2x(x1)i=0,,3(4,xi)(4,i=0,,3(xi)). we want to study under which conditions the ideal qn, j is equal to mjn. our aim is to find a set of generators for qn, j. for fqn, j, we have f(pn, xi) for each ij(modp), i{0,,pn1}. by (3) that means pn|f(i) for each such an i. equivalently, such a polynomial has the property that modulo pn it is zero at the pn1 residue classes of z / pnz which are congruent to j modulo p. without loss of generality, we proceed by considering the case j=0. we set m = m0=(p, x) and qn = qn,0=i0(modp)(pn, xi). let fqn, of degree m. we have(5)f(x)=q1(x)x+f(0) where q1z[x ] has degree equal to m1. since f(pn, xp), we have pn|f(p)=q1(p)p+f(0), so pn1|q1(p). by the euclidean algorithm,(6)q1(x)=q2(x)(xp)+q1(p) for some polynomial q2z[x ] of degree m2. since f(pn, x2p), we have pn|f(2p)=q2(2p)2p2+q1(p)2p+f(0). if p>2 then pn2|q2(2p), because pn|q1(p)2p+f(0). if p=2 then we can just say pn3|q2(2p). by the euclidean algorithm again, we haveq2(x)=q3(x)(x2p)+q2(2p) for some q3z[x ]. like before, if we set r2(x)=q2(2p)(xp)x+q1(p)x+f(0), we have r2mn if p>2, or r2qn if p=2. we define now the following family of polynomials : definition 3.1for each kn, k1, we setgp,0,k(x)=gk(x)h=0,,k1(xhp). notice that the polynomials gk(x), whose degree for each k is k, enjoy these properties : i)for every tz, gk(tp)=pkt(t1)(t(k1)). hence, the highest power of p which divides all the integers in the set { gk(tp)|tz } is pk+vp(k !). it is easy to see that k+vp(k!)=vp((pk)!).ii)gk(x)=(xkp)gk1(x).iii)since for every integer h, xhpm, we have gk(x)mk. we remark that k is the maximal integer with this property, since deg(gk)=k and gk(x) is primitive (since monic). for every tz, gk(tp)=pkt(t1)(t(k1)). hence, the highest power of p which divides all the integers in the set { gk(tp)|tz } is pk+vp(k !). it is easy to see that k+vp(k!)=vp((pk) !). since for every integer h, xhpm, we have gk(x)mk. we remark that k is the maximal integer with this property, since deg(gk)=k and gk(x) is primitive (since monic). recall that, by lemma 3.2, for every integer n we have qnmn. by property iii) above gkmn if and only if nk. by property i) we have gkqn if and only if k+vp(k!)n. from these remarks, it is very easy to deduce that, in the case pn, if gkqn then gkmn. in fact, if that is not the case, it follows from above that k p then rk1mn for k=1,,p and rk1qn for k = p+1,,m. let p be a prime and n a positive integer. let fqp, n,0=qn be of degree m. then for each 1km there exists qkz[x ] of degree mk such thatf(x)=qk(x)gk(x)+rk1(x) where rk1(x)h=1, moreover, for each such a k the following hold : i)pnvp((pk)!)|qk(kp), if vp((pk)!) p then rk1mn for k=1,,p and rk1qn for k = p+1,,m. qk(kp)gk(x)qn and if k m (this assumption is not restrictive, since xnqn):(8)f(x)=qmgm(x)+rm1(x)=qmgm(x)+k=1, the next proposition determines the primary components qn, j of ipn of (4) in the case pn. it shows that in this case the containment of lemma 3.2 is indeed an equality. proposition 3.1let pz be a prime and n a positive integer such that pn. proofit is sufficient to prove the statement for j=0 : for the other cases we consider the z[x]-automorphisms j(x)=xj, for j=1,,p1, which permute the ideals qn, j and mj. let qn = qn,0 and m = m0.the inclusion () follows from lemma 3.2. for the other inclusion (), let f(x) be in qn. we can assume that the degree m of f(x) is less than n, since xn is the smallest monic monomial in qn. (8) above, f(x) is in mn, since pnm divides qm, gmmm and rm1mn by lemma 3.3 (notice that m1<p). let pz be a prime and n a positive integer such that pn. it is sufficient to prove the statement for j=0 : for the other cases we consider the z[x]-automorphisms j(x)=xj, for j=1,,p1, which permute the ideals qn, j and mj. the inclusion () follows from lemma 3.2. for the other inclusion (), we can assume that the degree m of f(x) is less than n, since xn is the smallest monic monomial in qn. (8) above, f(x) is in mn, since pnm divides qm, gmmm and rm1mn by lemma 3.3 (notice that m1<p). remark 4 in the case pn, lemma 3.3 implies that qn is generated by { pnmgm(x)}0mn : it is easy to verify that these polynomials are in qn (using (3) again) and (8) implies that every polynomial fqn is a z - linear combination of { pnmgm(x)}0mn, since qm(mp) is divisible by pnm, for each of the relevant m. in the case pn, lemma 3.3 implies that qn is generated by { pnmgm(x)}0mn : it is easy to verify that these polynomials are in qn (using (3) again) and (8) implies that every polynomial fqn is a z - linear combination of { pnmgm(x)}0mn, since qm(mp) is divisible by pnm, for each of the relevant m. the following theorem gives a description of the ideal ipn in the case pn. in this case the containment of corollary 3.1 becomes an equality. then the ideal in z[x ] of those polynomials whose fixed divisor is divisible by pn is equal toipn=(p,i=0,,p1(xi))n. proofby proposition 3.1, for each j=0,,p1 the ideal qn, j is equal to mjn. so, by the last formula of the proof of corollary 3.1, we get the statement. let pz be a prime and n a positive integer such that pn. then the ideal in z[x ] of those polynomials whose fixed divisor is divisible by pn is equal toipn=(p,i=0,,p1(xi))n. by proposition 3.1, for each j=0,,p1 the ideal qn, j is equal to mjn. so, by the last formula of the proof of corollary 3.1, we get the statement. as a consequence, we have the following remark. let p be a prime and n a positive integer less than or equal to p. let fipn such that the content of f(x) is not divisible by p. then deg(f)np, since np = deg(i=0,,p1(xi)n). another well - known result in this context is the following : if we fix the degree d of such a polynomial f, then the maximum n such that fipn is bounded by nk1[d / pk]=vp(d !). if we drop the assumption pn, the ideal qn, j may strictly contain mjn, as we observed in remark 3. the next proposition shows that this is always the case, if p < n. this result follows from lemma 3.3 as proposition 3.1 does, and it covers the remaining case p < proposition 3.2let pz be a prime and n a positive integer such that p < n.,qn, n1gn1(x)) where, for each k = p,,n1, qn, k is an integer defined as follows : qn, k{pnvp((pk)!),ifvp((pk)!)<n,1,otherwise. we have to show that the polynomials qn, kgk(x), for k{p,,n1 }, lie in qn. this follows from property i) of the polynomials gk(x) and the definition of qn, m.now we prove the other containment (). let fqn be of degree m. if m < p then fmn (see lemma 3.3 and in particular (8)). we have(9)f(x)=k = p,,mqh(hp)gh(x)+rp1(x) where rp1(x)=k=1,,p1qk(hp)gh(x)mn and qmz, so that qm(mp)=qn, m. then, since qn, k = pnvp((pk)!)|qk(kp) if vp((pk)!)<n, it follows that the first sum on the right - hand side of the previous equation belongs to the ideal (qn, pgp(x),,qn, n1gn1(x)). for the last sentence of the proposition, we remark that the polynomials { qn, kgk(x)}k = p,,n1 are not contained in mn : in fact, for each k{p,,n1 }, by property iii) of the polynomials gk(x) we have that the minimal integer n such that qn, kgk(x) is contained in mn is nvp(k !) if vp((pk)!)=k+vp(k!)<n and it is k otherwise. in both cases it is strictly less than n (since vp(k!)1, if kp). let pz be a prime and n a positive integer such that p < n.,qn, n1gn1(x)) where, for each k = p,,n1, qn, k is an integer defined as follows : qn, k{pnvp((pk)!),ifvp((pk)!)<n,1,otherwise. we have to show that the polynomials qn, kgk(x), for k{p,,n1 }, lie in qn. this follows from property i) of the polynomials gk(x) and the definition of qn, m. let fqn be of degree m. if m < p then fmn (see lemma 3.3 and in particular (8)). we have(9)f(x)=k = p,,mqh(hp)gh(x)+rp1(x) where rp1(x)=k=1,,p1qk(hp)gh(x)mn and qmz, so that qm(mp)=qn, m. then, since qn, k = pnvp((pk)!)|qk(kp) if vp((pk)!)<n, it follows that the first sum on the right - hand side of the previous equation belongs to the ideal (qn, pgp(x),,qn, n1gn1(x)). for the last sentence of the proposition, we remark that the polynomials { qn, kgk(x)}k = p,,n1 are not contained in mn : in fact, for each k{p,,n1 }, by property iii) of the polynomials gk(x) we have that the minimal integer n such that qn, kgk(x) is contained in mn is nvp(k !) if vp((pk)!)=k+vp(k!)<n and it is k otherwise. in both cases it is strictly less than n (since vp(k!)1, if kp). remark 5the following remark allows us to obtain another set of generators for qn. we set(10)m=m(n, p)min{mn|vp((pm)!)n}. remember that vp((pm)!)=m+vp(m !). if pn then m=n and if p < then for each m{m,,n } we have vp((pm)!)n, since the function e(m)=m+vp(m !) is increasing. so for each such m we have qn, m=1, hence gm(gm(x)). so we have the equalities:(11)qn = mn+(qn, mgm(x)|m = p,,m)=(qn, mgm(x)|m=0,,m) where qn, m = pnm, for m=0,,p1, and for m = p,,m is defined as in the statement of proposition 3.2. the containment () is just an easy verification using the properties of the polynomials gm(x) ; the other containment follows by (9). we set(10)m=m(n, p)min{mn|vp((pm)!)n}. remember that vp((pm)!)=m+vp(m !). if pn then m=n and if p < then for each m{m,,n } we have vp((pm)!)n, since the function e(m)=m+vp(m !) is increasing. so for each such m we have qn, m=1, hence gm(gm(x)). so we have the equalities:(11)qn = mn+(qn, mgm(x)|m = p,,m)=(qn, mgm(x)|m=0,,m) where qn, m = pnm, for m=0,,p1, and for m = p,,m is defined as in the statement of proposition 3.2. the containment () is just an easy verification using the properties of the polynomials gm(x) ; the other containment follows by (9). we can now group together proposition 3.1, proposition 3.2 into the following one : proposition 3.3let pz be a prime and n a positive integer.,qn, mgm(x)) where m=min{mn|vp((pm)!)n } and for each m=0,,m, qn, m is an integer defined as follows : qn, m{pnvp((pm)!),m < m,1,m = m. let pz be a prime and n a positive integer,qn, mgm(x)) where m=min{mn|vp((pm)!)n } and for each m=0,,m, qn, m is an integer defined as follows : qn, m{pnvp((pm)!),m < m,1,m = m. it is clear what the primary ideals qj, for j=1,,p1, look like : qn, j=ij(modp)(pn, xi)=mjn+(qn, pgp(xj),,qn, mgm(xj))=(qn,0g0(xj),,qn, mgm(xj)). in fact, for each j=1,,p1, it is sufficient to consider the automorphisms of z[x ] given by j(x)=xj. moreover, (qn,0)=qn, j and (m0)=mj for each such a j, so that j permutes the primary components of the ideal ipn. the ideal ipn = pnint(z)z[x ] was studied in in a slightly different context, as the kernel of the natural map n : z[x]n, where the latter is the set of functions from z / pnz to itself. in that article a recursive formula is given for a set of generators of this ideal. for other works about the ideal ipn in a slightly different context, see,,. this ideal is important in the study of the problem of the polynomial representation of a function from z / pnz to itself. as a corollary we give an explicit expression for the ideal ipn in the case n = p+1. by proposition 3.2 the primary components of ipp+1 are(12)qp+1,j = mjp+1+(gp(xj)) for j=0,,p1.,p21(xi). we want to stress that the polynomial h(x) is not contained in the first ideal of the right - hand side of the statement. in a similar result is stated with another polynomial h2(x) instead of our h(x). indeed the two polynomials, as already remarked in, are congruent modulo the ideal (p,i=0,,p1(xi))p+1. proof of corollary 4.1like before, we set qp, p+1,j = qp+1,j. the containment () follows from corollary 3.1 and because the polynomial h(x) is equal to j=0,,p1gp(xj) and for each j=0,,p1 the polynomial gp(xj) is in qp+1,j by proposition 3.2. since qp+1,j, for j=0,,p1, are exactly the primary components of ipp+1 (see (4)), we get the claim.now we prove the other containment ().,p1qp+1,j. by (12) we have : f(x)cp, j(x)gp(xj)(modmjp+1) for some cp, jz[x ], for j=0,,p1.since the ideals { mjp+1=(p, xj)p+1|j=0,,p1 } are pairwise coprime (because they are powers of distinct maximal ideals, respectively), by the chinese remainder theorem we have the following isomorphism:(13)z[x]/(j=0p1mjp+1)z[x]/m0p+1z[x]/mp1p+1.we need now the following lemma, which tells us what is the residue of the polynomial h(x) modulo each ideal mjp+1:lemma 4.1let p be a prime and let h(x)=j=0,,p1 we haveh(x)gp(xk)(modmkp+1).prooflet k{0,,p1 } and set ik={0,,p1}{k}. for each jik we have gp(kj)(kj)p(modp). since gp(xk)mkp we have just to prove that tk(x)=1+jikgp(xj)mk. by formula (3) in remark 1 it is sufficient to prove that tk(k) is divisible by p. we havetk(k)1+jik(kj)p(modp)1+(s=1, we finish now the proof of the corollary.by the chinese remainder theorem, there exists a polynomial pz[x ] such that p(x)cp, j(x)(modmjp+1), for each j=0,,p1. then by the previous lemma p(x)h(x)cp, j(x)gp(xj)(modmjp+1) and so again by the isomorphism (13) above we havef(x)p(x)h(x)(modj=0,,p1mjp+1) so we are done since j=0,,p1mjp+1=(p,i=0,,p1(xi))p+1 (see the proof of corollary 3.1). the containment () follows from corollary 3.1 and because the polynomial h(x) is equal to j=0,,p1 the polynomial gp(xj) is in qp+1,j by proposition 3.2. since qp+1,j, for j=0,,p1, are exactly the primary components of ipp+1 (see (4)), we get the claim.,p1qp+1,j. by (12) we have : f(x)cp, j(x)gp(xj)(modmjp+1) for some cp, jz[x ], for j=0,,p1. since the ideals { mjp+1=(p, xj)p+1|j=0,,p1 } are pairwise coprime (because they are powers of distinct maximal ideals, respectively), by the chinese remainder theorem we have the following isomorphism:(13)z[x]/(j=0p1mjp+1)z[x]/m0p+1z[x]/mp1p+1. we need now the following lemma, which tells us what is the residue of the polynomial h(x) modulo each ideal mjp+1 : lemma 4.1let p be a prime and let h(x)=j=0, since gp(xk)mkp we have just to prove that tk(x)=1+jikgp(xj)mk. by formula (3) in remark 1 it is sufficient to prove that tk(k) is divisible by p. we havetk(k)1+jik(kj)p(modp)1+(s=1, by formula (3) in remark 1 it is sufficient to prove that tk(k) is divisible by p. we havetk(k)1+jik(kj)p(modp)1+(s=1, we finish now the proof of the corollary. by the chinese remainder theorem, there exists a polynomial pz[x ] such that p(x)cp, j(x)(modmjp+1), for each j=0,,p1. then by the previous lemma p(x)h(x)cp, j(x)gp(xj)(modmjp+1) and so again by the isomorphism (13) above we havef(x)p(x)h(x)(modj=0,,p1mjp+1) so we are done since j=0,,p1mjp+1=(p,i=0,,p1(xi))p+1 (see the proof of corollary 3.1). | we characterize the fixed divisor of a polynomial f(x) in z[x ] by looking at the contraction of the powers of the maximal ideals of the overring int(z) containing f(x). given a prime p and a positive integer n, we also obtain a complete description of the ideal of polynomials in z[x ] whose fixed divisor is divisible by pn in terms of its primary components. |
systemic embolization is an unfortunate sequel of infective endocarditis (ie) that can occur despite an appropriate therapy. cardiac embolization is seen in one - third to one - half of the patients, and the most common complication is heart failure (more common in aortic valve ie than mitral valve). coronary embolization is not uncommon, and micro - emboli are seen in up to two - third of patients at autopsy. despite the high incidence of coronary embolism, clinically significant transmural infarcts are very rare with ie. a 69-year - old male presented to the mount sinai saint luke 's emergency department with intermittent fever and increased urinary frequency for 1 week. his past medical history was significant for streptococcus sanguinis tricuspid valve endocarditis, bioprosthetic aortic valve replacement, coronary artery disease (status of post - triple vessel coronary artery bypass grafting), paroxysmal atrial fibrillation (afib), and type-2 diabetes mellitus. on physical examination, his vitals were notable for an irregular pulse of 112 beats / min, blood pressure of 136/77 mm hg, respiratory rate of 18/min, rectal temperature of 98f, and oxygen saturations of 98% on room air. there was a cardiac grade 2/6 ejection systolic murmur 6 cm of water at 45 position. laboratory studies were significant for glucose 204 mg / dl and troponin - i 0.05 ng / ml (normal < 0.034 ng / ml). urinalysis was positive for leukocyte esterase and pyuria with 3550 white blood cells / high - power field. electrocardiogram (ecg) on admission showed sinus rhythm with first degree av block and frequent premature atrial complexes. the patient was administered one dose of levofloxacin after drawing blood cultures which came back positive for gram - positive cocci. on the next day, his antibiotics were changed to vancomycin and gentamicin for suspected endocarditis. on day 1 of admission, the patient developed acute pulmonary edema and hypoxia following an episode of sub - sternal crushing chest pain. repeat ecg showed afib with rapid ventricular response and st - depressions in leads ii, avf, and v2v6[figure 1 ]. he was managed with nitroglycerine, furosemide, noninvasive positive pressure ventilation, and was transferred to cardiac care unit. immediate trans - thoracic echocardiogram (tte) did not show any vegetation or aortic insufficiency. his next troponin - i came back to 10.19 ng / ml and ck - mb was 163.8 ng / ml (0.54.7 ng / ml). he was treated for non - st elevation myocardial infarction with intravenous heparin, aspirin, clopidogrel, statin, and a beta blocker. the patient could not tolerate transesophageal echocardiography (tee) and had a repeat tte, which showed inferior and lateral akinesis, moderate systolic dysfunction, and probable vegetation on the noncoronary cusp of aortic valve. electrocardiogram demonstrating atrial fibrillation with rapid ventricular response and st - depressions in leads ii, avf, and v2v6 on day 3 of admission, blood cultures grew enterococcus faecalis. his troponin - i level peaked at 172.09 ng / ml. a presumptive diagnosis of enterococcal endocarditis of prosthetic aortic valve with embolic myocardial infarction from vegetation was established. eventually, the patient underwent tee. it showed moderately decreased left ventricular systolic function with an ejection fraction of 40%, regional wall motion abnormalities with mid and basal anterior and lateral akinesis, bioprosthetic aortic valve with an abscess (thickened intervalvular fibrosa), and small vegetation on the left coronary cusp. coronary angiography revealed new 99% occlusion of the left main coronary artery (lmca) and proximal left circumflex artery, chronic total occlusion of ostial left anterior descending artery (lad) and right coronary artery, patent saphenous vein graft to posterior descending artery, and patent left internal mammary artery graft to the lad artery. the patient improved clinically on medical management and stayed hemodynamically stable. on day 9 of admission, the patient underwent revision of aortic valve replacement, drainage of annular abscess, and lmca embolectomy. his aortic valve as well as the embolic debris was removed from lmca grew e. faecalis. subsequently, he was discharged to the skilled nursing facility in a stable condition where he completed 6 weeks of intravenous ampicillin. prosthetic valve endocarditis (pve) is a serious and potentially life - threatening infection. pve develops in 14% of patients during the 1 year following valve replacement, and in approximately 1% per year thereafter. it is usually hospital - acquired and common pathogens are coagulase - negative staphylococcus and s. aureus. pve developing 12 months after surgery is termed as late pve, which is usually community - acquired and common pathogens include streptococcal species and s. aureus. enterococcus faecalis is a nonmotile, facultative anaerobe with plasmid - encoded hemolysin called cytolysin, which is important for pathogenesis in animal models of infection. enterococcal species are the third leading cause of native valve endocarditis causing 510% of the early pve and 812% of late pve cases. enterococcal endocarditis has been reported in patients with systemic lupus erythematosus, heroin addicts, following legal abortion and complicating colonoscopy in heyde 's syndrome. only six cases have been reported of coronary embolism from ie resulting in sudden cardiac death secondary to lmca occlusion, left ventricular rupture, and lmca embolism after abortion. coronary embolism secondary to enterococcal endocarditis has been reported with papillary muscle rupture due to circumflex artery occlusion arising from native aortic valve vegetation. lmca embolism from vegetative material is extremely rare, and fatal reports have been described with native aortic valve and mitral valve endocarditis, and none of them were conclusively enterococcal. use of thrombolytics has been largely unfavorable due to increased risk of intracerebral hemorrhage which is mainly due to mycotic aneurysms and cerebral infarcts, which are sequels of ie. percutaneous transluminal coronary angioplasty carries a risk of re - occlusion due to a mobile embolus, risk of distal embolization, and development of mycotic aneurysm at the balloon dilation site. furthermore, inserting a stent in bacteremic patients is also controversial due to the risk of seeding of the stent. good prognosis can be achieved with surgery in patients with native valve endocarditis and pve, who have moderate to severe heart failure and in patients with late pve. patients with ie and large vegetation have lower mortality and embolic events if an approach with early surgical intervention is undertaken. in our patient, previous coronary artery bypass graft with good coronary flow resulted in a good prognosis despite occlusion of lmca by the vegetation. to our research, this is the first case of lmca embolism resulting from pve with e. faecalis. embolic coronary occlusion should be included in the differential diagnosis of chest pain and increased troponin levels in patients with confirmed or suspected ie because the therapeutic implications are complex and usual measures such as thrombolysis may be dangerous. | context : coronary embolization is potentially a fatal sequela of endocarditis. although the primary cause of acute coronary syndrome is atherosclerotic disease, it is imperative to consider septic embolism as an etiological factor.case report : herein, we report a case of ventricular fibrillation and st - segment depression myocardial infarction occurring in a patient who initially presented with fever and increased urinary frequency. coronary angiography revealed new 99% occlusion of the left main coronary artery (lmca). transesophageal echocardiography showed bioprosthetic aortic valve with an abscess and vegetation. histologic examination of the embolectomy specimen confirmed the presence of thrombus and enterococcus faecalis bacteria. subsequently, the patient was discharged to the skilled nursing facility in a stable condition where he completed 6 weeks of intravenous ampicillin.conclusion:we present a rare case of lmca embolism due to prosthetic valve endocarditis. the present report also highlights the diagnostic and therapeutic challenges associated with such patients. |
lentiviral vectors represent an extremely effective way of introducing dna into both dividing and quiescent cells (reviewed in refs. these vectors carry an enveloped rna genome which is reverse transcribed into dna upon cell entry, this resulting proviral dna : host / viral protein complex is then shuttled to the nucleus and ultimately integrated into the host genome, allowing for long - term expression. although integration was once thought to be a mandatory step for viral stability and expression, this has been successfully challenged by several authors who have reported efficient gene expression in vitro and in vivo using integration - defective lentiviral vectors(idlvs ; reviewed in ref. furthermore, in some studies, idlvs carrying a therapeutic expression cassette have shown efficacy in rodent disease models. idlvs are an attractive alternative to integrating vectors since they offer a reduced risk of insertional mutagenesis and replication - competent lentivirus generation, and additionally they may be less prone to gene silencing or chromatin - regulated expression. however, a disadvantage of using such vectors is that long - term expression may be limited to nondividing cells since expression from these vectors almost entirely originates from the unintegrated episomal forms of the virus that are rapidly lost by dilution in dividing cells. there are two classes of integrase mutations ; class i mutations specifically affect the integration process whereas class ii mutations affect integration and other viral processes such as packaging. the class i mutations can be in the coding sequence for the integrase gene, or at sites in the viral genome to which integrase attaches and mediates insertion (att sites). the most effective class i mutations are those within the catalytic triad, three residues at positions d64, d116, and e152 that are conserved absolutely among all retroelements. a single mutation in the d64 residue has been shown to have the largest single effect on integrase activity, and may abolish it completely. nevertheless, idlvs with a single mutation in d64 still possess a low level of residual integration activity (190-fold) in the idlvs compared with the parental vectors, confirming that the integration process had been attenuated (table 1). to confirm this in a biological assay, human hek293 t cells were transduced with each of the vectors and the percentage of gfp - positive cells was determined over time (figure 2). while the percentage of gfp - positive cells transduced with the parental eiav- and hiv-1integrating vectors remained stable at 60/70% respectively for the duration of the 12-day experiment, both the single and triple mutant hiv-1 and eiav idlvs showed a rapid decrease in the proportion of gfp - positive cells to 150-fold lower than in the cells transduced with the parental - integrating vector. the decrease in gfp - positive cells in the hiv-1 and eiav idlv transduced population is due to loss of unintegrated viral genomes through dilution associated with hek293 t cell division, and confirms the lack of effective integrase activity in the idlv vectors. the transduction and expression capabilities of the hiv-1 and eiav idlvs were compared with the parental vectors in differentiated nondividing skeletal muscle (c2c12) and neuronal (pc12) cells in vitro. transduced differentiated cells were assayed for gfp expression by fluorescence - activated cell sorting analysis. the percentage of gfp - positive cells and the mean fluorescence intensity (mfi) increased and subsequently stabilized at around 2 weeks for all vectors (figures 3 and 4). in the c2c12 differentiated cell line, the hiv-1 and eiav idlvs gave rise to a lower percentage of gfp - positive cells with a lower mfi compared with the parental - integrating vector (figure 3). in particular, the triple mutant eiav idlvs gave rise to a lower percentage of gfp - positive cells with a lower mfi compared with the single mutant, whereas there was no significant difference between the hiv-1 single and triple idlv mutants. interestingly, in the differentiated pc12 cells the level of gene expression mediated by the hiv-1 idlvs was comparable to the parental hiv-1 vector, although transduction efficiency was still reduced. in contrast, both transduction efficiency and expression level with both the single and triple eiav idlvs remained poor compared with the parental vector (figure 4). since gfp expression in differentiated pc12 cells mediated by the single and triple mutant hiv-1 idlvs was comparable to the parental hiv-1 vector we wanted to confirm that this was not due to residual integration activity. therefore, differentiated pc12 cells transduced with either the hiv-1 single or triple mutant idlvs or the parental hiv-1 vector were dedifferentiated at 2 weeks post - transduction (the time of maximal transduction and mfi). removal of nerve growth factor (ngf) was followed by an increase in the proportion of cells that were gfp - positive (figure 5) ; the exact cause of this is not known, but may be due to these cells becoming more metabolically active upon cell cycle re - entry. as the cells resumed division (i.e., only after the cells were first passaged, not upon removal of ngf), there was a concurrent loss of gfp expression in cells transduced with both hiv-1 idlvs, whereas the gfp expression in cells transduced with parental vector remained stable. the loss of gfp expression in dedifferentiated pc12 cells transduced with the hiv-1 idlvs confirms that expression was exclusively due to unintegrated idlvgenomes. we attempted to confirm this by integration - specific pcr to vector sequences but unfortunately this assay was not sufficiently sensitive or robust for this (data not shown). each of the eiav and hiv-1 single and triple integrase mutants and parental vectors shown in table 1 were stereotactically injected at a volume of 5 l into the striatum of the adult rat brain. although the hiv-1 parental vector had approximately sevenfold higher dna integration titer than the eiav parental vector (table 1), the parental eiav vector had significantly greater mean striatal fluorescence (figure 6). the expression of the gfp reporter gene was detected to some degree in both the eiav- and hiv-1 idlv - treated brains. however, the mean striatal fluorescence in the single and triple mutant eiav idlv - treated brains was poor and statistically lower than the fluorescence in the parental eiav vector - treated brain (figure 6). in contrast, the hiv-1 single integrase mutant idlv - treated brain showed a mean striatal fluorescence level that was comparable to the level observed with the parental hiv-1 vector. however, the triple integrase mutant hiv-1 idlv showed a relatively poor mean striatal fluorescence level that had a similar intensity to both of the eiav idlvs (figure 6b). the aim of this study was to compare the transduction and expression characteristics of well - known hiv-1 class i integrase mutant vectors with equivalent mutations in an eiav vector (figure 1). the mutations were made in the hiv-1 and eiav integrase genes in the so - called catalytic triad at amino acid positions d64, d116, and e152 ; these three residues are conserved absolutely across all reverse - transcribed elements, and are known to have the greatest effect on integration with no apparent effect on other viral processes. the characteristics of the hiv-1 and eiav idlvs with single (d64v) and triple (dde / vig or vaa, respectively) integrase mutations were compared with the equivalent parental integration - competent hiv-1 and eiav vectors. the dde / vig hiv triple integrase mutant was constructed as these residues have previously been shown to be completely integrase - defective. the dde / vaa eiav triple integrase mutant was used as this has also been shown to be integrase - defective. all vector genomes were constructed using identical configurations (spleen focus - forming virus promoter, enhanced green fluorescent protein (egfp) reporter gene, woodchuck hepatitis virus post - transcriptional regulatory element, central polypurine tract (cppt)/cts sequences, self - inactivating long terminal repeats (ltrs)). while the levels of rna genome and reverse transcriptase (rt) in the idlv vector preparations were similar compared with the parental vectors (table 1), as expected the dna integration titers were significantly reduced (by > 190-fold) in the idlvs compared with the parental vectors, confirming that the integration process had been attenuated (table 1). to confirm this in a biological assay, human hek293 t cells were transduced with each of the vectors and the percentage of gfp - positive cells was determined over time (figure 2). while the percentage of gfp - positive cells transduced with the parental eiav- and hiv-1integrating vectors remained stable at 60/70% respectively for the duration of the 12-day experiment, both the single and triple mutant hiv-1 and eiav idlvs showed a rapid decrease in the proportion of gfp - positive cells to 150-fold lower than in the cells transduced with the parental - integrating vector. the decrease in gfp - positive cells in the hiv-1 and eiav idlv transduced population is due to loss of unintegrated viral genomes through dilution associated with hek293 t cell division, and confirms the lack of effective integrase activity in the idlv vectors. the transduction and expression capabilities of the hiv-1 and eiav idlvs were compared with the parental vectors in differentiated nondividing skeletal muscle (c2c12) and neuronal (pc12) cells in vitro. transduced differentiated cells were assayed for gfp expression by fluorescence - activated cell sorting analysis. the percentage of gfp - positive cells and the mean fluorescence intensity (mfi) increased and subsequently stabilized at around 2 weeks for all vectors (figures 3 and 4). in the c2c12 differentiated cell line, the hiv-1 and eiav idlvs gave rise to a lower percentage of gfp - positive cells with a lower mfi compared with the parental - integrating vector (figure 3). in particular, the triple mutant eiav idlvs gave rise to a lower percentage of gfp - positive cells with a lower mfi compared with the single mutant, whereas there was no significant difference between the hiv-1 single and triple idlv mutants. interestingly, in the differentiated pc12 cells the level of gene expression mediated by the hiv-1 idlvs was comparable to the parental hiv-1 vector, although transduction efficiency was still reduced. in contrast, both transduction efficiency and expression level with both the single and triple eiav idlvs remained poor compared with the parental vector (figure 4). since gfp expression in differentiated pc12 cells mediated by the single and triple mutant hiv-1 idlvs was comparable to the parental hiv-1 vector we wanted to confirm that this was not due to residual integration activity. therefore, differentiated pc12 cells transduced with either the hiv-1 single or triple mutant idlvs or the parental hiv-1 vector were dedifferentiated at 2 weeks post - transduction (the time of maximal transduction and mfi). removal of nerve growth factor (ngf) was followed by an increase in the proportion of cells that were gfp - positive (figure 5) ; the exact cause of this is not known, but may be due to these cells becoming more metabolically active upon cell cycle re - entry. as the cells resumed division (i.e., only after the cells were first passaged, not upon removal of ngf), there was a concurrent loss of gfp expression in cells transduced with both hiv-1 idlvs, whereas the gfp expression in cells transduced with parental vector remained stable. the loss of gfp expression in dedifferentiated pc12 cells transduced with the hiv-1 idlvs confirms that expression was exclusively due to unintegrated idlvgenomes. we attempted to confirm this by integration - specific pcr to vector sequences but unfortunately this assay was not sufficiently sensitive or robust for this (data not shown). each of the eiav and hiv-1 single and triple integrase mutants and parental vectors shown in table 1 were stereotactically injected at a volume of 5 l into the striatum of the adult rat brain. one month later, the brains were removed for the evaluation of egfp expression. although the hiv-1 parental vector had approximately sevenfold higher dna integration titer than the eiav parental vector (table 1), the parental eiav vector had significantly greater mean striatal fluorescence (figure 6). the expression of the gfp reporter gene was detected to some degree in both the eiav- and hiv-1 idlv - treated brains. however, the mean striatal fluorescence in the single and triple mutant eiav idlv - treated brains was poor and statistically lower than the fluorescence in the parental eiav vector - treated brain (figure 6). in contrast, the hiv-1 single integrase mutant idlv - treated brain showed a mean striatal fluorescence level that was comparable to the level observed with the parental hiv-1 vector. however, the triple integrase mutant hiv-1 idlv showed a relatively poor mean striatal fluorescence level that had a similar intensity to both of the eiav idlvs (figure 6b). there continues to be considerable interest in idlvs which is unsurprising given their potential advantages for gene therapy for significantly reducing the risk of insertional mutagenesis and the potential to generate replication - competent lentivirus. interestingly, early reports using idlvs failed to show any significant associated expression suggesting that transgene expression from the episomal genome was extremely inefficient. however, more recently, unequivocal gene expression from cells transduced with hiv-1 idlvs has been demonstrated (reviewed in refs. 10,31). the reasons for the early failure of hiv-1 idlvs to produce significant gene expression point to design differences in the first generation vectors, including lack of cppt, self - inactivating ltrs or woodchuck hepatitis virus post - transcriptional regulatory element and the more recent use of stronger constitutive promoters or non - hiv vectors (such as feline immunodeficiency virus (fiv) and eiav), or combinations of these. in this study, we compared the gene transfer and expression characteristics of matched hiv-1 and eiav idlvs. as expected, in dividing hek293 t cells transduced with the hiv-1 or eiav idlvs, the number of gfp - positive cells was rapidly reduced as the nonintegrated genome was lost by dilution as the cell divides, confirming that integrase activity was attenuated in the idlv vectors. both hiv-1 and eiav single and triple mutant idlvs gave rise to a relatively lower percentage of gfp - positive cells (i.e., transduction efficiency) with lower mean egfp levels compared with the parental vectors in differentiated c2c12 cells. a previous study has shown that the hiv-1 single mutant (d64v) idlv gave a better transduction efficiency in this cell type than the parental hiv-1 vector. the discrepancy with our results may simply reflect the differences in the vector titration methods between these studies, however, as only the transduction efficiency (and not the mean fluorescence) was previously reported, this remains to be determined. interestingly, both the single and triple mutant hiv-1 idlvs - transduced differentiated pc12 neuronal cells to give an expression level that was comparable to the parental hiv-1 vector, albeit at a slightly lower transduction efficiency. moreover, when these cells were dedifferentiated and cell division restored, gfp fluorescence was lost over time, confirming that expression was due to episomally maintained nonintegrated genome. in the case of the single mutant hiv-1 idlv, this result was replicated in vivo in neurons following a stereotactic injection into the rat striatum with strong egfp expression that was equivalent to that seen in the rat brain treated with the parental hiv-1 vector, which has been observed previously for this and other integrase mutations. however, stereotactic injection of the triple mutant hiv-1 idlv that contains a completely mutated integrase catalytic triad did not lead to significant egfp expression in the rat brain. therefore, although both the single and triple hiv-1 idlvs expressed efficiently in nondividing neuronal pc12 cells in vitro, it appears that the additional mutations (ddevig) in the catalytic triad prohibit effective egfp gene expression in the rat brain, suggesting that results obtained with idlvs in vitro should be treated with caution, as they may not be replicated in vivo. in contrast to the hiv-1 idlvs, both the single and the triple mutant eiav idlvs led to poor egfp expression in vitro and in vivo in nondividing neuronal pc12 cells and in the rat brain. this is in agreement with previous studies using these eiav integrase mutations and other studies using nonhuman / primate lentivirus - based vectors (reviewed in ref. 31), and is in stark contrast to the robust expression and therapeutic efficacy demonstrated with hiv-1 and simian immunodeficiency virus (siv) idlvs. this raises the possibility that efficient expression from idlvs may be restricted to human and primate lentivirus - based vectors, in vivo at least. moreover, there seem to be differences in the properties between different nonhuman / primate lentiviral idlvs ; some are ineffective both in vitro and in vivo, such as the eiav idlvs shown here, whereas others such as fiv idlvs have been shown to function well in vitro but failed to show expression in vivo following subretinal injection in the neonate rat. these observations may be highlighting differences in key components of these different lentiviral vectors that are involved in nonintegrative lentiviral expression, and comparison of the similarities / differences of these vector systems with hiv-1/siv idlvs may shed light on the mechanism behind this phenomenon. only when this mechanism has been elucidated will it be possible to ascertain whether other gene therapy platforms can be modified to function in a nonintegrative form. so what could be behind the mechanism involved in non - integrative expression from hiv-1/siv vectors that is not present with other lentiviral vectors ? it could be that there are higher levels of episomal viral dna formed with hiv-1/siv vectors compared with other lentiviral vectors, and/or it could be due to some interaction between a cellular factor and the hiv-1/siv viral genome / protein complex specifically. in support of this, one study has implicated the cppt in the production of the hiv-1 circular vdna, and interestingly, there are differences between hiv-1/siv and both eiav and fiv ppt elements (the cppt and ppt sequences are identical in hiv-1 and some siv subtypes, but are different in eiav and fiv). however, when the cppt was removed from the hiv-1 vectors in the current studies, there was no significant reduction in expression in the pc12 cells in vitro (data not shown), this has also been demonstrated in vivo. although idlv reduce the potential risk of insertional mutagenesis, the magnitude of this benefit is difficult to determine because it is difficult to accurately calculate the level of residual integration, with studies ranging from 1:100 to 1:10,000 compared with the parental vector. the reason for this range is most likely due to the methods used to determine integration. pcr - based methods to quantitate the level of integrated virus tend to show the residual activity at the lower end of this scale (as our measurements of 200- to 400-fold also show) whereas measurements based on selection for a biological phenotype (such as an antibiotic marker or reporter gene) are at the upper end. this is perhaps unsurprising, since assays based on selection will be subject to a basal expression level (below which selection / detection will fail), so quantitation by this method will be an underestimate of the true integration rate. this is true in the current study if we used the facs data instead (figure 2) to calculate titer based on gfp expression. the titer of the idlvs then falls from 200- to 400-fold less than the parental - integrating vector based on integration titer to 500- to 1,000-fold less based on biological titer, presumably due to very low expressing integrants being below the level of detection. these data add to the growing body of literature that shows hiv-1 idlv is a viable alternative to the integrating vector in certain nondividing cells, at least in the short term. consistent with other studies using hiv-1 vectors with similar components, we have shown that the hiv-1 d64v mutant idlv produces comparable transgene levels to the parental vector in vitro and in vivo. the only apparent penalty for the loss of integrase activity seems to be a modest reduction in transduction efficiency, which might be overcome practically by simply increasing dose. however, the triple integrase mutant hiv-1 idlv is not effective in vivo which is surprising and, to our knowledge, this has been shown for the first time. conversely, although the parental eiav vector is more effective than the hiv-1 vector at transducing the rat striatum in vivo, the eiav idlvs, like fiv, are relatively ineffective. in fact, only human and primate lentiviral vector platforms have been shown so far to be amenable to these mutations in vivo to date, and this raises the possibility that this phenomenon may be restricted to these groups of lentiviruses. furthermore, of the in vivo studies using idlvs that have shown good expression or efficacy, only one used a non - hiv-1 idlv vector. used siv idlvs to show comparable immunization to the parental vector ; however, as immune response only (and not transgene level) was measured, this study did not show that siv idlv produced equivalent levels of transgene expression. it is therefore possible, that efficient idlv expression is a property of hiv-1 vectors exclusively, although this remains to be determined. in summary, we have shown that effective expression from idlvs is dependent on the type of tissue targeted, the integrase mutation(s), and the nature of lentiviral vector platform. further work into the precise mechanisms involved will be important to determine whether the properties of the hiv-1 idlv could be expanded to other gene therapy vector platforms in the future. phf2 g and pony8.5nsg, both contain an internal spleen focus - forming virus ltr promoter driving egfp expression, a woodchuck hepatitis virus post - transcriptional regulatory element, a cppt, and self - inactivating ltr. mutations in the eiav and hiv integrase gene to generate the d64v single residue change and the ddevaa and ddevig triple residue changes were introduced into the pesyngp and psyngpgag - pol plasmids, respectively by site - directed mutagenesis. cell lines. the human embryonic kidney cell line hek293 t (atcc, manassas, va) and the mouse muscle myoblast cell line c2c12 (ecacc, porton down, uk) were cultured in dulbecco 's modified eagle 's medium supplemented with 10% fetal bovine serum, 2 mmol / l glutamine and penicillin / streptomycin. the rat adrenal pheochromocytoma cell line pc12 (ecacc) was cultured in rpmi 1640 supplemented with 10% fetal bovine serum and penicillin / streptomycin, and the cells were adhered to collagen iv - coated plasticware before transduction. we decided to arrest cell growth by differentiation rather than by chemical induction as this more closely mirrors nondividing cells in vivo. c2c12 cells were irreversibly differentiated by culturing in low serum medium containing 2% horse serum for 7 days, and pc12 cells were reversibly differentiated by supplementing media with 100 ng / ml ngf for 7 days (which induces a neuronal phonotype), then adding fluorodeoxyuridine to 20 mol / l for a further 3 days to eliminate undifferentiated cells. differentiation was reversed by removing ngf for 1 week before splitting to induce cell cycling. t, c2c12, and pc12 were seeded at 9, 2, and 3 10 cells / well respectively in 12-well plates. cells were transduced in duplicate in the presence of 8 g / ml polybrene for 6 hours at multiplicity of infections of 0.6 (for dividing hek293 t) and 30 (for c2c12 and pc12 cells). for the idlvs, vector volumes for transduction were calculated by using the parental integration titer normalized to the rna copy number of the respective idlv. hiv-1 and eiav vectors were produced by transient transfection of vector genome plasmid, gag - pol packaging plasmid, and envelope plasmid (and for hiv-1 vector only, prev plasmid) into hek293 t using fugene-6 (roche diagnostics, burgess hill, uk) in 10-layer cell factories (nunc, rochester, ny) as described previously (wong., 2005). virus was pseudotyped with the g glycoprotein of vesicular stomatitis virus envelope and concentrated by double centrifugation (6,000 g for 1824 hours, then 20,000 g for 1.5 hours). all of the vectors were characterized with respect to integration (strength titer) in hek293 t cells, rna copy number, and rt activity. to determine the integration titer in hek293 t cells, dna was extracted from the transduced cells after > 12 population doublings to ensure dilution of unintegrated copies to undetectable levels. integrated vector genomes were quantified by quantitative pcr using a gfp - specific primers and probe set. to determine the rna copy number, rna was extracted from neat virus using the qiaamp viral rna mini kit (qiagen, valencia, ca). rna copy number was quantified by quantitative rt - pcr using the same gfp - specific primers and probe set as above. the rt activity in each vector was determined using the performance - enhanced rt assay. rt was released from vector particles with a mild detergent, and used to synthesize cdna using ms2 bacteriophage rna as a template. this cdna product was then quantified by quantitative pcr using an ms2-specific primers and probe set. the product was normalized to an internal control vector sample of known integration titer to generate a duplicate wells were trypsinized and cells measured for gfp expression using flow cytometry at regular timepoints. dead cell exclusion was performed using to - pro-3 and data was acquired on a facscalibur (becton dickinson, oxford, uk). facs analysis was performed using winlist software (verity software house, topsham, me). the adult wistar rats were handled according to procedures approved by the uk home office and the university of bristol research ethics committee. a total volume of 5 l of each vector was injected into the striata of the adult rat brain using a hamilton syringe fitted with a 33 gauge needle at 0.2 l / minute. coordinates used were 0 mm anterior - posterior, 3.5 mm lateral, and 4.75 mm dorsoventral. after 1 month, all rats were killed and perfused with 400 ml 4% paraformaldehyde in 0.1 mol / l phosphate - buffered saline before removing the brains into 4% paraformaldehyde for 1624 hours and cryoprotecting in 30% sucrose / phosphate - buffered saline. the brains were mounted in oct (sakura, alphen aan den rijn, netherlands) and into 40 mol / l sections using a leica 3050 cryostat (leica microsystems, milton keynes, uk). every fifth 40 mol / l section was assessed for gfp fluorescence, and all positive sections were photographed using identical image - capturing parameters. total gfp fluorescence in each section was then measured using adobe photoshop cs3 following a published methodology, from the section in which it was first detected to the section when the signal was lost ; these measurements were plotted against section number, and the area - under - the - curve (subtracting background fluorescence) was then calculated in order to quantify total fluorescence in each striatum. statistical analysis. statistical differences between mean total fluorescence measurement for each vector group was determined using the student 's t - test (unpaired, two - tailed). | the interest in integrase - defective lentiviral vectors (idlvs) stems from their potential advantage of large cloning capacity and broad cell tropism while avoiding the possibility of insertional mutagenesis. here, we directly compared the transducing potential of idlvs based on the equine infectious anemia virus (eiav) to the more commonly described hiv-1 idlvs. idlvs were constructed by introducing equivalent single / triple mutations into the integrase catalytic triad. we show that both the single and the triple mutant hiv-1 idlvs transduce the pc12 cells, but not the c2c12 cells, with similar efficiency to their parental hiv-1 vector. in contrast, the single and triple eiav idlvs did not efficiently transduce either differentiated cell line. moreover, this hiv-1 idlv - mediated expression was independent of any residual integration activity because reporter expression was lost when cell cycling was restored. four weeks following stereotactic administration into adult rat brains, only the single hiv-1 idlv mutant displayed a comparable transduction profile to the parental hiv-1 vector. in contrast, neither eiav idlv mutants showed significant reporter gene expression. this work indicates that the transducing potential of idlvs appears to depend not only on the choice of integrase mutation and type of target cell, but also on the nature of the lentiviral vector. |
mistletoe, a semiparasitic plant, is widely distributed across the globe and has been used as a constituent of traditional medicine in northeast asia for centuries. viscum album l., known as european mistletoe, and loranthus parasiticus, known as mulberry mistletoe, are mainly used for traditional medicine in the republic of korea. mistletoe possesses various beneficial effects, such as anticancer, antiobesity, neuroprotection, antioxidant, and anti - inflammation activities. the extract of viscum album l., known as iscador, has been used in anticancer therapy in europe because it possesses strong anticancer action. such effects of mistletoe are associated with various bioactive compounds, including lectins, viscotoxins, triterpenes, sesquiterpene lactones, flavonoids, and phenolic compounds. nonetheless, the biological effect of loranthus parasiticus is still unknown with the exception of its neuroprotective effects. the inflammatory response is associated with the degranulation of immunoglobulin e- (ige-) sensitized mast cells or basophilic cells. these cells express fcri receptors known as the high - affinity ige receptor located on the plasma membrane. when ige - sensitized mast cells are stimulated by antigens, the cells liberate various inflammatory mediators, including tumor necrosis factor- (tnf-), interleukin-4 (il-4), prostaglandin e2 (pge2), prostaglandin d2 (pgd2), and leukotriene c4 (ltc4) with -hexosaminidase, a biomarker of degranulation, through activation of the fcri signaling cascade [810 ]. moreover, pgd2 and ltc4 are involved in chronic inflammation in asthma or allergic rhinitis [11, 12 ]., we found that the extract of loranthus parasiticus (lpe) possessed antiallergic activity in ige - mediated allergic responses in mast cells and demonstrate how lpe inhibits allergic responses in the above cells. in conclusion, the results may provide further information for the development of a phytomedicine for allergic diseases. mem- medium, 1x dpbs, fetal bovine serum (fbs), penicillin, and streptomycin were purchased from ge healthcare life sciences (hyclone, logan, ut, usa). the ez - cytox cell viability assay kit was obtained from daeillab service co. (seoul, korea). specific antibodies against phospho - protein kinase b (akt ; # 9271), phospho - cytosolic phospholipase a2 (cpla2 ; # 2831), phospho - extracellular signal - regulated kinase 1/2 (erk ; # 9101), phospho - c - jun n - terminal kinase 1/2 (jnk ; # 9251), phospho - src family protein kinase (lyn ; # 2731), phospho - p38 (# 9211), phospho - protein kinase c (pkc ; # 2055), phospho - phospholipase c1/2 (plc1/2 ; # 2821, # 3871, resp.), and phospho - spleen tyrosine kinase (syk ; # 2710) and cyclooxygenase-2 (cox-2 ; # 4842) were obtained from cell signaling technology, inc. specific antibodies against phospho - feline yes - related protein (fyn ; orb128087) and -actin (sc-47778) were obtained from biorbyt ltd. (dallas, tx, usa), respectively. a specific antibody against 5-lipoxygenase (5-lo ; 10007820) and eia kits for ltc4, pgd2, and pge2 were obtained from cayman chemical co. (ann arbor, mi, usa). dinitrophenyl - human serum albumin (dnp - hsa), dnp - ige, folin - ciocalteu reagent, caffeic acid, diethylene glycol, quercetin, and 4-nitrophenyl n - acetyl--d - glucosaminide (p - nag) were purchased from sigma - aldrich co. (st. louis, mo, usa). lpe was prepared according to a modification of a process reported previously ; loranthus parasiticus was obtained from the yeongcheon oriental herbal market (yeongcheon, korea) and then identified by dr. ki - hwan bae, a professor emeritus at the college of pharmacy, chungnam national university (daejeon, korea). loranthus parasiticus (1 kg) was boiled in distilled water (10 liter) for approximately 3 h at 115c. the aqueous extract was filtered through a testing sieve (aperture 500 m and 150 m). the filtered extract was filtered through a 60 m nylon net filter (millipore, ma, usa) and deposited overnight. the supernatant was lyophilized, and then the dried pellet was stored at 20c until use. the powder of lpe was dissolved in 10% dimethyl sulfoxide (dmso) solution for all experiments. the amounts of total phenolic compounds and flavonoids in lpe were evaluated following previously reported methods. lpe powder was dissolved using 20 mm pbs buffer (ph 7.4) to a final concentration of 100 mg / ml. the solution (0.33 ml) was mixed with 2.5 ml of distilled water and then incubated with 0.16 ml of folin - ciocalteu reagent for 5 min. the above solution was further incubated for 30 min in darkness after treatment with 10% sodium bicarbonate solution (0.3 ml). the absorbance at 760 nm was measured using a microplate reader (spectramax i3, molecular devices, ca, usa). separately, to determine amounts of total flavonoids in lpe, 0.4 ml of lpe was added to 4 ml 90% diethylene glycol containing 0.4 ml of 1 n naoh, and then the mixture was incubated for 1 h. the absorbance of the solution at 420 nm was measured using a microplate reader. rbl-2h3 cells, a mast cell line originating from rat basophilic leukemia, were cultured in mem- medium including 10% fbs and antibiotics (100 u / ml penicillin and 100 g / ml streptomycin) at 37c in a humidified atmosphere of 5% co2. all the experiments contain a control group as a vehicle control group containing 0.1% dmso. cell viability was evaluated by measuring the mitochondrial - dependent conversion from wst-1 to water - soluble tetrazolium salt. in brief, rbl-2h3 cells were seeded on a 96-well plate (1 10 cells / well) in mem- medium containing 10% fbs at 37c overnight. the above cells were washed with 1x dpbs and then incubated with 50 ng / ml dnp - ige. after 24 h, ige - sensitized cells were preincubated with lpe (0 to 400 g / ml) in mem- medium with 1% fbs for 1 h, simultaneously mixed with 0.1 g / ml dnp - hsa and 10 l ez - cytox reagent, and then further incubated for 4 h. the cell viability of the above cells was determined by a microplate reader (450 nm). supernatant (25 l) was added to 50 l p - nag (10 mm) in 0.1 m sodium citrate buffer (ph 4.5) and then incubated for 1 h at 37c. the reaction was finished by 0.1 m sodium carbonate buffer (ph 10.0). the absorbance was measured at 405 nm using a microplate reader. to determine the amounts of tnf- or il-4 in cultured media, ige - sensitized cells were preincubated with lpe in mem- medium with 1% fbs for 1 h and then stimulated with dnp - hsa for 4 h. all cultured media were centrifuged (17,000 g) for 10 min at 4c, and then the samples were stored at 80c until use. il-4 and tnf- were evaluated by elisa kits according to the manufacturer 's instruction. to measure the levels of pgd2, pge2, or ltc4 in cultured media, ltc4, pgd2, and pge2 and were measured by eia kits according to the manufacturer 's instruction. blotted membranes were visualized using the ecl plus kit as a chemiluminescent reagent (bio - rad, hercules, ca, usa) with an imaging system (chemidoc touch imaging system, bio - rad, hercules, ca, usa). the density levels of target proteins identified by a protein standard size marker (biofact, daejeon, korea) were compared to those of a loading control (-actin). the density of target protein bands was measured using imagej software (version 1.49v for windows, nih, usa). one - way analysis of variance (anova) was used for multiple comparisons (graphpad prism version 5.03 for windows, san diego, ca, usa). if there was a significant variation between treated groups, the dunnett test was applied. differences at the p < 0.05 and p < 0.01 levels were considered statistically significant. first, we investigated whether lpe includes phenolic compounds and flavonoids because these compounds from various mistletoes are known to possess various beneficial effects, such as antioxidant, neuroprotection, and anticancer effects. lpe contained total phenolic compounds (10.72 0.06 mg / g dry weight, the mean sd values of triple determinations) and total flavonoids (56.20 0.40 mg / g dry weight, the mean sd values of triple determinations). these results indicate that lpe contains phenolic compounds and flavonoids that may be closely associated with the beneficial actions of loranthus parasiticus. because we found that lpe included total phenolic compounds and flavonoids, we investigated whether lpe can inhibit degranulation of ige - activated mast cells. when ige - sensitized rbl-2h3 cells were preincubated with various concentrations of lpe (0 to 400 g / ml) prior to antigen challenge (0.1 g / ml dnp - hsa), lpe inhibited the release of -hexosaminidase, a common biomarker of degranulation, in a concentration - dependent manner with an ic50 value of 184.5 g / ml (figure 1(a)). in addition, 400 g / ml lpe dramatically suppressed ige - mediated degranulation to a similar level as the control without significant cytotoxicity (figure 1(b)). therefore, these results indicate that lpe possesses antiallergic activity at noncytotoxic concentrations by inhibiting degranulation of ige - activated mast cells. proinflammatory mediators are released from granules in ige - activated mast cells upon stimulation with antigens [9, 18 ]. in addition, the mediators are closely associated with the progression of allergic diseases, such as asthma, allergic rhinitis, and atopic dermatitis [810, 18 ]. therefore, we investigated the effect of lpe on the production of proinflammatory cytokines, such as tnf- and il-4, and eicosanoids, such as pge2, pgd2, and ltc4. when ige - sensitized rbl-2h3 cells were preincubated with lpe before antigen challenge, lpe significantly inhibited the formation of tnf- (ic50, 84.27 g / ml, figure 2(a)), il-4 (ic50, 93.43 g / ml, figure 2(b)), and ltc4 (ic50, 43.27 g / ml, figure 3(b)). in addition, lpe suppressed the biosynthesis of pge2 (ic50, 84.10 g / ml, figure 3(a)) and pgd2 (figure 3(c)) in a dose - dependent manner up to 200 g / ml, whereas 400 g / ml lpe gradually increases the levels of pge2 and pgd2. it seems that the effects of lpe at 400 g / ml may lead to activation of activity or / and expression of pge2 and pgd2 synthases. therefore, further studies are required to develop lpe as a phytomedicine for allergic therapy. taken together, these findings suggest that lpe inhibits the formation of allergic inflammatory mediators, including proinflammatory cytokines and eicosanoids, but exhibits mild side effects on formation of pgd2 and pge2 at a high concentration (400 g / ml). consequently, lpe may block acute or chronic inflammation caused by allergic inflammatory mediators in allergic diseases. next, we assessed the effect of lpe on enzymes responsible for biosynthesis of eicosanoids, such as pge2, pgd2, and ltc4, which induce chronic inflammation in allergic diseases [10, 19, 20 ]. to address the issue, we examined the effect of lpe on phosphorylation of cpla2, a rate - limiting enzyme of the arachidonate cascade, and 5-lo, a rate - determining enzyme of leukotriene biosynthesis, and the expression of cox-2, a rate - controlling enzyme of prostaglandin biosynthesis. as shown in figure 4, when ige - sensitized rbl-2h3 cells were preincubated with various concentrations of lpe for 4 h before antigen exposure, lpe inhibited phosphorylation of 5-lo and expression of cox-2 but not phosphorylation of cpla2. these results indicate that lpe inhibits the biosynthesis of eicosanoids, including pge2, pgd2, and ltc4, through the regulation of 5-lo and cox-2 activation in prostaglandin and leukotriene biosynthesis, respectively. finally, because lpe suppressed the rate - limiting enzymes involved in prostaglandin and leukotriene biosynthesis in the late phase (4 h), we further examined the rate - limiting and intermediate proteins related with the fcri signaling cascade in the early phase (10 min) because the activation of eicosanoid biosynthesis is implicated in the fcri signaling cascade in ige - activated mast cells [17, 21 ]. as shown in figure 5(a), when ige - sensitized rbl-2h3 cells preincubated with lpe were activated by antigen for 10 min, lpe reduced the phosphorylation level of syk but not fyn and lyn, which are initial proteins in the fcri signaling cascade. furthermore, lpe significantly inhibited the phosphorylation level of plc1/2 and pkc, which are related to the degranulation process (figure 5(b)), and decreased the phosphorylation levels of erk, jnk, p38, and akt, which are related to expression of proinflammatory cytokines (figure 5(c)). these results suggest that lpe can block activation of the fcri signaling cascade by suppressing the activity of syk in ige - activated mast cells. the action of loranthus parasiticus in allergic reaction is unknown, although it has some beneficial effects. thus, the present study demonstrates that loranthus parasiticus has antiallergic properties in ige - activated mast cells based on in vitro tests. in addition, such effects of loranthus parasiticus are caused by total phenolic compounds or / and flavonoids, because triterpenes, sesquiterpene lactones, or flavonoids derived from loranthus parasiticus are associated with numerous beneficial effects. phenolic compounds and flavonoids attenuate allergic responses in ige - activated mast cells [14, 17 ]. nevertheless, the effects of components in loranthus parasiticus on allergic reactions have not been reported. one possible mechanism for the antiallergic activities of lpe may be related to a direct suppression of activation of the fcri signaling cascade in ige - activated mast cells because the degranulation initiation of ige - activated mast cells is closely associated with the activation of the fcri receptor located on the plasma membrane of the cells [7, 8 ]. consequently, ige - activated mast cells liberate various inflammatory mediators, such as il-4, tnf-, histamine, prostaglandins, and leukotrienes [9, 10, 18, 19 ]. in support of this, in our study, when ige - sensitized mast cells were preincubated with lpe prior to antigen challenge, lpe decreased il-4, tnf-, pgd2, pge2, and ltc4 production. in addition, lpe inhibited activation of syk, a rate - limiting intermediate protein of the fcri signaling cascade. moreover, lpe also suppressed activation of the plc1/2-pkc pathway, which is related to degranulation process, and akt, p38, erk, and jnk, which are associated with cytokine expression, in ige - activated mast cells. therefore, the activation of both the plc1/2-pkc pathway and intermediate proteins is directly associated with activation of the fcri signaling cascade. taken together, the antiallergic action of lpe is closely associated with inhibiting syk activation in the fcri signaling cascade. therefore, lpe may directly regulate activation of the fcri signaling cascade through inhibition of syk activation in ige - activated mast cells. another possible mechanism for the antiallergic activities of lpe may be associated with suppression of arachidonate cascade activation in ige - activated mast cells because the above cells can produce various proinflammatory lipid mediators, such as ltc4, pgd2, and pge2 [810 ], and release them from numerous granules [11, 12 ]. moreover, these lipid mediators lead to chronic inflammation in allergic diseases, such as asthma and allergic rhinitis [7, 10 ]. therefore, the regulation of eicosanoid formation is another important factor for the antiallergic properties of lpe. consistently, lpe reduced biosynthesis of pge2, pgd2, and ltc4 and suppressed expression of cox-2, a rate - limiting enzyme for prostaglandin biosynthesis, and activation of 5-lo, an initial enzyme for leukotriene biosynthesis, in ige - activated mast cells. these findings suggest that lpe inhibits the formation of eicosanoids through regulation of rate - limiting enzymes, such as cox-2 and 5-lo. in addition, lpe may regulate other enzymes related with eicosanoid biosynthesis with the exception of cpla2. such effects of lpe may contribute to the enhancement of its antiallergic properties in allergic responses. in this study, we revealed, for the first time, a novel role of lpe in ige - mediated allergic reactions. we found that lpe has antiallergic efficacy in ige - activated mast cells and contains numerous total phenolic compounds and flavonoids that are potentially responsible for antiallergic actions. the mechanisms of its antiallergic properties include various targets, such as syk, akt, erk, jnk, p38, plc1/2, pkc, 5-lo, and cox-2. lpe can be used to develop a functional food or a phytomedicine for alleviating allergic diseases. furthermore, it is necessary to identify the active compounds in loranthus parasiticus that possess antiallergic action. | the mistletoe loranthus parasiticus has been used as a compound for traditional medicine in northeast asia for a long time and is known to possess neuroprotective action. nonetheless, the effect of loranthus parasiticus on allergic responses remains unknown. in the present study, we evaluated whether the water extract of loranthus parasiticus (lpe) could inhibit ige - mediated allergic responses in rbl-2h3 cells. lpe inhibited the release of -hexosaminidase (ic50, 184.5 g / ml) and the formation of tumor necrosis factor- (ic50, 84.27 g / ml), interleukin-4 (ic50, 93.43 g / ml), prostaglandin e2 (ic50, 84.10 g / ml), prostaglandin d2, and leukotriene c4 (ic50, 43.27 g / ml) in a concentration - dependent manner. moreover, lpe inhibited phosphorylation of syk, plc1/2, pkc, erk, jnk, p38, and akt. in the late phase, lpe decreased 5-lipoxygenase phosphorylation and cox-2 expression but not cpla2 phosphorylation. additionally, lpe included total phenolic compounds (10.72 mg / g dry weight) and total flavonoids (56.20 mg / g dry weight). these results suggest that the phenolic compounds or flavonoids contained in lpe may be associated with antiallergic activity. the phenolic compounds and flavonoids in lpe are antiallergic phytochemicals capable of inhibiting the activation of the fcri signaling cascade in mast cells. such effects may provide further information for the development of a phytomedicine for allergic diseases. |
pulmonary tuberculosis (tb) can cause parenchymal destruction by up - regulation of several proteases and dysregulation of protease control.1 the histopathological abnormalities after treatment for pulmonary tb include fibrosis, bronchiectasis, and brochial stenosis, all of which can cause pulmonary function abnormalities.23 previous studies have evaluated the changes in pulmonary function before, during and after pulmonary tb treatment.417 between 48.7% and 76% of patients had pulmonary function abnormalities after completing pulmonary tb treatment.910,1315,17 although some studies have shown obstructive defects as the main abnormality, recent studies have shown that abnormalities could be obstructive, restrictive, or mixed defects.1415,17 in a study performed on gold miners in south africa, forced expiratory volume in the first second (fev1) and forced vital capacity (fvc) were found to be worse six months after tuberculosis treatment and were stabilized 13 to 18 months after treatment.16 the impairment in pulmonary function after completing pulmonary tb treatment is related to long - term respiratory symptoms, which affect quality of life.1112,18 it is important to identify patients with risk factors for pulmonary function deterioration after the completion of pulmonary tb treatment. however, little is known about the trends in the changes in pulmonary function and the risk factors associated with pulmonary function deterioration. therefore, we conducted a study to investigate the trends and predictors of the changes in pulmonary function in patients with pulmonary tuberculosis after the completion of treatment. from january 2000 to december 2008, 2,789 patients with culture - confirmed pulmonary tb were identified at the national taiwan university hospital, a tertiary care referral center with 2,000 beds in northern taiwan. among these patients, ninety - eight patients were excluded from further analysis due to the presence of the following conditions before the diagnosis of pulmonary tb : (1) pulmonary co - morbidities, including asthma, chronic obstructive airway disease (copd) confirmed by spirometry, pneumoconiosis, interstitial lung disease, history of respiratory failure requiring mechanical ventilation, lung cancer, and lung resection ; (2) congestive heart failure (new york heart association functional class > 2) ; (3) autoimmune disease ; and (4) human immunodeficiency virus infection.19 the remaining 115 patients with 162 pulmonary function test results were analyzed. tests performed within five years after the end of anti - tuberculosis treatment were selected to investigate the predictors of pulmonary function. this study was approved by the international review board of the national taiwan university hospital. mycobacteriological studies were performed using standard procedures.2022 briefly, sputum specimens were decontaminated with naoh - citrate - n - acetyl - l - cysteine and then neutralized and centrifuged. the specimen sediments were inoculated onto lwenstein - jensen (lj) slants and into mycobacterium growth indicator tubes (mgit) using a fluorometric bactec technique (bactec mgit 960 system ; becton - dickinson diagnostic instrument systems ; sparks, md). the lj slants were incubated in a co2 incubator for an initial week and in ambient air for another seven weeks at 35c. the pulmonary function tests were performed using masterscreen, (jaeger, germany) or vmax 6200 (sensormedics corp., usa) spirometers in our pulmonary function laboratory, according to the recommendation by the american thoracic society.7,2324 daily calibration was performed for each machine before measuring pulmonary function. patient data, including age, sex, weight, height, medication history, and smoking status, were collected via standard questionnaires prior to pulmonary function testing. each patient performed at least three acceptable forced expiratory maneuvers, which fulfilled the criteria of repeatability.24 the reference equations for fev1, fvc, and fev1/fvc were obtained from a previous study of 354 non - smoking healthy chinese patients (online supplement 1) and were used to calculate the predicted values of corresponding pulmonary function parameters.25 normal pulmonary function should have fev1 80%, fvc 80%, and fev1/fvc 70% of predicted values. for abnormal pulmonary function, an obstructive defect was defined as fev1/fvc 2) ; (3) autoimmune disease ; and (4) human immunodeficiency virus infection.19 the remaining 115 patients with 162 pulmonary function test results were analyzed. tests performed within five years after the end of anti - tuberculosis treatment were selected to investigate the predictors of pulmonary function. this study was approved by the international review board of the national taiwan university hospital. mycobacteriological studies were performed using standard procedures.2022 briefly, sputum specimens were decontaminated with naoh - citrate - n - acetyl - l - cysteine and then neutralized and centrifuged. the specimen sediments were inoculated onto lwenstein - jensen (lj) slants and into mycobacterium growth indicator tubes (mgit) using a fluorometric bactec technique (bactec mgit 960 system ; becton - dickinson diagnostic instrument systems ; sparks, md). the lj slants were incubated in a co2 incubator for an initial week and in ambient air for another seven weeks at 35c. the pulmonary function tests were performed using masterscreen, (jaeger, germany) or vmax 6200 (sensormedics corp., usa) spirometers in our pulmonary function laboratory, according to the recommendation by the american thoracic society.7,2324 daily calibration was performed for each machine before measuring pulmonary function. patient data, including age, sex, weight, height, medication history, and smoking status, were collected via standard questionnaires prior to pulmonary function testing. each patient performed at least three acceptable forced expiratory maneuvers, which fulfilled the criteria of repeatability.24 the reference equations for fev1, fvc, and fev1/fvc were obtained from a previous study of 354 non - smoking healthy chinese patients (online supplement 1) and were used to calculate the predicted values of corresponding pulmonary function parameters.25 normal pulmonary function should have fev1 80%, fvc 80%, and fev1/fvc 70% of predicted values. for abnormal pulmonary function, an obstructive defect was defined as fev1/fvc < 70%, a restrictive defect as an fev1/fvc ratio 70% and fvc < 80%, and a mixed defect as fev1/fvc < 70% and fvc < 80% of predicted values. from patients ' medical records, we retrieved demographic data, smoking history, underlying co - morbidities, radiographic findings, laboratory results, pulmonary tb treatment courses, and results of pulmonary function tests. the standard anti - tuberculosis regimen consisted of oral isoniazid, rifampicin, and ethambutol, plus pyrazinamide daily during the first two months and isoniazid plus rifampicin daily during the following four months. according to previous studies, non - tuberculosis mycobacteria (ntm) are present in the respiratory tracts of 7.1% to 7.3% of patients with pulmonary tuberculosis.2627 it was unknown whether these ntm isolates would cause pulmonary function deterioration. therefore, we recorded the frequency of ntm isolates (0, 1 or 2) from sputum samples collected after beginning anti - tuberculosis treatment and before pulmonary function testing. radiographic scores (rs) of chest radiographs9 that were taken before and after anti - tuberculosis treatment were recorded (in accordance with a previous study) to define the extent of the disease. briefly, each lung was divided into three areas, and each area was rated on a four - point scale (0 to 3) for the extent of infiltration. continuous variables were reported as means (standard deviation) and categorical variables as numbers (percentage). changes in pulmonary function over time after the completion of anti - tuberculosis treatment have been shown to be biphasic (non - linear).16 therefore, we first established three generalized additive models for fev1, fvc, and fev1/fvc, with the gaussian distribution selected.7 in addition to time after the completion of pulmonary tb treatment, we included 3 other covariates into these models : age, body weight, and height. locally weighted scatterplot smoothing (loess) was selected as the smoothing technique for the smooth functions of the four covariates. from the partial prediction curves of the smooth functions of time after the completion of pulmonary tb treatment, we determined the time point at which the worst pulmonary function occurred. some subjects who were enrolled in this study performed repeated pulmonary function tests over time. the pulmonary function test results were not independent of each other, and a generalized linear model could not be applied for analysis. therefore, we performed statistical analyses using generalized estimating equation (gee) models with a stepwise variable selection procedure to identify the predictors of pulmonary function (fev1 and fvc) and the predictors of obstructive defect development within 5 years after completion of pulmonary tb treatment. the time interval (t) between the date of each pulmonary function test and the timing of the worst pulmonary function (obtained by loess smoothing models) was included in the analysis. other parameters with the potential to influence pulmonary function after completion of pulmonary tb treatment were also included in the analysis ; these parameters were demographic data, smoking history, underlying co - morbidities, radiographic findings, laboratory results, and pulmonary tuberculosis treatment courses. a two - sided p value < 0.05 was considered to be significant. all analyses were performed using sas software (version 9.1.3, sas institute inc., cary, nc, u.s.a.). a total of 115 patients underwent 162 pulmonary function tests after they completed pulmonary tb treatment within the study period. twenty - eight patients had more than one pulmonary function test result, and the time between repeated pulmonary function tests was at least six months (figure 1). the median interval between the completion of pulmonary tuberculosis treatment and a pulmonary function test was 16 months (range : 0 to 112 months). among these 115 patients, 106 (92.2%) underwent 140 (86% of all pulmonary function tests) pulmonary function tests within 5 years. table 1 summarizes the baseline clinical characteristics of these 106 patients and the results of the 140 pulmonary function tests. the mean age at the first pulmonary function test was 59.3 years (range : 19 to 90). ninety - four (67.1%) pulmonary function tests were abnormal, with obstructive defects being the most common problem (68 tests, 48.6%). twenty - eight patients (26.4%) had at least one concurrent ntm isolate ; these patients contributed 39 pulmonary function test results (table 1). the mean age at which the patients underwent their first pulmonary function test was older in those patients with ntm isolates (p<0.001 by student 's t - test, compared to those without ntm isolates). otherwise, they had no significantly different characteristics compared to those patients without ntm isolates. from partial prediction curves, we observed trends of biphasic changes in fev1, fvc, and the fev1/fvc ratio with time after completion of pulmonary tuberculosis treatment (figure 2a c). the predictors of fev1 within 60 months after completion of anti - tuberculosis treatment are shown in table 2 (for statistical details, please refer to online supplement 2). the time variable (t), defined as the absolute time interval between the date of the pulmonary function test and the timing of worst fev1 (i.e., 18 months), patients who had smear - positive disease, longer treatment duration, higher rs before treatment, or less rs improvement after treatment had worse fev1 after completion of anti - tuberculosis treatment. from the end of the anti - tuberculosis treatment to the nadir of fev1, the estimated monthly reduction in fev1 was 18.83 ml (0.017 l / month 1 month + 0.022 l / year 1 month/12 = 0.01883 l) after adjusting for the other parameters. the predictors of fvc within 60 months after completion of anti - tuberculosis treatment are listed in table 3. patients had worse fvc after completion of anti - tuberculosis treatment if they had longer treatment duration, a change in regimen during treatment, higher rs before treatment, or less rs improvement after treatment. we also investigated the risk factors of obstructive defects (fev1/fvc ratio < 70) after completion of treatment for pulmonary tb (table 4). patients with smear - positive disease had a higher risk of obstructive defects after completing anti - tuberculosis treatment (odds ratio 2.546, p = 0.045). a total of 115 patients underwent 162 pulmonary function tests after they completed pulmonary tb treatment within the study period. twenty - eight patients had more than one pulmonary function test result, and the time between repeated pulmonary function tests was at least six months (figure 1). the median interval between the completion of pulmonary tuberculosis treatment and a pulmonary function test was 16 months (range : 0 to 112 months). among these 115 patients, 106 (92.2%) underwent 140 (86% of all pulmonary function tests) pulmonary function tests within 5 years. table 1 summarizes the baseline clinical characteristics of these 106 patients and the results of the 140 pulmonary function tests. the mean age at the first pulmonary function test was 59.3 years (range : 19 to 90). ninety - four (67.1%) pulmonary function tests were abnormal, with obstructive defects being the most common problem (68 tests, 48.6%). twenty - eight patients (26.4%) had at least one concurrent ntm isolate ; these patients contributed 39 pulmonary function test results (table 1). the mean age at which the patients underwent their first pulmonary function test was older in those patients with ntm isolates (p<0.001 by student 's t - test, compared to those without ntm isolates). otherwise, they had no significantly different characteristics compared to those patients without ntm isolates. from partial prediction curves, we observed trends of biphasic changes in fev1, fvc, and the fev1/fvc ratio with time after completion of pulmonary tuberculosis treatment (figure 2a c). the predictors of fev1 within 60 months after completion of anti - tuberculosis treatment are shown in table 2 (for statistical details, please refer to online supplement 2). the time variable (t), defined as the absolute time interval between the date of the pulmonary function test and the timing of worst fev1 (i.e., 18 months), patients who had smear - positive disease, longer treatment duration, higher rs before treatment, or less rs improvement after treatment had worse fev1 after completion of anti - tuberculosis treatment. from the end of the anti - tuberculosis treatment to the nadir of fev1, the estimated monthly reduction in fev1 was 18.83 ml (0.017 l / month 1 month + 0.022 l / year 1 month/12 = 0.01883 l) after adjusting for the other parameters. the predictors of fvc within 60 months after completion of anti - tuberculosis treatment are listed in table 3. patients had worse fvc after completion of anti - tuberculosis treatment if they had longer treatment duration, a change in regimen during treatment, higher rs before treatment, or less rs improvement after treatment. we also investigated the risk factors of obstructive defects (fev1/fvc ratio < 70) after completion of treatment for pulmonary tb (table 4). patients with smear - positive disease had a higher risk of obstructive defects after completing anti - tuberculosis treatment (odds ratio 2.546, p = 0.045). our results showed that after completing pulmonary tb treatment, patients experienced gradual declines in pulmonary function until approximately 18 months. the risk factors for pulmonary function deterioration in association with tb included smear - positive disease, extensive pulmonary involvement with poor radiographic improvement after treatment, prolonged duration of treatment, and change in regimen. several co - morbidities could affect pulmonary function test results, including chronic lung diseases, severe congestive heart failure, autoimmune diseases, and hiv infection. autoimmune diseases could have pulmonary involvement, possibly leading to significant changes in pulmonary function.28 in addition, patients with hiv infection have a high risk of contracting respiratory diseases such as pneumocystis jirovecii infection and recurrent bacterial pneumonia.29 therefore, in our study, we excluded patients with these co - morbidities to give a more accurate description of the changes in pulmonary function and trends after completion of pulmonary tb treatment. the time course for changes in pulmonary function after completing anti - tuberculosis treatment has never been well - studied. one previous study showed that the annual decrease in fev1 was 35.3 ml in patients with obstructive defects within 15 years after the end of anti - tuberculosis treatment.30 however, the anti - tuberculosis treatment in this study was less effective than the modern four - drug combination regimen, and exposure to other noxious environmental factors may have contributed to changes in pulmonary function during long follow - up time periods. in a study performed in cape town, south africa by hnizdo., the pulmonary function of coal miners with pulmonary tb reached a nadir at approximately 6 months after completing anti - tuberculosis treatment (with a monthly fev1 reduction of 56.8 ml) and stabilized approximately 7 to 12 months later.16 our results confirmed the biphasic change in pulmonary function over time after the end of anti - tuberculosis treatment, with a monthly fev1 reduction of 18.83 ml within the first 18 months. the greater declines in pulmonary function that were noted in the coal miners with tb were probably due to the concomitant presence of pneumoconiosis. in addition, without adjusting for smoking history or disease extent in the analysis, it is difficult to determine the contribution of each factor to the abnormalities in pulmonary function. smear - positive disease, which suggests either a high mycobacterial load or endobronchial involvement, has been shown to be an important predictor of pulmonary function deterioration and obstructive defects after the completion of pulmonary tb treatment.31 in accordance with previous studies, we found that disease extent was a predictor of pulmonary function deterioration after the completion of pulmonary tb treatment.10,1213 however, the previous studies did not consider the timing of the pulmonary function test. smoking, which is the most important risk factor for copd, increases the risk of active pulmonary tuberculosis.3234 the impact of smoking on pulmonary function abnormalities after pulmonary tb infection is unknown, and previous studies have shown inconsistent results.910,15,17 in our study, smoking history was not a significant predictor of pulmonary function deterioration. the reason for this finding might be that most subjects with a history of smoking in our study had quit smoking before or immediately after their diagnosis with pulmonary tb, and subjects with copd, as confirmed by spirometry, were excluded. it might also be the case that short - term changes in pulmonary function are mainly caused by post - tuberculosis pulmonary inflammation, which masks pulmonary function deterioration due to smoking. it is not uncommon for patients with pulmonary tb to have ntm isolation from respiratory specimens.2627 the clinical significance of ntm isolation, especially with respect to pulmonary function, is unclear in pulmonary tuberculosis patients ; it might be the result of colonization, specimen contamination or true infection.35 patients with multiple ntm isolates were more likely to be symptomatic and had longer treatment times for pulmonary tb. cavitations were more frequently visualized by chest radiographs in patients with ntm isolates.26 further study is needed to clarify the clinical significance of ntm colonization in changes in pulmonary function after completion of anti - tuberculosis treatment. first, the study was limited by retrospective data and a relatively small sample size. without longitudinal follow - up data, second, selection bias might exist because our study population was treated in a tertiary medical center. nevertheless, we believe that this is a minor concern because more than 90% of the patients with pulmonary tb were ambulatory and sought primary care in the outpatient department. third, only 7.3% of our patients underwent pulmonary function tests after completing anti - tuberculosis treatment. patients who received pulmonary function tests could have had more prominent respiratory symptoms and worse pulmonary function compared to patients who did not undergo such tests. nevertheless, our results showed a prominent loss of pulmonary function after pulmonary tb infection. pulmonary function follow - up analysis is recommended in patients with pre - existing lung disease who are at risk of pulmonary decompensation after pulmonary tuberculosis. in conclusion, after completion of pulmonary tb treatment, the risk factors for pulmonary function deterioration include smear - positive disease, extensive disease before treatment, prolonged treatment duration, and less radiographic improvement after treatment. for patients with significant respiratory symptoms and multiple risk factors, the pulmonary function test should be followed up to monitor the progression of functional impairment and responses to treatment, especially within the first 18 months after completing anti - tuberculosis treatment. we would like to thank fu - chang hu and chia - chi cheng for their assistance in statistical computation. | objectives : the present study aimed to investigate the trends in changes in pulmonary function and the risk factors for pulmonary function deterioration in patients with pulmonary tuberculosis after completing treatment.introduction:patients usually have pulmonary function abnormalities after completing treatment for pulmonary tuberculosis. the time course for changes in pulmonary function and the risk factors for deterioration have not been well studied.methods:a total of 115 patients with 162 pulmonary function results were analyzed. we retrieved demographic and clinical data, radiographic scores, bacteriological data, and pulmonary function data. a generalized additive model with a locally weighted scatterplot smoothing technique was used to evaluate the trends in changes in pulmonary function. a generalized estimating equation model was used to determine the risk factors associated with deterioration of pulmonary function.results:the median interval between the end of anti - tuberculosis treatment and the pulmonary function test was 16 months (range : 0 to 112 months). the nadir of pulmonary function occurred approximately 18 months after the completion of the treatment. the risk factors associated with pulmonary function deterioration included smear - positive disease, extensive pulmonary involvement prior to anti - tuberculosis treatment, prolonged anti - tuberculosis treatment, and reduced radiographic improvement after treatment.conclusions:after the completion of anti - tuberculosis tb treatment, several risk factors predicted pulmonary function deterioration. for patients with significant respiratory symptoms and multiple risk factors, the pulmonary function test should be followed up to monitor the progression of functional impairment, especially within the first 18 months after the completion of anti - tuberculosis treatment. |
piwi - interacting rnas (pirnas) are germline - specific small rnas that function in rna silencing. in drosophila, pirnas arise mostly from transposable elements and associate with germline - specific argonaute proteins (piwi proteins), aubergine (aub), piwi, and ago3, to guide piwi proteins to their targets (saito., 2006, 2009 ; vagin., 2006 ; aravin., 2007 ;, 2007 ; gunawardane., 2007 ; nishida., 2007). mutations in the piwi genes lead to de - repression of transposable elements (cox., 1998 ; schmidt. kalmykova., 2005 ; klattenhoff., 2007 ; li., 2009). mutations in the pirna - producing genomic loci, the pirna clusters, show a similar phenotype (brennecke., 2007). thus, both piwi and pirnas are necessary for transposon silencing in the germline. through this mechanism, the piwi pirna complex, also called the pirna - induced silencing complex (pirisc), protects the integrity of the germline genome from invasive transposable dna elements and ensures oogenesis and spermatogenesis. piwi - interacting rnas are produced through two mechanisms, the primary processing pathway and the amplification loop (or the ping - pong cycle ; aravin. primary pirnas are first produced through the primary processing pathway ; then, the majority of primary pirnas are amplified by the ping - pong cycle to maintain a high expression level of pirnas in the germline. maternally deposited pirnas also serve as primary pirnas to induce the ping - pong cycle in embryos. primary pirnas are mostly antisense to transposon mrnas, show a strong bias for uracil at the 5 end (1u bias) and are favorably loaded onto aub and piwi (li., 2009 ; malone., primary pirnas in ovarian somatic cells require the female sterile (1) yb (yb) gene for their accumulation (olivieri., 2010 ; yb is necessary for self - renewal of germline and somatic stem cells in ovaries (szakmary., 2009). germ cells do not express yb and, therefore, yb is unnecessary for primary pirna production in germ cells. zucchini (zuc) and armitage (armi) are necessary for primary pirna production in both ovarian somas (haase., 2010 ; olivieri., 2010 ; saito., 2010) and germ cells (malone., 2009) vasa, maelstrom (mael), krimper (krimp), armi, and spindle - e (spn - e) are necessary for primary pirna accumulation in testes (nagao., 2010). the ping - pong cycle performs pirna amplification and transposon silencing, simultaneously ; this is because transposon mrnas serve as substrates for the aub the aub - slicer activity, the endonuclease activity required for cleaving the target mrnas (gunawardane., 2007 ; nishida., 2007), determines the 5 end of mature pirnas in the sense orientation. the ago3pirna complex, in turn, cleaves antisense transcripts of transposons and the resultant pirnas are loaded onto aub. other factors required for the amplification loop include vasa, tudor (tud), krimp, spn - e, and mael (findley., 2003 ; tud is one of the tud domain - containing proteins expressed in fly gonads (boswell and mahowald, 1985 ; thomson and lasko, 2004 ; arkov., 2006). the tud domains in tud specifically interact with the symmetrical dimethylarginines (sdmas) in aub and ago3 (kirino. complex contains pirna intermediate - like rna molecules but, in the complex, aub and ago3 are free from mature pirnas. thus, tud might serve as a platform for the amplification loop (nishida., 2009). spn - e has a tud domain (gillespie and berg, 1995 ; ponting, 1997) ; however, it does not seem to associate with aub and ago3 (nishida., 2009). whether krimp has the ability to associate with piwi members through their sdmas remains undetermined. until now, the pirna biogenesis factors that have been identified in drosophila are localized in the cytoplasm and are preferentially accumulated in specific cytoplasmic perinuclear structures, the nuage, and yb bodies in germ and somatic cells, respectively. hence, both nuage and yb bodies are considered to be the locations for pirna biogenesis (findley., 2003 ; qi., 2011 ; for review see siomi., 2011). saito., 2010). how zuc localization to mitochondria is mechanistically involved in pirna biogenesis accumulated evidence has shown that in ovaries, nuage localization of the pirna factors occurs in a molecular hierarchical fashion. the current understanding is that vasa, a dead - box rna helicase that is detected specifically in the germ cells, is the first to be located to the nuage (findley., 2003) : vasa localization is followed by spn - e, aub, ago3, and mael. whether this hierarchy is female - specific or occurs also in males remains unexamined. to address this question, we performed immunofluorescence analyses using monoclonal antibodies against individual pirna factors and found that the nuage localization hierarchy was gender - specific ; an obvious difference was seen for krimp. krimp is placed between aub and ago3 in ovaries, but is placed at the same level with aub, but upstream of ago3 in testes. these results suggest that the functional requirement of krimp in pirna biogenesis may be different between male and female gonads. the yellow white (y w) and oregon - r strains were used as wild - type (wt) strains. the tud allele used was tud bw sp / cyo l(2)dts513 (drosophila genetic resource center stock number : 106505). the aub alleles used were aub cn bw / cyo and aub cn bw / cyo. aub cn bw / cyo and aubcn bw / cyo were crossed to yield aub heterozygous mutant flies ; aub / aub (nishida., 2007). ago3/tm6b tb and ago3/tm6b tb were crossed to yield ago3 heterozygous mutant flies ; ago3/ago3 (li., the krimp allele used was w ; pbac{wh}krimp / cyo (bloomington drosophila stock center ; 18990). w ; pbac{wh}krimp / cyo and w ; df(2r)bsc309/cyo (bloomington drosophila stock center ; 23692) were crossed to yield krimp transheterozygote mutant flies ; krimp / df. transheterozygotes krimp / df exhibited male and female sterility and an extent of loss in krimp perinuclear staining similar to that exhibited by homozygous krimp (lim and kai, 2007, and data not shown). hence, krimp was used as a loss - of - function allele to characterize krimp phenotypes in this study. an anti - tudor monoclonal antibody was raised specifically against the c - terminus of the protein. a 327 amino acid fragment at the c - terminal end of tudor (amino acid 2189 to the end) was fused with glutathione s - transferase (gst) and used to immunize mice. an anti - krimp monoclonal antibody was raised specifically against the n - terminus of the protein. a 200 amino acid fragment at the n - terminal end of krimp was fused with gst and used to immunize mice. anti - tudor and anti - krimp monoclonal antibodies were produced essentially as described previously (ishizuka., 2002) and purified from culture supernatant of hybridoma cells under standard procedures using thiophilic - superflow resin (bd biosciences). western blot analysis was performed as described previously (miyoshi., 2005). anti - aub and anti - ago3 antibodies were used as described previously (nishida. testes and ovaries were stained with the various antibodies as previously described (nishida. an anti - aub antibody was directly labeled using the hilyte fluor 555 labeling kit - nh2 (dojindo molecular technologies). anti - ago3 (1:5,000 dilution ; nagao., 2010), anti - tud (1:250 dilution ; nishida., 2009), and anti - krimp (1:250 dilution) antibodies were used as primary antibodies. alexa 488-conjugated or alexa 546-conjugated anti - mouse igg (molecular probes ; 1:1,000 dilution) were used as secondary antibodies. all immunohistochemical analyses were performed at least three times and representative images are shown in the figures. the yellow white (y w) and oregon - r strains were used as wild - type (wt) strains. the tud allele used was tud bw sp / cyo l(2)dts513 (drosophila genetic resource center stock number : 106505). the aub alleles used were aub cn bw / cyo and aub cn bw / cyo. aub cn bw / cyo and aubcn bw / cyo were crossed to yield aub heterozygous mutant flies ; aub / aub (nishida., 2007). ago3/tm6b tb and ago3/tm6b tb were crossed to yield ago3 heterozygous mutant flies ; ago3/ago3 (li., the krimp allele used was w ; pbac{wh}krimp / cyo (bloomington drosophila stock center ; 18990). w ; pbac{wh}krimp / cyo and w ; df(2r)bsc309/cyo (bloomington drosophila stock center ; 23692) were crossed to yield krimp transheterozygote mutant flies ; krimp / df. transheterozygotes krimp / df exhibited male and female sterility and an extent of loss in krimp perinuclear staining similar to that exhibited by homozygous krimp (lim and kai, 2007, and data not shown). hence, krimp was used as a loss - of - function allele to characterize krimp phenotypes in this study. an anti - tudor monoclonal antibody was raised specifically against the c - terminus of the protein. a 327 amino acid fragment at the c - terminal end of tudor (amino acid 2189 to the end) was fused with glutathione s - transferase (gst) and used to immunize mice. an anti - krimp monoclonal antibody was raised specifically against the n - terminus of the protein. a 200 amino acid fragment at the n - terminal end of krimp was fused with gst and used to immunize mice. anti - tudor and anti - krimp monoclonal antibodies were produced essentially as described previously (ishizuka., 2002) and purified from culture supernatant of hybridoma cells under standard procedures using thiophilic - superflow resin (bd biosciences). western blot analysis was performed as described previously (miyoshi., 2005). anti - aub and anti - ago3 antibodies were used as described previously (nishida. testes and ovaries were stained with the various antibodies as previously described (nishida., 2007 ; an anti - aub antibody was directly labeled using the hilyte fluor 555 labeling kit - nh2 (dojindo molecular technologies). 2010), anti - tud (1:250 dilution ; nishida., 2009), and anti - krimp (1:250 dilution) antibodies were used as primary antibodies. alexa 488-conjugated or alexa 546-conjugated anti - mouse igg (molecular probes ; 1:1,000 dilution) were used as secondary antibodies. all immunohistochemical analyses were performed at least three times and representative images are shown in the figures. to ensure that we had a constant supply of anti - tud and anti - krimp antibodies for this study, we produced tud and krimp monoclonal antibodies. we used recombinant tud and krimp peptide fragments expressed in and purified from e. coli to immunize mice. western blotting of total ovary and cultured schneider 2 (s2) lysates showed that the antibodies reacted with proteins of the expected sizes of tud and krimp (figures 1a, b, respectively). the tud band in s2 lysate migrated slightly faster than that in ovary lysate (figure 1a). this may reflect post - translational modification of tud protein, although clear evidence for this is still required. we also performed western blotting on tud and krimp mutant ovaries (figures 1a, b). the bands corresponding to tud and krimp were not present in the mutants, indicating a high specificity of the antibodies for the individual antigens. (a) western blotting was performed on schneider 2 (s2) and total ovary lysates using an anti - tud monoclonal antibody raised in our laboratory. a band of the size predicted for tud was detected in both lysates. in tud mutant (tud / tud) ovaries, the bands corresponding to tud disappeared, indicating that the antibody was specific to the antigen. (b) western blotting was performed on s2 and total ovary lysates using an anti - krimp monoclonal antibody. in krimp mutant (krimp / df) ovaries, the bands corresponding to krimp disappeared, indicating that the antibody was specific to krimp. we previously examined the subcellular localization of tud in aub mutant ovaries using an anti - tud antibody that was a kind gift from a. nakamura (cdb - riken, japan ; nishida., 2009). here, we used the newly produced anti - tud antibody to repeat this immunofluorescence study on both control and aub mutant ovaries. very little tud was localized to the nuage when aub was not expressed (figure 2a), confirming the previous observation that aub is necessary for tud nuage localization (nishida., 2009). we immunostained for tud in ago3 mutant ovaries and detected tud in the nuage (figure 2a), suggesting that ago3 is dispensable for tud nuage localization. thus, the nuage localization of aub, but not of ago3, is necessary for tud nuage localization. aub localization to nuage is not affected by loss of ago3 expression (figure 2a), although ago3 localization to nuage is affected by loss of aub expression (figure 2a). hence, for nuage localization, ago3 requires aub but aub does not require ago3. (a) ovaries : the control (a), and aub (aub / aub) (b), and ago3 (ago3/ago3) (c) mutant ovaries were stained with anti - tud antibody ; the control (d), and aub (aub / aub) (e) ago3 (ago3/ago3) (f) mutant ovaries were stained with anti - aub antibody ; and the control (g), and aub (aub / aub) (h), and ago3 (ago3/ago3) (i) mutant ovaries were stained with anti - ago3 antibody. (b) testes : the control (a), and aub (aub / aub) (b), and ago3 (ago3/ago3) (c) mutant testes were stained with anti - tud antibody. enlarged images of the regions that are boxed with white dotted lines in (b) (a c) are shown in (b) (d f), respectively. the control (g), and aub (aub / aub) (h), and ago3 (ago3/ago3) (i) mutant testes were stained with anti - aub antibody ; and the control (j) ; and aub (aub / aub) (k) ; and ago3 (ago3/ago3) (l) mutant testes were stained with anti - ago3 antibody. scale bars in all testis images indicate 20 m. all immunohistochemical analyses were performed at least three times and representative images are shown. the tud signal was detected in the nuage in ago3 mutant testes (figure 2b) the same as in control. in aub mutant testes, however, the tud signal was hardly detected in the nuage ; instead, it appeared to be in the nucleus in particular in spermatogonia cells (figure 2b). aub was localized to the nuage in ago3 testes (figure 2b), confirming that aub nuage localization does not require ago3 in both female and male gonads. we then investigated how the subcellular localization of aub and ago3 was affected by loss of tud function. in ovaries, aub was detected only slightly in the nuage in tud mutant flies (figure 3a), as has been reported previously (nishida., 2009). in contrast, ago3 was localized to the nuage, the same as in the control. these results suggest that under conditions where tud function is absent, ago3 finds an alternative way to be localized to the nuage in a tud - independent manner. another possible interpretation is that a small amount of aub in the nuage is sufficient for ago3 to be localized to the nuage. also in testes, ago3 localization was barely affected by loss of tud function, whereas aub localization was drastically changed (figure 3b) ; aub was sparsely localized in the nuage and instead was dispersed in the cytosol. these results indicate that the requirement and dispensability of tud for nuage localization of aub and ago3, respectively, are similar in male and females. loss of tud function affects the subcellular localization of aub and ago3 in ovaries and testes similarly. (a) ovaries : tud heterozygous and homozygous mutant ovaries were immunostained with anti - tud, anti - aub, and anti - ago3 antibodies [(a) : tud hetero / anti - tud ; (b) : tud homo / anti - tud ; (c) : tud hetero / anti - aub ; (d) : tud homo / anti - aub ; (e) : tud hetero / anti - ago3 ; (f) : tud homo / anti - ago3 ]. mutant and control testes were immunostained with anti - aub and anti - ago3 antibodies [(a) : tud hetero / anti - aub ; (b) : enlarged image of the region boxed with a white line in (a) ; (c) : tud homo / anti - aub ; (d) : enlarged image of indicated region in (c) ; (e) : tud hetero / anti - ago3 ; (f) : enlarged image of indicated region in (e) ; (g) : tud homo / anti - ago3 ; (h) : enlarged image of indicated region in (g) ]. scale bars in all testis images indicate 20 m. all immunohistochemical analyses were performed at least three times and representative images are shown. we also examined how loss of krimp affects the localization of aub and ago3 in the germline. in krimp mutant ovaries, ago3 lost its ability to localize to the nuage, although aub was accumulated in the nuage the same as in the control (figure 4a). thus, krimp is necessary for nuage localization of ago3, but not of aub. tud nuage localization was maintained even after krimp expression was lost (figure 4a). in contrast, in krimp mutant testes, aub, ago3, and tud nuage localization was abolished (figure 4b). the requirement of krimp for aub and tud nuage localization is different in ovaries and testes. loss of krimp function affects the subcellular localization of aub and ago3 in ovaries and testes differently. (a) ovaries : the control and krimp mutant ovaries were immunostained with anti - aub, anti - ago3, and anti - tud antibodies [(a) : control / anti - aub ; (b) : krimp mutant / anti - aub ; (c) : enlarged image of the part boxed with a white line in (b) ; (d) : control / anti - ago3 ; (e) : krimp mutant / anti - ago3 ; (f) : enlarged image of indicated region in (e) ; (g) : control / anti - tud ; (h) : krimp mutant / anti - tud ; (i) : enlarged image of indicated region in (h) ]. (b) testes : the control and krimp mutant testes were immunostained with anti - aub, anti - ago3, anti - tud, and anti - krimp antibodies [(a) : control / anti - aub ; (b) : krimp mutant / anti - aub ; (c) : control / anti - ago3 ; (d) : krimp mutant / anti - ago3 ; (e) : control / anti - tud ; (f) : krimp mutant / anti - tud ; (g) : control / anti - krimp ; (h) : krimp mutant / anti - krimp ]. scale bars in all testis images indicate 20 m. all immunohistochemical analyses were performed at least three times and representative images are shown. an earlier study showed that krimp is localized to the nuage in ovaries (lim and kai, 2007). in the present study, we confirmed this localization (figure 5a) using our newly produced anti - krimp antibody (figure 1b). we then examined by immunostaining of ovaries and testes how krimp nuage localization is affected by loss of other pirna factors. we found that loss of ago3 and tud expression in ovaries did not affect krimp localization (figure 5a). however, in aub mutant ovaries, krimp signals were hardly detected in the nuage ; instead, krimp accumulated to unknown cytoplasmic structures (figure 5a). thus, aub, but not ago3 or tud, is required for krimp nuage localization. the krimp signal of immunostaining was lower in aub and tud mutant testes than that in the control, while krimp expression was unchanged by loss of ago3 expression (figure 5b). western blotting confirmed a severe reduction in the expression level of krimp in aub and tud mutant testes (figure 5c). thus, the requirement of tud in krimp nuage localization is different between males and females. why loss of aub and tud expression causes a severe reduction in the krimp expression requires further investigation. however, ago3 was able to accumulate in the nuage, possibly because aub was, at least partially, localized to the nuage in tud mutant testes. it could be postulated that ago3 nuage localization necessitates either aub or krimp in the nuage. (a) ovaries : the control (a) and aub (b), ago3 (c), and tud (d) mutant ovaries were immunostained with an anti - krimp antibody. (b) testes : the control (a) and aub (b), ago3 (c), and tud (d) mutant testes were immunostained with an anti - krimp antibody. (c) western blotting shows that the expression of krimp is hardly detected in aub and tud mutant testes. the nuage localization of pirna factors, aub, tud, krimp, and ago3, in ovaries and testes from wt and the pirna factor mutant flies was summarized (figure 6a) and their molecular hierarchy was compared (figure 6b). their expression patterns in wt ovary and testis were also summarized (figure 6c). an obvious difference in the hierarchy is the location of krimp between aub and ago3 in ovaries but at the same level as aub and upstream of ago3 in testes. krimp expression in testis is limited to developmentally early stage cells, such as germline stem cells, gonialblasts, and spermatogonia (figure 6c) and this may be the cause of the gender difference observed in the location of krimp in the molecular hierarchy. molecular hierarchy of the pirna factors in their localization to nuage in ovaries and testes. (a) the nuage localization of pirna factors, aub, tud, krimp, and ago3 in wild - type (wt) and their mutant ovaries and testes : n.d. : none detected. since none of the pirna factors in this figure were detected in the nucleus of wt ovaries and testes, therefore they were considered to be cytoplasmic proteins. in tud mutant ovaries and testes, aub is only weakly accumulated in the nuage ; thus, the nuage is indicated with a thinner line. (b) the nuage localization hierarchy : aub is placed upstream of ago3 in both ovaries and testes. however, krimp shows gender - specificity ; in ovaries krimp lies between aub and ago3, in testes krimp is placed at the same level as aub in testes. however, ago3 can be localized to the nuage without tud function in both males and females. it is likely that ago3 nuage localization occurs in both aub - dependent and tud - independent manners. (c) the expression patterns of aub, tud, krimp, and ago3 in ovaries and testes. gscs, germline stem cells ; cbs, cystoblasts ; ncs, nurse cells ; oo, oocyte ; gbs, goniablasts ; sg, spematogonia. we reported previously that, in testes, ago3 functions in the amplification loop with aub for producing transposon - derived pirnas (nagao., 2010). however, most ago3-associated testis pirnas corresponding to suppressor of stellate [su(ste) ] genes that function in silencing the stellate gene, are antisense - oriented and are also detected in pirnas associated with aub (nagao., 2010). in addition, the vast majority of ago3-associated pirnas derived from the at - chx locus on chromosome x, some of which may function in silencing vasa, are also antisense - oriented and are found among aub - associated pirnas (nagao., 2010). the pirnas that show characteristics similar to those of at - chx and su(ste) pirnas have so far not been found in ovaries (nagao., 2010). our current finding, that krimp is located in a different position in the nuage localization hierarchy in ovaries and testes, may further support the idea that some mechanisms of pirna biogenesis differ in ovaries and testes. our interpretation is that krimp may be one of the important factors for producing at - chx and su(ste) primary pirnas and may enabling their loading into not only aub, but also ago3, in testes. in ovaries, krimp is placed between aub and ago3 in the hierarchy and this may be why primary pirnas are specifically loaded into aub, but not into ago3. krimp has a tud domain and, thus, it is reasonable to postulate that krimp associates with aub and/or ago3 through their sdma modifications in both ovaries and testes. however, the mode of their associations could be different in the two tissues : the extent of sdma modification of aub and ago3 may be different and/or the sdma requirements in the association between krimp and aub / ago3 may be different. these possible differences could be the cause of the gender difference for krimp in the pirna factor hierarchy. we are currently investigating the biochemical function of krimp in pirna biogenesis and its relationship with aub and ago3, as well as the modification status of aub and ago3 in both ovaries and testes, which will help further our understanding of pirna biogenesis in general and of the gender differences in the molecular mechanisms in particular. | piwi - interacting rnas (pirnas) are germline - specific small non - coding rnas that form pirna - induced silencing complexes (piriscs) by associating with piwi proteins, a subclade of the argonaute proteins predominantly expressed in the germline. piriscs protect the integrity of the germline genome from invasive transposable dna elements by silencing them. multiple pirna biogenesis factors have been identified in drosophila. the majority of pirna factors are localized in the nuage, electron - dense non - membranous cytoplasmic structures located in the perinuclear regions of germ cells. thus, pirna biogenesis is thought to occur in the nuage in germ cells. immunofluorescence analyses of ovaries from pirna factor mutants have revealed a localization hierarchy of pirna factors in female nuage. however, whether this hierarchy is female - specific or can also be applied in male gonads remains undetermined. here, we show by immunostaining of both ovaries and testes from pirna factor mutants that the molecular hierarchy of pirna factors shows gender - specificity, especially for krimper (krimp), a tudor - domain - containing protein of unknown function(s) : krimp is dispensable for piwi protein aubergine (aub) nuage localization in ovaries but krimp and aub require each other for their proper nuage localization in testes. this suggests that the functional requirement of krimp in pirna biogenesis may be different in male and female gonads. |
we conducted a prospective rct in a single trauma center, the neurosurgery department of shahid rajaee hospital, affiliated to shiraz university of medical sciences, shiraz, iran based on a previously published protocol.5 patient enrollment began in september 2010 and will continue until a sample size of approximately 328 is reached.5 ethical approval was obtained prior to enrollment. written informed consent patients meeting the following criteria were included : age of 18 years or older, tsci between t1-l1, hemodynamic stability, evidence of spinal cord / conus medullaris compression and/or mri signal change, and hospital admission before 24 hours of injury. subjects meeting any of the following were excluded : major and current psychiatric illness, significant concurrent traumatic brain injury, major concurrent medical disease, pre - injury major neurologic deficits or disease, ankylosing spondylitis, penetrating thoracolumbar injuries, pregnant females, life - threatening injuries preventing early cord decompression, criminals under indictment, or incarceration, substance abuse, an american spinal injury association (asia) impairment scale (ais) grade of e, no cord compression on mri, spinal shock, any cognitive deficit, inability to provide informed consent, and an injury involving more than 2 adjacent vertebral levels. the diagnosis of tsci was based upon history and asia criteria. on admission after resuscitation and neurologic assessment, patients were assessed for suitability according to predefined inclusion / exclusion criteria. a preoperative mri and ct scan selected patients were randomly divided into 2 groups : early, undergoing decompressive surgery less than 24 hours from injury, and late, undergoing surgery between 24 - 72 hours. specifications of surgical intervention, such as direction of approach and number of levels decompressed were decided by a single attending spinal surgeon. one of 5 surgical procedures was used : 1) long - segment including : pedicle screws inserted at 5 levels ; 2 levels above, 2 levels below, and in the fractured vertebra as a fixation point. 2) long - segment non - including : pedicle screws inserted at 4 levels ; 2 levels above, 2 levels below, but not inside the fractured vertebra. 3) short - segment including : pedicle screws inserted at 3 levels ; one level above, one level below, and in the fractured vertebra. 4) short - segment non - including : pedicle screws inserted at 2 levels ; one level above and one level below, but not inside the fractured vertebra. 5) 360 procedure : a 2-stage operation involving a posterior decompression followed by an anterior approach 1 - 2 weeks afterwards. posterior decompression was followed by insertion of titanium vertebral body replacer or tricortical iliac graft. the screw placement was evaluated 8 - 24 hours after each operation by spiral ct scan and revised immediately if the location or alignment were not acceptable. patients received intravenous methylprednisolone (30mg / kg bolus over 15 minutes, and 5.4mg / kg / h infusion over 23 hours 45 minutes if they arrived 0.05) (table 1). flow diagram of traumatic spinal cord injury patient selection, randomization, and follow - up. american spinal injury association impairment scale (ais) grade improvement at 12 months : a) early versus b) late surgery in traumatic spinal cord injury patients. for cases with complete tsci, no improvement of mean asia motor score was observed in either group over 12 months. for incomplete tsci, both groups showed improvement on follow - up visits (figure 3). in the early surgery group, the mean motor score improved from 77(22) on preoperative examination, to 92(12) on 12-month follow - up visit. in the late surgery group, a change from 68(22) to 82(16) was observed. two cases of deep vein thrombosis were observed, one in each group. in the late surgery group, there were 2 cases of wound infection, one case of csf leak, one case of meningitis, and one case with decubitus ulcer. a total of 250 screws were used for surgical fixation, 6 of which required revision. in the early surgery groups there was one right - sided t6 revision and a bilateral revision at t9. in the late surgery group, there was one right - sided t12 revision and also one left t9 and right t7 revision both performed in the same patient. all revisions were performed on the same day and were due to lateral placement of screws without cord compression. there was one case of bilateral rod fracture, and screw pulled - out in the late surgery group one year after surgery. table 3 demonstrates mean restoration / rebuilding of vertebral height and reduction of kyphotic angle one week after surgery and loss of restoration / rebuilding of height and reduction of kyphotic angle after one year. there were no significant differences in radiologic outcomes between groups. mean american spinal injury association (asia) motor score at baseline and over 1-month, 3-month, 6-month, and 12-month postoperative (post - op) follow - up visits in incomplete traumatic spinal cord injury (sci) and early versus late surgery groups. no change in motor score was observed in patients with complete traumatic spinal cord injury. description of restoration / rebuilding of vertebral height and reduction of kyphotic angle one week after surgery, and one year loss of restoration / rebuilding between groups among traumatic spinal cord injury (sci) patients. despite the few prospective studies on surgical decompression in acute sci, clinical benefits of early or late surgery remain controversial. a prospective analysis indicated no clinical and neurological benefits of surgical decompression between the first 72 hours after cervical sci and later than 5 days post - injury.3 the relatively late spinal cord decompression, and the 21% loss to follow - up of patients were 2 concerns for the above - mentioned study. however, another level-2 evidence study suggested that surgical decompression earlier than 8 hours would provide better neurological outcome, shorter hospital stay, and lower frequency of secondary complications in comparison with undergoing surgical intervention 3 - 15 days after thoracolumbar sci.11 recently, a prospective cohort compared the effectiveness of early (< 24 hours) versus late (24 hours) decompressive surgery after cervical tsci, the results of which favor early surgery.1 a smaller prospective canadian study2 supported these results, reinforcing the association between early surgery and improved neurologic outcomes. our main purpose in conducting this rct was to evaluate the efficacy of early versus late surgical intervention in the setting of thoracic / thoracolumbar tsci, as carried out in retrospective studies.12,13 we considered a 24-hour cutoff recommended for early surgical sci decompression.14 we assigned patients with acute t1-l1 tsci with documented spinal cord compression to the early / late decompression groups. most of the reported literature on tsci comes from western countries ; however, these injuries are potentially more disabling in developing countries.15,16 feasibility studies have shown that due to transport and life saving measures, only 23.5 - 51.4% of tsci patients can undergo an operation within the first 24 hours of injury.17,18 these figures are even more limited in developing countries due to lack of fundamental resources.13 the benefit of surgery timing is unclear, and only a minority of patients may actually receive a 24 hour surgery time frame with current measures in developing countries. considering the above along with the benefit to society from the scientific answer to timing of surgery, the positive impact that this knowledge may have in allowing for appropriate resource utilization and future planning outweighs the risk of furthermore, paraplegia is more common than tetraplegia in tsci patients in developing countries,19 possibly due to delayed transportation and limited access to full capability facilities for managing cervical tsci. therefore, most tsci patients referred to our trauma centers are cases with thoracolumbar injury. the spinal cord is more sensitive to trauma than lumbar nerve roots,20 and cord injury is more prevalent than root injury in spine trauma.13 we found an increase in ais grade in both groups. despite similar baseline characteristics, surgical profiles, and vertebral restoration profiles, a 2-grade improvement was observed in 3 patients undergoing early versus one undergoing late surgery. although this limited observation is not statistically justifiable, it is in line with recent findings favoring early surgical intervention in sci. the lower number of complications observed among the early surgery group also follows this trend. the surgical timing in acute spinal cord injury study1 showed that decompression < 24 hours was associated with improved neurologic outcomes defined as at least a 2-grade ais improvement over 6-month follow - up. however, the authors tempered the conclusions given the intrinsic limitations of the cohort study design. high - dose steroid therapy is the only pharmacologic therapy shown to have efficacy on neurologic recovery in sci. considering the effect of methylprednisolone administration, all our patients were treated according to the recommendations of the nascis-2 study.21 therefore, although methylprednisolone administration may be a major confounding factor for differences in recovery, we believe its effects to be balanced among our study groups. the asia motor score is considered a more reliable predictor of functional outcome after sci than sensory score.22 no change in motor score was observed in any of our patients with complete tsci. the ais change in these patients was solely attributable to an improved sensory level. regarding the level of tsci, there may be a predominant component of lower motor neuron, rather than upper motor neuron injury among most of our patients. the chance of neural recovery would be potentially greater than the proximal cervical and upper thoracic spinal cord.23 improved ais grade may therefore be ascribed to root escape. furthermore, regarding the efficacy of treatment, the mid - thoracic region is the most difficult to evaluate. in cases of small caudal improvement in the injured thoracic cord, the large distance from the mid - thoracic to cervical and lumbar enlargements makes the efficacy of small positive changes to the motor system almost unrecognizable.24 a retrospective evaluation of t2-t11 sci showed that surgical decompression has no apparent neurologic benefit in complete tsci.12 also, a retrospective evaluation of blunt t12-l1 tsci revealed no correlation between decompression timing and motor improvement.13 with further regard to asia motor score, determination of a functionally meaningful threshold to document the benefit of therapeutic intervention depends on the level and severity of tsci, as well as degree of spontaneous recovery with conventional treatment.25 in our study protocol, a 5-point or greater improvement in summed asia motor score was considered significant.5 on 12-month follow - up of incomplete tsci, we observed a mean improvement of 15(10) scores in early, and a 14(6) score improvement in late surgery patients. also of note is the fact that the absolute difference in the number of asia motor points between groups is not as important as whether a statistically valid difference is present and if the magnitude of the difference confers with clinical benefit and improved functional outcome.26 one of the strengths of the current study is that all surgical procedures were performed under supervision and decision of a single attending. nevertheless, a uniform standard approach could not be considered due to the distinct quality of each case. also, separate randomization of complete and incomplete t1-l1 tsci enables a comparison of outcome measures in these groups with long - term follow - up and a low dropout rate. neurological examination of our patients is prone to inter - observer variability as patient assessment and follow - up were not performed by a single examiner. a further limitation is the small number of cases preventing us from employing powerful statistical analyses. we limited our results to mainly presenting descriptive data acquired from the groups thus far. the role of surgical intervention in t1-l1 tsci can more clearly be determined through advancement and completion of our rct. in conclusion, the primary results of our rct shows an overall improvement in ais score and motor score in both groups of early and late surgery. two - grade improvements in ais were seen in 3 out of 16 in early, and one out of 19 in late surgery patients. regarding complete tsci, no improvement in motor score was found in either group ; ais change in this group was solely due to increased sensory scores. an increase in the number of enrolled patients allowing for powered statistical analysis will shed more light on the role of surgery timing in the management of acute t1-l1 sci. moosavi ns, emami z, vaccaro ar, rahimi - movaghar v. long - term follow - up study of patients with spinal cord injury. butt mf, dhar sa, farooq m, hussain a, mir ba, halwai ma,. neurological outcome following delayed fixation of unstable thoracolumbar spinal injuries with short segment posterior fixation. farrokhi mr, motallebi h. comparison of outcome of surgical and nonsurgical methods in the treatment of unstable traumatic lesions of the lower cervical spine. | objective : to assess the efficacy of surgical decompression < 24 (early) versus 24 - 72 hours (late) in thoracic / thoracolumbar traumatic spinal cord injury (tsci).methods : a randomized controlled trial (rct) of 35 t1-l1 tsci patients including early (n=16) and late (n=19) surgical decompression was conducted in the neurosurgery department of shahid rajaee hospital from september 2010. pre- and postoperative american spinal injury association (asia) impairment scale (ais), asia motor / sensory scores, length of hospitalization, complications, postoperative vertebral height restoration / rebuilding and angle reduction, and 12-month loss of height restoration / rebuilding and angle reduction were evaluated.results:sixteen patients (46%) had complete tsci. no ais change was seen in 17 (52%) patients. complete tsci patients had no motor improvement. the ais change in this group was solely due to increased sensory scores. for incomplete tsci, the mean motor score improved from 77 (22) to 92 (12) in early, and from 68 (22) to 82 (16) in late surgery. one deep vein thrombosis was observed in each group. there were 2 wound infections, one csf leak, one case of meningitis, and one decubitus ulcer in the late surgery group. six screw revisions were required.conclusion:our primary results show overall ais and motor score improvement in both groups. motor improvement was only observed in incomplete tsci. two - grade improvements in ais were seen in 3 early, and one late surgery patient. |
from a worldwide perspective, the life span of humans has been prolonged, and the proportion of elderly people in the total population has increased. various changes occur in the human body with age. in elderly men, lower urinary tract symptoms (luts), erectile dysfunction (ed), and decreased libido often appear concurrently. several community - based studies have shown a strong correlation among sexual dysfunction, increasing age, and the severity of luts. this coexistence of sexual problems with luts and benign prostatic hyperplasia (bph) further affects quality of life (qol). luts and ed share increased prevalence as age increases, and in many cases, the two are synchronously present. thus, continuous efforts have been made to disclose the common pathophysiology between these two conditions. no definite explanations have been given to clarify the pathophysiological mechanism of the correlation between luts and ed, but several hypotheses have been proposed [2 - 5 ]. one possible cause is overactivation of the autonomic nervous system and increased tension of the sympathetic nervous system. according to this hypothesis, luts increases sympathetic nervous system activity, which induces the occurrence of urinary storage symptoms due to the contraction of smooth muscles in the prostate gland and urinary bladder. ed occurs as the result of smooth muscle contractions in the corpus spongiosum, and the myosin light chain is phosphorylated because of increased rho - kinase activity, which can cause smooth muscle layer contraction. it has been proposed that endothelial dysfunction in the prostate gland and corpus spongiosum and hormonal imbalance are physiological causes for the correlation between luts and erectile dysfunction. changes in lifestyle due to nocturia and rapid eye movement sleep disorder may affect the occurrence of erectile dysfunction. also, the result of our preliminary study showed that age, international prostate symptom score (ipss), nocturia, and the uroflow rate correlated significantly with the 5-item version of the international index of erectile function (iief-5). even in cases in which age was controlled for, qol was found to have a significant correlation with sexual function. prostate gland volume was not significantly correlated with sexual function, implying that erectile function is not affected by subjective luts even with a relatively greater prostate size. irwin reported that overactive bladder (oab) was significantly associated with increased prevalence of ed and that sexual activity, sexual enjoyment, and sexual satisfaction were reduced as the result of urinary symptoms. in recent years, many studies have reported that administration of alpha - blockers improves luts due to bph and sexual dysfunction. these findings suggest that rudimentary treatments of luts, a risk factor for erectile dysfunction, would be mandatory to effectively improve sexual function in patients with bph who concurrently have luts and ed. the primary treatment regimen for bph is to administer alpha - blockers, for which the main mode of action is based on smooth muscle relaxation in the prostate and the resulting improvement in the urinary flow rate and voiding symptoms. however, a synchronous improvement of irritative and voiding symptoms may be the most effective treatment regimen for luts / bph. it is expected that the concomitant administration of alpha - blockers and anticholinergics would improve luts more effectively in patients with luts / bph. these effects are also expected to have a positive effect in secondarily improving sexual function. thus, the aim of this study was to examine the effects on erectile function of a combination of the alpha - blocker tamsulosin to improve luts and the antimuscarinic agent solifenacin to improve irritative symptoms of the bladder. male patients aged 40 years or older who had concurrent luts / bph and ed were enrolled in this study. inclusion criteria were an ipss total score > 12, an ipss - qol score > 3, and an iief-5 score 4 mg / dl ; 6) severe renal dysfunction or hepatic dysfunction ; 7) residual urine > 100 ml ; and 8) ineligibility for the current study as judged by the investigators. sixty male patients between the ages of 41 and 76 years with luts / bph and ed were divided into two groups by using a table of random sampling numbers : group 1 (the alpha - blocker treatment group ; n=30) and group 2 (the alpha - blocker+solifenacin treatment group ; n=30). in group 1, an alpha - blocker (tamsulosin 0.2 mg q.d.) was solely administered for 3 months. in group 2, an alpha - blocker and an antimuscarinic drug (tamsulosin 0.2 mg and solifenacin 5 mg q.d) were concomitantly administered during the same period. in patients who were suspected of having a significant bladder outlet obstruction (postvoiding residual urine > 100 ml or maximum flow rate 100 ml or maximum flow rate 100 ml or qmax < 5 ml / s) and who had luts / bph ; we therefore found significantly improved luts with no complications such as acute urinary retention. common side effects following the use of anticholinergic (or antimuscarinic) agents are dry mouth, constipation, blurred vision, headache, and dry eye, but almost no reports have described the effects on sexual function. some concern exists that the use of anticholinergic (or antimuscarinic) agents in patients with bph may lead to aggravation of voiding difficulty or the development of urinary retention. to date, urologists have not actively used anticholinergic (or antimuscarinic) agents, but many types of antimuscarinic agents have been used in recent years to treat oab in a clinical setting. as related experiences have accumulated, these drugs have been selectively used for patients with luts / bph. almost no reports have addressed the effects of antimuscarinic agents on sexual function in luts / bph patients. in a study of 39 men with bph and luts in whom previous alpha - blocker therapy had failed, kaplan found normal sexual function in 27 (63%) and 29 (67%) patients before and after administration, respectively. no reports have indicated that the administration of anticholinergic (or antimuscarinic) agents significantly impairs sexual function. in our series, a comparison of iief-5 scores before and after concomitant drug treatment with solifenacin and tamsulosin revealed no statistically significant changes. thus, it can be inferred that administration of anticholinergic (or antimuscarinic) agents had no significant effect on sexual function. in patients with luts / bph, concomitant treatment with alpha - blockers and antimuscarinics may lead to improvement in luts, but this treatment can not be expected to improve sexual function. in patients with luts / bph and ed, concomitant use of tamsulosin and solifenacin was effective for improving luts. however, sexual function was not significantly improved by the concomitant administration of alpha - blockers and antimuscarinics. | purposeto examine the effects on erectile function of concomitant treatment with an alpha - blocker (tamsulosin) and an antimuscarinic agent (solifenacin) in patients with lower urinary tract symptoms (luts)/benign prostatic hyperplasia (bph).materials and methodsfifty - seven male patients with luts / bph were assessed for the degree of luts and erectile function. in group 1 (tamsulosin) and group 2 (tamsulosin and solifenacin), changes in the international prostate symptom score [ipss : total scores, storage symptoms (st), voiding symptoms (vd), and quality of life (qol) ], prostate - specific antigen, trans - rectal ultrasonography, urine flowmetry, residual urine, and a 5-item version of the international index of erectile function (iief-5) were assessed after a 3-month treatment period. in both groups, it was determined whether treatment was associated with changes in luts and erectile function and whether improvement in the ipss was correlated with the iief-5. comparative analysis was also done to examine the linear relationship between improved ipss scores and iief-5 scores.resultsa comparison of the degree of improvement in all the parameters indicated that both groups showed significant improvement in total ipss, ipss - st, ipss - vd, and ipss - qol (p<0.05). a comparison of the degree of improved sexual function associated with improved luts in each patient showed significant improvement in the iief-5 score associated with the degree of improvement in the ipss - st domain in group 1, but no significant associations were found in group 2. in cases in which tamsulosin was administered, the iief-5 score significantly improved as the ipss - st domain score improved. in the group in which tamsulosin and solifenacin were concomitantly administered, improvement of the ipss - st domain score had no significant effect on the iief-5 score.conclusionsin patients with luts / bph, tamsulosin and solifenacin combination therapy was effective for luts, but erectile function was not significantly improved. therefore, although effective for improving luts, combination therapy with an alpha - blocker and an antimuscarinic agent was not effective for improving erectile function. |
preoperative vital signs, blood tests, urinalysis, electrocardiogram, physical exam, and chest x - ray findings were all normal as well. as premedication, 0.25 mg of intramuscular atropine sulfate was injected. after the patient arrived in the operating room, electrocardiography, pulse oximetry, and noninvasive blood pressure monitoring pre - anesthetic blood pressure was 130/80 mmhg, heart rate was 120 beats / min, and spo2 was 99%. intravenous injection of thiopental sodium 125 mg and recuronium bromide 20 mg was performed for induction. during the maintenance phase of general anesthesia, 1.5 vol% of sevoflurane, 1.5 l / min of air, 1.5 l / min of oxygen, and 0.5 g / kg / min of remifentanil hcl were used. after intubation, we applied oxytetracycline hcl / polymyxin b sulfate (terramycin ophthalmic ointment, pfizer, jakarta, indonesia) eye ointment to both eyes, and taped the eyelids closed with paper surgical tape. we applied the same ointment to the upper lip to prevent lip injuries due to the use of a davis - crowe mouth gag during tonsillectomy. vital sign were stable throughout the operation, and the surgery ended with no specific problems. removing the mouth gag, we found swelling of the patient 's upper lip (fig. there was no evidence of injury due to surgical procedures, and there was no edema or urticaria in any other body part. vital signs were stable, and there was no wheezing or change in the inspiratory pressure. we assumed that the symptoms were due to an allergic reaction of unknown etiology and injected 2 mg of chlorpheniramine maleate and 2 mg of dexamethasone disodium phosphate. even though there was no swelling other than in the eyelids and upper lip, we could not completely rule out laryngeal edema and thus decided to extubate after confirmation. after suctioning the oral secretions, the cuff of the endotracheal tube was deflated and we could confirm an air leak around the tube. re - intubation and tracheostomy sets were prepared in case of unexpected airway obstruction, and the endotracheal tube was removed after complete recovery of spontaneous respirations. after extubation, the patient 's respirations were normal and his vital signs remained stable. we consulted ophthalmologists and dermatologists, and these specialists concluded that, since we had seen follicular reaction and injection in the conjunctivae, with no corneal lesions or visual disturbances, the symptoms were due to an allergic reaction of the eyelids, conjunctivae, and lips, which would recover as time passed. we explained this to the patient 's parents, and decided to treat him conservatively using steroid eye drops and intravenous steroids and antihistamines. the patient was moved to the general ward, and complained of no specific symptoms other than some itching sensations in the lips and periorbital area. on postoperative day 2, the edema was almost completely relieved, and the patient was discharged. instillation of eye ointment or taping of the eyelids closed is performed to prevent possible ocular injuries during general anesthesia. the latter method is simple and effective, but there has been a case report of periorbital edema as an allergic reaction to the tape. in the case discussed here, edema was present not only in the eyelids and conjunctivae, but also in the upper lip, to which the ointment had also been applied. accordingly, we assumed that the edema had occurred due to the ointment, rather than the tape. allergic reaction of the eyes due to contact with external allergens causes conjunctival injection, follicular conjunctivitis, periorbital edema, and pruritus. the signs that the patient in our case showed are consistent with an allergic reaction, considering that he suffered from conjunctival injection, conjunctival follicles, and periorbital edema. certainly, allergic reaction to the muscle relaxant, inhalation anesthetic, thiopental sodium, or remifentanil is to be considered. however, allergy related to the drugs frequently used during anesthesia is usually accompanied by systemic reactions such as hypotension, tachycardia, hypoxia, high airway resistance, and more extensive skin reactions [8 - 10 ]. physical irritation during anesthetic induction or surgery can cause edema as well. however, in this case there was no need to forcefully compress the patient 's face with a mask, because ventilation was well maintained during the induction phase, and all body parts other than the mouth were covered with surgical drapes. the operator mentioned that he had not compressed the patient 's face during the operation. there is little chance that edema had occurred due to position or physical irritation, since the surgery was performed in a supine position with the head elevated 10 degrees. in an allergic reaction, the mainstay of treatment is conservative management using antihistamines, mast - cell stabilizing drugs, and steroids, and it is important to find the causative agent to prevent recurrence. in this case, our therapeutic methods appear to have been proper, since the use of steroids and antihistamines promptly relieved the patient 's symptoms. eye ointments may be effective for protection of the eyes during surgery because they remain for a long time after instillation. however, a study showed that there was no significant difference in protective effect between taping of the eyes after applying ointment and taping only. furthermore, eye ointments can cause postoperative foreign body sensation and visual blurring, as well as periorbital edema and conjunctival injection, which were present in our case. thus, routine instillation of eye ointments during surgical procedures should be considered with caution, even though it is performed for protection of the eyes. in the case discussed here, edema of the eye and upper lip, conjunctival injection, and follicular conjunctivitis occurred after tonsillectomy and adenoidectomy. this response is assumed to have occurred due to an allergic reaction to eye ointment applied for protection of the eyes and lips. therefore, eye ointment for protection of the eyes under general anesthesia should be used carefully and with more strict criteria. | patients undergoing non - ocular surgery under general anesthesia may suffer from ocular complications because of the loss of protective reflexes. simple taping of the eyelids closed, the instillation of ointments into the conjunctival sac, and the use of protective goggles have been recommended for eye protection. we observed a case of a 6-year - old child undergoing tonsillectomy and adenoidectomy who experienced severe edema after application of an ointment (terramycin ophthalmic ointment) to the orbits and upper lip. after several days of steroid and antihistamine administration, the edema of the orbits and lip returned to normal. therefore, we suggest that unexpected edema in the orbits and lip following surgery can be due to hypersensitivity to the ointment. |
specific mirnas have been found to be expressed in cell type - specific manner, at specific developmental stages, and in disease states including cancer [1, 2 ]. during the initiation and progression of cancer, mirnas have been observed to act as oncogenes or tumor suppressors [3, 4 ]. while some mirnas are overexpressed in cancers, the majority appear to be lost and often localize to fragile sites. differences in the mirna expression profiles of normal compared to cancerous tissue of the endometrium, breast and ovary have been documented [611 ]. mirnas can affect the expression of a large number of proteins, including those involved in pathways relevant to cancer, such as apoptosis, migration and metastatis. thus, mirnas hold promise as biomarkers for several types of cancer [12, 13 ]. epithelial to mesenchymal transition (emt) is a normal process that occurs during development in which individual cells or groups of cells become motile. the same process is thought to be used by cancer cells during tumor progression to enable them to become more motile and thus more metastatic. emt involves reprogramming of the cells by transcription factors such as zeb1, sip1 (zeb2), twist, snail, and slug. a hallmark of emt is loss of e - cadherin expression, loss of polarity, acquisition of mesenchymal markers, and increased motility [16, 17 ]. both zeb1 and the closely related zeb2 bind e - box like sequences in the e - cadherin promoter, recruit the corepressor ctbp and thereby repress e - cadherin. zeb1 expression is confined to cells of mesenchymal origin, while normal epithelial cells and low grade carcinomas do not express zeb1. however, we and others have shown that in high grade, aggressive carcinomas that have undergone emt, zeb1 can be expressed, leading to loss of e - cadherin [1922 ]. several mirnas have been implicated in the process of emt, among them are the members mir-200 family [2325 ]. this family contains five members (mir-200a, -200b, -200c, -141 and, -429) which are highly homologous. originally, mir-200c was reported to directly bind zeb1 and cause degradation of the mrna, resulting in an upregulation of e - cadherin. subsequently, other reports have shown that all members of the mir-200 family, since they share a high degree of homology especially in their seed sequence, are capable of repressing both zeb1 and zeb2 [2729 ]. we have demonstrated that mir-200c represses not only zeb1/2, but a program of transcripts normally expressed only in cells of mesenchymal origin. since members of the mir-200 family are responsible for repressing zeb1 and zeb2 as well as other mesenchymal genes, these mirnas are considered mir-200 family members are therefore thought to be expressed in an epithelial cell - specific manner in normal tissues. recently, the ability of zeb1 to transcriptionally repress expression of mir-200 family members has been documented [31, 32 ]. this double negative feedback loop between mir-200 family members and zeb1 allows for plasticity between the epithelial and mesenchymal states. in this paper, we focus on the role of mir-200c in breast, ovarian, and endometrial cancers. the mutual repression between zeb1 and mir-200c is functional in some, but not all cells that we have tested. we demonstrate that the decrease in migration and invasion observed when mir-200c is reintroduced to cancer cells that lack it is independent of restoration of e - cadherin. lastly, we have previously demonstrated that class iii beta tubulin (tubb3) is directly controlled by mir-200c. expression of tubb3 is known to be a common mechanism of resistance to microtubule - targeting agents in many types of cancer. here, we present conclusive data that repression of tubb3 is the mechanism whereby mir-200c restores sensitivity to paclitaxel. taken together, these data demonstrate that loss of mir-200c is a marker for chemoresistance and aggressiveness in breast, ovarian, and endometrial cancers. hec50 cells, representing the more aggressive type ii endometrial cancers, were cultured in dmem with 10% fbs, and 2 mm l - glutamine. mda - mb-231 are a triple negative breast cancer cell line and were grown in media containing 5% fbs, hepes, nonessential amino acids, l - glutamine, penicillin, streptomycin, and insulin. the identity of all the cell lines was confirmed by dna profiling using the identifiler kit (applied biosystems). lipofectamine 2000 (invitrogen) was combined with pre-200c (mirna mimic) or scrambled negative control (ambion) at a concentration of 60 nm and incubated in serum free rpmi for 20 minutes prior to addition to hey cells. cells were incubated at 37c for 4 hours before replacement of fbs to 10%. protein and rna were harvested 48 hours posttransfection. tubb3 (from fernando cabral, university of texas - houston medical school) was cloned into pci - neo. transient transfection of 3.3 g of tubb3 plasmid or empty vector (pci - neo, promega) per well in a 6-well plate was performed using lipofectamine 2000. transduction of cells was performed using smartvectortm shrna lentivral particles (thermo scientific dharmacon). each cell line was transduced with 3 separate lentiviral constructs targeting zeb1 as well as two controls : smartvector empty vector particles and smartvector firefly luciferase control particles. the former is a negative control and does not correlate with gene silencing and the latter is a positive control targeting firefly luciferase plasmids pgl2 and pgl3. all vectors are packaged and contain a turbogfp and an scmv promoter, as well as a puromycin - resistance selectable marker. mda231 and hec50 cells were plated at 3000 cells / well and 1500 cells / well, respectively, in triplicate using 96 well plates. the following day, media was replaced with 80 l of fresh media containing 10 g / ml polybrene (sigma). the amount of viral particles / well was determined using the following calculation : (moi cn)/vt, where moi (multiplicity of infection) = 10 tu / cell, cn = number of cells / well, and vt = stock viral titer of 10 tu/l. viral particles were added in a total volume of 20 l to each well. the following day, transduction media was removed and wells were rinsed with pbs and replaced with regular media. once confluent, cells were trypsinized and replated in 48 well plates. at this point, antibiotic selection was initiated and cells were ultimately expanded and maintained using 1 g / ml of puromycin (sigma). whole cell protein extracts were denatured and 50 g separated on 8% sds page gels and transferred to pvdf membranes. the membranes were blocked in 5% milk in tbs - t, and then probed overnight at 4c. doug darling, university of kentucky, 1 : 1500 dilution), e - cadherin (clone nch-38 from dako, 1 g / ml), tubb3 (rabbit polyclonal prb-435p from covance, 1 : 5000 dilution), and -tubulin (clone b-5 - 1 - 2 from sigma, 1 : 20000 dilution). after incubation with appropriate hrp - conjugated secondary antibody, bands were detected using western lightning chemiluminescence reagent plus (perkin elmer). rna was harvested from cells using trizol (invitrogen) as per the manufacturer 's instructions. prior to generating cdna, mrna was treated with dnase1 (invitrogen) for 15 minutes at room temperature. rna was reverse transcribed into cdna in a reaction containing reaction buffer, dntps, rnase inhibitor (applied biosystems), random hexamers, and 200 u of mulv - rt (applied biosystems). the reaction proceeded at 22c for 10 minutes, then at 37c for one hour. for normalization, real time rt - pcr was performed on the cdna using eukaryotic 18s rrna endogenous control primers and fam - mgb probe (applied biosystems). taqman microrna reverse transcription kit was used to generate cdna for real time rt - pcr reaction in conjunction with a mir-200c specific primer and probe (abi, assay i d 002300). the reverse transcription primer for mir-200c is a hairpin primer which is specific for the mature mirna and will not bind to the precursor molecules. reported values are the means and standard errors of 3 biological replicates. the relative mrna or mirna levels were calculated using the comparative ct method (ct). briefly, the cycle threshold (ct) values for the rrna were subtracted from ct values of the target gene to achieve the ct value. the 2 was calculated for each sample and then each of the values was then divided by a control sample to achieve the relative mirna levels (ct). the assays were performed on mda - mb-231 stable empty vector or shzeb1 # 2, or hey cells transiently transfected with the mir-200c mimic for 48 hours. bd biocoat control insert chambers 24-well plate with 8 micron pore size and bd biocoat matrigel invasion chambers were used for migration and invasion assays, respectively. after starvation, cells were removed from the plate and 50000 hey cells or 250000 mda - mb-231 cells were plated in 0.5 ml media with 0.5% fbs in the upper chamber. in the lower chamber 0.8 ml of 50% conditioned media plus 50% complete media containing an additional 10% fbs hey cells were incubated for 24 hours and mda - mb-231 cells for 48 hours at 37c. migrating or invading cells on the lower surface of the membranes were stained with diff - quik stain (fisher) and counted manually using imagepro plus software (mediacybernetics inc.). adhesion assays were performed using innocyte ecm cell adhesion assays (calbiochem) for collagen iv, fibronectin, basement membrane complex and laminin. to each well 50000 cells the plates were incubated for 1 hour at 37c, and fluorescence was read with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. the relative fluorescent units were plotted, and the error bars represent standard error of the mean over four replicates. hey cells were plated into 6-well plates at a density of 2000 cells per well. twenty - four hours after plating, the cells were treated with 0, 1, 2, 3, 4, or 5 nm paclitaxel (sigma) in triplicate. the cells were incubated at 37c for 8 days before fixing and staining with crystal violet. photos were taken of the plates and the images analyzed using imagej software (nih). the average number of colonies and the average total area was plotted, with error bars representing the standard error of the mean over the three replicates. hec50 cells were transfected with the mir-200c mimic as described previously for the hey cells. twenty - four hours after transfection, cells were treated with 0, 15, 20 or 25 nm paclitaxel (sigma). twenty - four hours after treatment, the cell death elisa (roche) which recognizes mono- and oligonucleosomes in the cytoplasm of dying cells was performed as per manufacturer 's instructions. cells were plated into 6-well plates (4000 hey cells / well and 6000 mda - mb-231 or hec50 cells / well). at time points indicated, cells were trypsinized and counted using the vi - cell cell viability counter (beckman coulter). just as members of the mir-200 family can repress zeb1 by degradation of its transcript, zeb1 can repress expression of the mir-200 family members by binding to e - boxes within their promoter regions [31, 32 ] ; see figure 1(a). directly increasing mir-200c levels in hey cells (aggressive serous ovarian cell line) by transfection of a mir-200c mimic (pre-200c) results in repression of zeb1 expression (figure 1(b)). although zeb1 is a repressor of e - cadherin, we did not observe e - cadherin expression induced by the repression of zeb1 in these cells (data not shown). however there are several other mechanisms through which e - cadherin can be lost including methylation of the promoter [34, 35 ] and chromosomal deletion [36, 37 ]. in contrast, we have previously shown that transient transfection of the mir-200c mimic into mda - mb-231 (an aggressive triple negative breast cancer cell line) and hec50 (an aggressive type 1 endometrial cancer cell line) causes a marked repression of zeb1 and a restoration of e - cadherin expression. presently, we stably transduced lentiviral shrnas targeting zeb1 into these two cell lines (hec50 and mda - mb-231). while two of the shrnas did not decrease zeb1 protein, shrna # 2 caused an almost complete repression of zeb1 expression resulting in re - expression of e - cadherin in both cell lines (figures 2(c) and 2(d)). intriguingly, while knock down of zeb1 in mda - mb-231 cells causes the expected increase in mir-200c levels (indicative of the reciprocal regulation), no such increase is observed in hec50 cells. this suggests that while reciprocal repression of mir-200c and zeb1 occurs in some cell lines, it does not occur in all. it has been previously shown that the mir-200 family members cause a decrease in cell migration and invasion [27, 29, 30 ]. we observe a decrease in migration and invasion in the mda - mb-231 cells in which zeb1 has been knocked down, resulting in an increase in mir-200c levels. in the mda - mb-231 cells there is 52% decrease in migration and a 50% decrease in invasion in the shzeb1 # 2 containing cells in which zeb1 is completely knocked down versus luciferase control (figures 2(a) and 2(b)). we show here that the same holds true in the aggressive ovarian cancer hey cell line. this cell line is highly migratory and invasive, and reintroduction of mir-200c to these cells results in an 83% decrease in migration and a 7986% decrease in invasion compared to negative controls (figures 2(c) and 2(d)). however, it is interesting to note that the effect on migration and invasion caused by mir-200c is independent of the e - cadherin status of the cells, since unlike the mda - mb-231 cells, hey cells do not regain e - cadherin expression in response to decreased zeb1 levels. while e - cadherin protein affects epithelial cell - cell contact, we also wished to determine if mir-200c affects adhesion to substrates as measured by fluorescent adhesion assays. hey cells transiently transfected with the mir-200c mimic showed a small but statistically significant decrease in adhesion to basement membrane complex (bmc) and laminin (figures 3(a) and 3(b)). there is also a trend towards decreased adhesion to collagen iv (figure 3(c)) ; however, this did not reach statistical significance. no difference in adhesion to fibronectin was observed (figure 3(d)). since there was an affect on adhesion to bmc and laminin in ovarian cancer cells with high mir-200c levels, we performed the adhesion assay with the hec50 and mda - mb-231 cells in which zeb1 had been stably knocked down. we again see a decrease in adhesion to bmc and laminin in the mda - mb-231 cells ; however, only the decrease in bmc binding is statistically significant (figures 3(e) and 3(f)). in contrast to the hey and mda - mb-231 cells, there was no decrease in adhesion to either substrate in the hec50 cells in which zeb1 is knocked down (data not shown), but there is not a concomitant increase in mir-200c, as shown in figure 1(d). we have previously demonstrated that mir-200c expression causes increased chemosensitivity to microtubule targeting agents such as paclitaxel. while the elisa cell death assay that we have used previously to demonstrate this property of mir-200c is a short - term assay, we confirm here, in a relatively long - term clonogenic assay, that there is increased sensitivity of hey cells to paclitaxel when transfected with pre-200c (figure 4(a)). we observe a 4955% decrease in total area and a 6770% decrease in the number of colonies in the pre-200c treated cells versus the negative control with 5 nm paclitaxel treatment (figures 4(b) and 4(c)). as the assay is conducted over a relatively long period of time, the maximum dose of paclitaxel used is relatively small compared to what is used in the assays that look at acute toxicity (i.e., 24 hours). at doses of paclitaxel of 10 nm and higher, we have previously implicated the ability of mir-200c to directly target tubb3 (class iii beta tubulin) as being the mechanism responsible for the increased chemosensitivity to microtubule targeting agents. tubb3 is normally only expressed in neuronal cells ; however aberrant expression of tubb3 in several different types of cancers has been shown to cause resistance to paclitaxel [3843 ]. to definitively test whether tubb3 is responsible for the mir-200c - mediated increase in chemosensitivity to paclitaxel, we transfected cells with a tubb3 construct lacking its 3 utr (containing the mir-200c binding site) which is therefore not able to be targeted by mir-200c. transfection of this exogenous tubb3 construct does not affect the transfection of the mir-200c mimic, nor its ability to downregulate zeb1 and upregulate e - cadherin (figures 5(a) and 5(b)) in hec50 cells. when the hec50 cells are transfected with an empty vector (no exogenous tubb3) in addition to mir-200c mimic, there is a statistically significant increase in sensitivity to paclitaxel as measured in a cell death elisa ; see figure 5(c). however, when the cells are transfected with the tubb3 expression vector lacking its 3 utr, the enhanced sensitivity to paclitaxel is lost ; see figure 5(d). therefore, expression of exogenous tubb3 lacking the mir-200c target site reverses the chemosensitivity to paclitaxel caused by increased mir-200c expression. it can be argued that cells with increased proliferation would be more sensitive to microtubule poisons and that could be an alternative explanation for the observed chemosensitivity upon restoration of mir-200c. we therefore performed proliferation assays in the three cell types and found decreased proliferation in all three (figures 6(a), 6(b), and 6(c)). since the decrease in proliferation is observed in all three cell types, including the hec50s where there was no increase in mir-200c levels, it is likely that the effects on proliferation occur via zeb1 and not by mir-200c. the fact that the increase in chemosensitivity is found in cells that are proliferating more slowly than the negative controls demonstrates that increased proliferation is not the mechanism behind the increase in chemosensitivity. in this paper we build on our previous work to further characterize the role that loss of mir-200c plays in generating an aggressive cancer phenotype. zeb1 is normally only expressed in cells of mesenchymal origin ; however, its aberrant expression is observed in cancers that have undergone emt. zeb1 (and the closely related zeb2) transcripts are targeted by mir-200c and the other mir-200 family members. interaction of any of the mir-200 family members with the zeb transcripts results in degradation and inhibition of translation. therefore the maintenance of mir-200c expression in normal epithelial cells serves to prevent zeb1 and zeb2 from being expressed. since both zeb1 and zeb2 repress genes involved in polarity, repression of these proteins serves to maintain polarity, an important epithelial cell characteristic. we have recently shown that in addition to repressing zeb1 and 2, mir-200c represses a program of transcripts normally only expressed in cells of mesenchymal and neuronal origin, such as fibronectin (fn1), neurotrophic tyrosine kinase (ntrk2), quaking 1 (qki), and tubb3. thus, mir-200c maintains epithelial cell characteristics not only by maintaining polarity via repression of zeb1 and zeb2, but also by repressing additional non - epithelial genes. it has been recently demonstrated that mir-200c and zeb1 regulate each other in a double - negative feedback loop [31, 32 ]. the mir-200 family of mirnas is expressed in two clusters, one on choromosome 1p36.33 and the other on chromosome 12p12.31. therefore, in cells that have undergone emt, zeb1 and 2 not only serve to repress genes involved in polarity, but also repress the mir-200 family and thereby release the repression of many genes characteristic of the mesenchymal phenotype. central to the double feedback loop between mir-200c and zeb1 is tgf-. during tgf- -induced emt, there is an increase in ets1 which binds to and activates the promoter of zeb1. therefore, in a tumor microenvironment, increased tgf- levels are thought to result in an increase of zeb1 transcription to a point where it can overcome the repression caused by mir-200c. as zeb1 protein begins to be made, it can then repress the mir-200 family members, resulting in progression through emt. we have previously shown that restoration of mir-200c in hec50 endometrial cells and mda - mb-231 breast cancer cells causes repression of zeb1 and re - expression of e - cadherin protein. here we show that transfection of mir-200c mimic into hey cells, an aggressive serous ovarian cell line, also causes a dramatic repression of zeb1 ; however no expression of e - cadherin was observed. to test whether the double - negative feedback loop is intact in the hec50 and mda - mb-231 cells, these cells were infected with lentivirus expressing an shrna against zeb1. in both cell lines, efficient knock down of zeb1 was achieved, as was re - expression of e - cadherin. in mda - mb-231 cells, zeb1 knock down resulted in an increase in mir-200c levels, as would be expected from the negative feedback loop. however, this was not the case in hec50 cells, where there was no increase in mir-200c. whether the break in the negative feedback loop is an anomaly of this particular cell line remains to be tested. the mechanism behind the phenomenon is also unknown ; however, it does offer an opportunity to dissect the contribution of zeb1 versus that of mir-200c to the phenotype of the cells. for example, significantly decreased proliferation was observed in the hey cells transiently transfected with the mir-200c mimic as well as in the mda - mb-231 and hec50 cells that have zeb1 stably knocked down, although mir-200c levels did not rise in the hec50s. therefore it is likely that the decrease in proliferation is due to the lack of zeb1, not an increase in mir-200c. conversely, the decrease in adhesion to the basement membrane complex and laminin was only observed in the hey and mda - mb-231 cells, and not the hec50s, suggesting that this phenotype is a function of mir-200c expression rather than zeb1. we and others have previously shown that restoration of mir-200c to cancer cells that do not express it causes a decrease in invasion and migration [27, 30 ]. here we show that knock down of zeb1 in mda - mb-231 cells, which causes an increase in mir-200c, negatively affects migration and invasion. furthermore, we show that restoration of mir-200c in hey ovarian cancer cells results in a dramatic decrease in migration and invasion even though e - cadherin is not restored in these cells, despite complete repression of zeb1. loss of e - cadherin expression can result from mechanisms other than zeb1 transcription repression, including chromosomal deletion and promoter hypermethylation [3437 ]. possibilities for its continued absence in these cells include promoter methylation such that even when repression by zeb1 is relieved, e - cadherin will not be expressed, or perhaps levels of another transcriptional repressor such as snail or twist remain high and repress e - cadherin. regardless of the mechanism, the effects of mir-200c on invasion and migration appear to be independent of e - cadherin status. while e - cadherin is involved in epithelial cell - cell adhesion and its expression has been shown to negatively affect migration and invasion [45, 46 ], increased mir-200c is able to decrease migration and invasion on its own. we have previously observed that restoration of mir-200c affects genes involved in cell motility and invasion such as arhgdib, ntrk2, ephb1, and fn1. we demonstrate that mir-200c causes a decrease in adhesion to basement membrane complex, laminin, and perhaps collagen type iv. this observation is particularly relevant to ovarian cancer because the cancerous cells adhere to sites within the peritoneal cavity. during the progression of cancer there is switching of the expression patterns of the cell surface adhesion molecules, such as the cadherins and integrins [4749 ]. although the change in the number of adherent cells appears modest, this might play a significant role in developing a potential treatment for ovarian cancer. the ability of ovarian cancer cells to spread and adhere to the peritoneal cavity is one of the major phenotypes of this disease. a small change in ability of the cells to adhere might reflect a great decrease in the tumor burden and/or increase the ability to debulk the tumor. these results are independent of e - cadherin expression since the decrease in adhesion is observed in both the mda - mb-231 cells (where e - cadherin expression is regained with increased mir-200c) and in hey cells, where it is not. clonogenic assays reveal that there is an increase in chemosensitivity to paclitaxel with increased mir-200c levels. indeed, acquired resistance to paclitaxel in ovarian cancer cells has been shown to be associated with emt, resulting in an aggressive phenotype. clinically, aberrant expression of tubb3 (not normally expressed in epithelial cells) has been found to be associated with resistance to taxanes [3841 ]. we have previously shown that tubb3 is a direct target of mir-200c and suggested that its repression by mir-200c is the mechanism behind the ability of mir-200c to increase chemosensitivity to microtubule targeting agents. here, we perform the definitive experiment to prove that mir-200c - mediated tubb3 downregulation is indeed the cause of the enhanced chemosensitivity. we utilized exogenous tubb3 lacking its 3utr such that it can not be targeted by mir-200c and show that resistance to paclitaxel is maintained even in the presence of mir-200c. in contrast, endogenous tubb3 is reduced when mir-200c is added, resulting in enhanced chemosensitivity to paclitaxel. microtubule targeting agents such as paclitaxel work more efficiently in cells that are rapidly dividing. consequently, it could be argued that the increase in chemosensitivity caused by mir-200c is due to increased proliferation. however, we show that increase of mir-200c or direct knockdown of zeb1 results in decreased proliferation in three different types of cancer cells. it is therefore the downregulation of tubb3, not an increase in proliferation that is responsible for the enhanced chemosensitivity to taxanes observed with restoration of mir-200c to resistant cancer cells. mir-200c expression serves to maintain the epithelial phenotype in well - differentiated, low - grade, breast, ovarian, and endometrial cancer cells. furthermore we find that not all of mir-200c 's actions can be attributed to the restoration of e - cadherin via targeting of zeb1. we further prove that mir-200c - mediated repression of tubb3 is the cause of enhanced chemosensitivity to microtubule targeting agents. lastly we demonstrate that not all cells exhibit the double negative feedback loop between mir-200c and zeb1 and that this can be exploited to identify the distinct roles of mir-200c as compared to zeb1. | we focus on unique roles of mir-200c in breast, ovarian, and endometrial cancers. members of the mir-200 family target zeb1, a transcription factor which represses e - cadherin and other genes involved in polarity. we demonstrate that the double negative feedback loop between mir-200c and zeb1 is functional in some, but not all cell lines. restoration of mir-200c to aggressive cancer cells causes a decrease in migration and invasion. these effects are independent of e - cadherin status. additionally, we observe that restoration of mir-200c to ovarian cancer cells causes a decrease in adhesion to laminin. we have previously reported that reintroduction of mir-200c to aggressive cells that lack mir-200c expression restores sensitivity to paclitaxel. we now prove that this ability is a result of direct targeting of class iii beta - tubulin (tubb3). introduction of a tubb3 expression construct lacking the mir-200c target site into cells transfected with mir-200c mimic results in no change in sensitivity to paclitaxel. lastly, we observe a decrease in proliferation in cells transfected with mir-200c mimic, and cells where zeb1 is knocked down stably, demonstrating that the ability of mir-200c to enhance sensitivity to paclitaxel is not due to an increased proliferation rate. |
breast cancer is the most common malignancy and the second major cause of mortality and morbidity in western women. the systemic outgrowth and spread of the cancer cells through a process known as metastasis is the main cause of deaths in these patients. recently, disease - related mortality and metastasis have declined as a result of early diagnosis and application of adjuvant therapy. mammographic screening, surgery, radiotherapy, chemotherapy, antibody therapy, and endocrine therapy facilitate the suppression of the metastatic dissemination of local tumor. however, these treatments target the tumor cells and disregard the auxiliary cells present in the surrounding microenvironment that is also referred to as the stromal cells. these auxiliary cells, including myoepithelial cells, fibroblasts, myofibroblasts, endothelial cells, inflammatory cells, and bone - marrow - derived cells (bmdcs) such as macrophages, mast cells, neutrophils, and lymphocytes, are widely recognized to collaborate with cancerous cells and other host cells to create a tumor - permissive microenvironment capable of providing continuous support for tumor growth, progression, angiogenesis, invasion, and metastasis [3, 4 ]. metastasis is the systemic dissemination of tumor cells at sites distinct from the primary lesion. it is a multistep process that involves detachment of cells from the primary tumor, followed by survival in the blood vessels or lymphatic system and finally development of secondary tumor. it is a poorly understood aspect of carcinogenesis that requires the clarification of the underlying cellular and molecular events that control the metastatic cascade from onset to colonization. it is undisputed that metastasis of tumor cells is mediated by the reciprocal interplay between tumor cells and stromal cells and the extracellular matrix (ecm). in this paper, we discuss the different steps of breast cancer progression and delineate the importance of the myoepithelial cell layer disruption for invasion of tumor cells. we also address the tumor promoting effect of the stromal cells in each step of the metastatic cascade of breast cancer. the development of breast cancer involves the progression via a series of intermediate hyperplastic lesions with and without atypia (atypical ductal hyperplasia, atypical lobular hyperplasia, and usual ductal hyperplasia) followed by subsequent evolution into in situ carcinoma, for example, ductal carcinoma in situ (dcis) and lobular carcinoma in situ (lcis), invasive carcinomas, and metastatic cancers (figure 1) [69 ]. in atypical hyperplasia, the breast cells are abnormal in number, size, shape, appearance, and growth pattern that may be seen as an excessive growth of cells of the ducts (atypical ductal hyperplasia) or the cells of the lobules (atypical lobular hyperplasia). in usual ductal hyperplasia, the breast tissue has an increased number of benign cells within the duct. dcis is thought to be a precursor of invasive ductal carcinoma, in which tumor cells are confined to the lumen of the mammary duct. lobular carcinoma in situ consists of a noninvasive increase in the cells of the milk - producing lobules of the breast. normal breast ducts are composed of a layer of epithelial cells physically separated from the normal microenvironment by a basement membrane and myoepithelial cell layer [10, 11 ]. in situ carcinoma is characterized by intact myoepithelial cell layer and basement membrane, and proliferation of epithelial cells [10, 11 ]. when the breast tissue undergoes focal disruption of the myoepithelial cell layer and degradation of the underlying basement membrane, tumor cells invade surrounding tissues and migrate to distant organs, eventually leading to metastasis [1012 ]. despite the dramatic improvement in our ability to detect carcinomas in situ (dcis), our understanding of the pathophysiology of this disease and factors involved in its progression to invasive carcinoma lags far behind. the normal breast tissue is comprised of two major compartments, the epithelium and the stroma. myoepithelial cells together with luminal cells constitute the epithelium of the ducts and of the lobule of the mammary gland. the anatomical position of myoepithelial cells between the stroma and the luminal epithelial cells from which cancer arises facilitates proper communication between both compartments. they express a number of tumor suppressor proteins (maspin), ecm structural proteins (fibronectin, collagen), proteinase inhibitors (tissue inhibitor of metallopreoteinase-1, timp-1), and angiogenic inhibitors (thrombospondin-1) [13, 14 ]. they also downregulate the expression of matrix metalloproteinases (mmps) in fibroblasts and tumor cells, contribute significantly to basement membrane production, and accumulate ecm rather than degrade it [14, 16, 17 ]. the aforementioned functions suggest that the normal myoepithelial cell layer is a natural paracrine tumor suppressor that physically and functionally inhibits tumor growth, invasion, and angiogenesis. this tumor suppressive phenotype was identified based on the ability of myoepithelial cells to secrete paracrine factors (such as bfgf, tgf-, and il-6) that inhibit the growth and invasion of breast cancer cells in coculture assays in vitro [1820 ]. collective evidence suggests that myoepithelial cells also function as autocrine tumor suppressor that is supported by their resistance to transformation and their tendency to transform to tumors of low malignancy [19, 21 ]. due to their tumor suppressor potential, myoepithelial cells myoepithelial cells surround both normal ducts and precancerous lesions of the breast, for example, dcis. however, dcis myoepithelial cells differ from their normal counterparts in their ability to polarize luminal cells in three - dimensional collagen assays [16, 23 ], which indicates that tumor - derived myoepithelial cells are unable to transmit the necessary and correct signals to luminal cells. moreover, myoepithelial cells isolated from normal tissue have distinct gene expression pattern as compared to dcis myoepithelial cells. the former express high levels of laminin, tenascin, thrombospondin, cytokeratins, oxytocin receptor and tropomyosin, whereas dcis myoepithelial cells show overexpression of proteases (cathepsins, mmp-2, and prss11), protease inhibitors (thrombospondin 2, serping1, cystatin c, and timp3), and collagens. a common diagnostic feature of breast cancer progression from in situ to invasive tumor is the aberration of the fully differentiated myoepithelial cell layer suggesting that dissolution of the myoepithelial cell layer is an absolute prerequisite for tumor invasion. however, it is unknown what mechanisms lead to focal myoepithelial cell layer disruption and its contribution to tumor progression. studies by man and sang revealed that focal myoepithelial cell layer disruptions are associated with higher leukocyte infiltration supporting the release model which is proposed by polyak el al. to describe the role of stromal and myoepithelial cells in invasion onset. the tumor microenvironment or the stroma is composed of extracellular and cellular tissue network that surrounds and interacts with tumor cells. the cellular part includes fibroblasts, myofibroblasts, endothelial cells, adipocytes, and various immune cells [25, 26 ]. these cells are surrounded by an ecm that is a dynamic three - dimensional structure composed of many components including collagens, laminin, and fibronectin. the ecm is also a rich source of matrix metalloproteinases (mmps) and soluble growth factors that affect neoplastic dissemination. a specialized ecm, called basement membrane, is made of several glycoproteins and proteoglycans and separates the epithelial and endothelial cell layers from the surrounding microenvironment. a well - organized basement membrane acts as a gatekeeper of invasive phenotype providing a physical support, a signaling intermediate between different compartments and a regulator of cell behavior. during tumor development, cancer cells become in direct contact with a remodeled stroma that was long considered to be a passive responder to the malignant transformation. the significance of the tumor microenvironment as an active contributor in promoting and initiating breast cancer development is proposed. the difference in molecular signatures between stromal cells from tumors and normal tissues bear witness that stromal cells provide cues for tumorigenesis. in contrast to normal fibroblasts, cancer - associated fibroblasts (cafs) enhance tumor growth and metastasis through the production of growth factors and ecm proteins and modulating immune polarization. they also have different gene signatures related to paracrine signaling, transcriptional regulation, extracellular matrix, cell - cell interaction and cell adhesion / migration such as wnt1 inducible signaling pathway protein 1 (wisp1), kruppel like factor 4 (klf4), tgf2, fibulin1 (fbln1), plasminogen activator inhibitor 2 (pai2), and tissue plasminogen activator (plat) [29, 31 ]. recently, they induced hepatocyte growth factor (hgf) production by fibroblasts to support their own growth and progression. it is noteworthy to mention that a model which delineates the role of tumor microenvironment in breast cancer initiation and progression has been proposed. this model or reverse warburg effect suggested that tumor cells can induce an oxidative stress on neighboring fibroblasts which promotes stromal autophagy associated with caveolin-1 loss and elevated cytokine production. this, in turn, results in production of nutrients that can nourish anabolic tumor cells [33, 34 ]. tumor stroma also includes myofibroblasts, which are activated fibroblasts with -smooth muscle actin (-sma) expression. in human tissue sections with invasive breast cancer, higher proportion of myofibroblasts were associated with higher - grade upregulation of ki-67, vegf, and bfgf, and shorter overall survival and relapse - free survival. tumor invasion and angiogenesis was shown to be promoted by -sma - positive myofibroblasts and not by -sma - negative fibroblasts. stromal myofibroblasts promote tumorigenesis of oral squamous cell carcinomas, for example, by secreting activin a. in the tumor - stroma interactive microenvironment transforming growth factor - beta 1 (tgf - beta 1) for example, mir-21 was recently shown to participate in tgf - b1-induced myofibroblast transdifferentiation by targeting and downregulating programmed cell death 4 (pdcd4) gene [38, 39 ]. furthermore, cancer - cell - derived tgf - b release proangiogenic vascular endothelial growth factor a (vegfa) from the myofibroblasts in esophageal squamous cell to regulate angiogenesis. implicated with angiogenesis, coculture of adipocytes with cancer cells resulted in increased invasiveness of cancer cells and modified phenotype of the adipocytes characterized by lower lipid accumulation, decreased expression of adipocyte markers, and overexpression of proteases (mmp-11) and proinflammatory cytokines (il-6, il-1). the stromal compartment also contains various bone - marrow - derived cells (bmdcs) such as macrophages, mast cells, neutrophils, and lymphocytes that are recruited by the primary tumor cells to increase tumor cell migration, angiogenesis, and invasion. at sites of focal disruptions of the myoepithelial cell layer in breast tissues, immune infiltrates were observed at the invasive front suggesting a potential role of these cells in malignancy and subsequent metastatic spread [10, 11 ]. to metastasize, tumor cells are required to escape the primary tumor, intravasate the blood stream or lymphatic circulation, survive in the vasculature, extrude from the blood vessels or lymphatic system, arrest at distant sites, and develop into secondary mass. at each step of the metastatic cascade, stromal cells appear to be crucial players in the transition from benign to invasive and finally metastatic disease (figure 2) [5, 43 ]. the metastatic cascade is a quite inefficient process meaning that failure to complete any step will quench the whole process. until now, it is unclear which step is the key rate - limiting one that contributes to the inefficiency of the metastatic lesion. metastatic colonization has been suggested as a major rate - limiting step because intravenous injection of cancer cells resulted in about 90% arrest and extravasation with only 0.1% of cells growing at the secondary site [44, 45 ]. extravasation was also thought to be a key rate - limiting step in metastasis with highly metastatic cells extravasating faster than poorly metastatic ones. however, a study by koop. showed that extravasation is independent of the metastatic ability and highly metastatic ras - transformed cells extravasate at the same rate of control fibroblasts. similar to extravasation, intravasation was proposed to be a major rate - limiting step. in vivo data showed that intravasation can be a major rate - limiting step in the metastatic cascade. wyckoff. showed that metastatic cells exhibited a faster entry into the vasculature than poorly metastatic cells. the authors found that hep-3 cells had higher metastatic rate than ht-1080 cells due to the lower efficiency of the latter cells in intravasation and metastatic colonization. collectively, intravasation and growth at secondary sites represent major rate - limiting steps in the metastatic cascade. however, the rate - limiting step may vary depending on the tumor type. under normal physiological conditions, the surrounding microenvironment imposes proper tissue architecture maintained by basement membrane alignment and intercellular communication. this interplay between epithelial cells and surrounding stroma maintains organ homeostasis that serves as a protective constraint against malignant transformation. during tumor development, cancerous cells circumvent the normal controls regulating the activity of ecm proteases. in response to these proteolytic enzymes, the basement membrane undergoes gradual degradation and structural changes causing violation of normal tissue boundaries and conduits for malignant cell egress. to invade, tumor cells should lose cell - cell and cell - ecm interactions mediated by integrins and cadherins. invasion is also accompanied by proteolytic degradation of surrounding tissue mediated by proteases, motility of tumor cells mediated by chemokines and growth factors, and recruitment of stromal cells. they are primarily responsible for the synthesis, deposition, and remodeling of the basement membrane and ecm through the production of collagen (i, iii, iv, and v), fibronectin, and laminin. they are also an important source of paracrine growth factors (hgf, egf, fgf2, and tgf), proteolytic enzymes (mmp-1, mmp-7), and cytokines (il6, cxcl12) that can affect cell proliferation, survival, morphology, and death [48, 49 ]. during tumorigenesis, fibroblasts maintain an activated phenotype characterized by the expression of -smooth muscle actin (-sma) and are referred to as carcinoma - associated fibroblasts (cafs) or myofibroblasts. in breast carcinomas, about 80% of stromal fibroblasts acquired the caf - activated phenotype. during tumor development, cafs exhibit a higher proliferation index and become the predominant cell population in the stroma. once the basement membrane is degraded, cafs are accumulated causing expansion of tumor stroma and increased deposition of ecm through expression of stress fibers and -sma. this phenotype is termed desmoplasia and is associated with recruitment of inflammatory cells and activation of angiogenesis. several studies demonstrated a direct involvement of fibroblasts in initiation of cancer. in a xenograft mouse model, kuperwasser. showed that the upregulation of transforming growth factor- (tgf) or hepatocyte growth factor (hgf) in mouse fibroblasts stimulated the initiation of benign and malignant lesions in the breast epithelium. in addition, a gene expression profiling study of all cell types in normal and neoplastic breast demonstrated that overexpression of cxcl12 in myofibroblasts was correlated with epithelial cell proliferation and invasion. another study of xenograft mouse model coinjected with mcf-7 breast cancer cells and cafs or normal fibroblasts showed that xenografts infused with cafs had enhanced growth than xenografts injected with normal fibroblasts. to reconcile, these data reveal the major role of cafs in tumor initiation and progression. an initial reaction of the host to the tumor development is the recruitment of leukocytes and subsequent local inflammation. as a physiological response to tissue injury, inflammatory cells are recruited to the injured site to support tissue repair and remodeling through the production of growth factors and cytokines such as tnf-, ccl2, cxcl8, ccl5, tgf- and so forth. in normal conditions, the inflammatory response is resolved once the tissue is repaired [54, 55 ]. however, the tight regulation of inflammation is overridden during malignant transformation recalling the historic view of tumors as persistent inflammatory response characterized by activated leukocytes and secretion of several cytokines and chemokines (including tumor necrosis factor (tnf), interleukins, and interferons) elaborates the formation of tumor - promoting microenvironment. such protumor stroma is incentive not only for primary tumor development but also for metastatic dissemination into systemic circulation. neoplastic transformation is regulated by dynamic reciprocity between epithelial cells, activated stromal cells and ecm components. if the changes in the microenvironment occur prior or concomitant with epithelial cell changes is still debatable. recently, a gene expression profiling study on laser captured breast stroma and epithelia showed 90% change in the stromal gene expression at the transition from normal to dcis, and only 10% stromal alterations in dcis compared to invasive disease [57, 58 ]. another study used mrna in situ hybridization of breast tissue with different stages of cancer to examine the expression of angiogenic factors and stromal components. a similar expression profile was observed in carcinoma in situ, invasive cancer, and metastatic disease. these studies suggest that stromal changes induced by the emerging epithelial lesions precede invasion and that cancer cells invade into an abnormal breast microenvironment with growth - promoting effects. intravasation or penetration of tumor cells into the vasculature involves the movement of cancer cells through the ecm, the basement membrane, and finally through the endothelium of the blood vessel or lymphatic duct. the underlying molecular mechanisms governing intravasation are not clear as all studies have focused on later steps in the metastatic cascade. detailed evaluation of all steps in tumor metastasis is crucial to understand the cellular mechanisms controlling neoplastic dissemination. in a murine breast cancer model, yang it augments epithelial - to - mesenchymal transitions and promotes the rate of hematogenous intravasation. another possible mechanism that executes the migration of tumor cells across the vessel wall was shown by intravital imaging studies of experimental mammary carcinomas [61, 62 ]. these studies showed that carcinoma cells intravasate through the blood vessels due to chemoattractive gradients generated by perivascular macrophages that are recruited by the tumor cells to the injured site. in breast carcinomas, macrophages and cancerous cells form a paracrine loop involving epidermal growth factor (egf) and colony stimulating factor-1 (csf-1) to augment chemotaxis and intravasation. egf produced by macrophages promotes migration of neoplastic cells into hematogenous vasculature through its interaction with egf receptor expressed on breast cancer cells. tumor cells, in turn, express csf-1 which acts as a potent chemoattractant for csf-1 receptor positive macrophages. this crosstalk lends credence to the collaborative work between tumor microenvironment and neoplastic cells at the site of intravasation. once malignant cells have invaded the angiogenic vasculature, they are subject to harsh microenvironment characterized by hemodynamic shear forces, surveillance of immune cells, and lack of substratum. to bypass these perils, tumor cells use platelets as a shield. through their tissue factor, tumor cells bind coagulation factors (viia and x) on the platelets creating an embolus that arrests in the capillaries [65, 66 ]. these aggregates protect the cancerous cells from immune - cell - mediated lysis and decrease the shear forces of the blood circulation, thus increasing their survival, arrest, and extravasation. whether macrophages can infer protective effects on neoplastic cells in the bloodstream as they do during invasion and intravasation is not yet known. to explain the metastatic phenotype, pawelek and chakraborty proposed the fusion of macrophages or other bmdcs with cancer cells forming a hybrid capable of surviving in the circulation and homing to secondary sites. should the fusion theory be accepted in human cancers, bmdcs would have a beneficial role in the survival of tumor cells in the vasculature. after survival and arrest in the circulation, tumor cells must escape out of the blood and lymphatic vessels in a process known as extravasation. to do so, tumor cells induce disruptions in the endothelial junctions that allow tumor cells to bind to the subendothelial ecm and extrude into target organs. vascular permeability is mediated by activated src kinases in endothelial cells, which once exposed to vascular endothelial growth factor (vegf) from tumor cells promotes endothelial retraction, resulting in movement of cancer cells towards surrounding tissues. to identify the role of macrophages in metastasis, qian. used an animal model of breast cancer metastasis and an intact ex vivo lung imaging system to show that modified host macrophages are required for proper metastatic seeding and growth. the authors found that macrophage ablation dramatically decreased the number of tumor cells observable in the lungs. moreover, lung - resident macrophages were visualized to physically interact with tumor cells as soon as they extravasate through the vessel walls, thus promoting the rate of extravasation and metastatic dissemination. the prometastatic macrophages are characterized by cell surface expression of csf1r, cd11b, f4/80, with high levels of ccr2, cx3cr1, and vegfr, absence of gr1 and low cd11c [70, 71 ]. metastasis is regulated not only by changes in tumor cells but also by reciprocal interactions with the surrounding microenvironment. in his seed and soil hypothesis, paget for example, breast cancers metastasize to lungs, bone, liver, and brain, whereas advanced prostate cancers colonize the bone as the predominant site. since the circulatory patterns provide only partial explanation for the tissue tropism aspect of metastasis, several molecular and cellular mechanisms have been proposed. an emerging paradigm suggests that primary tumor cells may secrete factors capable of inducing a fertile microenvironment, termed premetastatic niches, that favors the seeding and proliferation of metastatic cells at unique sites. for example, (adamts1) and matrix metalloproteinase-1 (mmp1) participate in a paracrine signaling cascade that includes the release of metastasis membrane - bound epidermal - growth - factor (egf-) like growth factors, amphiregulin (areg), heparin - binding egf (hb - egf), and transforming growth factor alpha (tgf alpha) from tumor cells resulting in a downregulation of osteoprotegerin expression in osteoblasts and therefore modulating brain microenvironment in favor of osteoclastogenesis and bone metastasis. recent work have identified the following mediators of extravasation and brain colonization : the cyclooxygenase cox2, the epidermal growth factor receptor (egfr) ligand, hbegf, and the alpha 2,6-sialyltransferase, st6galnac5, which were found to enhance breast cancer passage through the blood brain barrier and to facilitate their adhesion to brain endothelial cells. egfr ligands and cox2 are also linked to breast cancer infiltration of the lungs [76, 77 ]. recently, breast cancer cells infiltrating the lungs were shown to support their own metastasis - initiating ability by expressing tenascin c (tnc). tnc is an extracellular matrix protein of stem cell niches that promotes the survival and outgrowth of pulmonary micrometastases via upregulation of stem cell signaling components, musashi homolog 1 (msi1), and leucine - rich repeat - containing g protein - coupled receptor 5 (lgr5) until the tumor stroma takes over as a source of tnc. through the production of growth factors (vegfa) and chemokines (s100a8, s100a9), tumor cells induce the recruitment of bmdcs, and endothelial progenitor cells to the premetastatic niche. the bone marrow progenitors are vegf receptor-1 (vegfr1) positive and can migrate and proliferate in response to tumor - derived vegf. these vegfr1 cells also express integrin vla-4 and tend to form clusters induced by integrin - fibronectin interactions, the latter of which is synthesized by resident fibroblasts. matrix metalloproteinase-9 (mmp-9) secreted by bmdcs degrades the basement membrane and liberates the matrix - sequestered vegfr1 ligand, the vegfa, promoting the homing of more vegfr1 cells into the niche. through the production of vegfa, tgf, and tumor necrosis factor- (tnf), tumor cells enhance the expression of the chemoattractants s100a8 and s100a9 in lung endothelium and myeloid cells, which in turn promote tumor cell homing and adhesion to the metastatic site [81, 82 ]. fibroblast - specific protein-1 (fsp-1/s100a4), a fibroblast - specific marker, is highly expressed on tumor - associated fibroblasts and is released upon stimulation of fibroblasts by tumor cells. injection of fsp-1-positive fibroblasts into these mice restored the ability of mammary adenocarcinoma cells to develop tumors and generate metastasis suggesting a potential role of tumor - associated fibroblasts in the metastatic dissemination. another study found that only metastasized melanoma cells were affected by fibroblasts suggesting that fibroblasts might be important in creating the permissive soil that supports tumor cell growth at distant sites. recently, o'connell and colleagues found that s100a4 fibroblasts provide the proper metastatic niche to support metastatic colonization. through production of vegfa and tenascin - c, fibroblasts can promote angiogenesis as well as provide protection against apoptosis, respectively. following the implantation of tumor cells, persistent growth of metastasis is maintained by the establishment of sufficient blood supply capable of providing the necessary oxygen, growth factors, nutrients, and metabolites. blood vessels are composed of vascular basement membrane, endothelial cells and specialized smooth muscle cells, the pericytes. the induction of a tumor vasculature termed the angiogenic switch requires basement membrane assembly, recruitment and proliferation of endothelial precursors, and pericytes attachment. initially, the vascular basement membrane is degraded by several mmps produced by stromal cells, endothelial cells, or tumor cells. this basement membrane degradation causes the release of endothelial cells to migrate and proliferate, the liberation of matrix - sequestered growth factors such as vegf, basic fibroblast growth factor (bfgf), and platelet - derived growth factor (pdgf) and the disassembly of the pericytes that line the blood vessels. in response to vegf, vegfr2 endothelial progenitor cells are recruited to the metastatic site through vegfa signaling to contribute to vessel formation. analysis of these progenitor cells shows upregulation of several angiogenic molecules (vegf, fgf, pdgf, cxcl1, etc.) moreover, vegfr1 bmdcs has been shown to produce several angiogenic factors and are required to provide stability to the neovessels. inhibition of vegfr1 bmdcs either during primary tumor or after the formation of premetastatic niche caused the prevention of endothelial cell migration and metastasis. thus, the recruitment of vegfr2 endothelial progenitor cells into vessels requires the incorporation of vegfr1 bmdcs to support neovascularization [60, 73 ]. macrophages, for example, are a good source of angiogenic factors such as vegf and mmp-9. in a mouse model of highly aggressive metastatic mammary carcinoma, lin and pollard found that tumor - associated macrophages may provide essential cues to press the angiogenic switch. colony stimulating factor-1 (csf-1) deletion caused failure in macrophage homing to the malignant stroma that was associated with attenuated angiogenic responses, decreased neoplastic progression and inhibition of pulmonary metastasis. the coinjection of cafs and mcf-7 breast cancer cells into nude mice resulted in the recruitment of bone - marrow - derived endothelial progenitors in response to caf - derived stromal cell derived factor (sdf1/cxcl12) stimulating angiogenesis and tumor formation. another proangiogenic mechanism of cafs involves the release of several factors such as vegf and fgf which can positively contribute to vascularization. it is evident that metastasis is a multistep process where each stage requires an intricate interplay between cancerous cells and cells of the microenvironment. this tumor - host crosstalk supports the notion that cotargeting cancer cells and tumor stromal cells will be a viable approach for mammary cancer prevention and treatment. researchers are dedicated to explore the stromal cells as an effective target for anticancer therapeutics. such host - targeted therapies should be directed towards bmdcs, fibroblasts, and endothelial cells that home to the metastatic site to support tumor dissemination and outgrowth. it is important to inhibit the mobilization and proliferation of the stromal cells and to disrupt tumor - stroma interactions mediated by paracrine factors. to achieve these goals, several agents have been investigated and they fall into several categories including protease inhibitors (e.g., mmp inhibitors), antiadhesive molecules (e.g., anti - integrin peptides or antibodies), signal pathway modulators (e.g., tyrosine kinase pathway inhibitors), antifibrotic drugs (e.g., pirfenidone), and antiangiogenic molecules (e.g., vegf and bfgf antagonists) [92, 93 ]. the trials were conducted only on patients with advanced disease, and mmpis used were broad spectrum mmpis which can block bad mmps as well as good ones. avastin / bevacizumab, a monoclonal antibody targeted against all isoforms of vegf - a, has recently been withdrawn from the fda list for the treatment of breast cancer. unlike trastuzumab which is a her-2-targeted antibody, avastin delayed tumor progression with no improvement in overall survival. wu. have shown that targeting galectin-1 to significantly inhibit caf - conditioned medium - induced tumor cell migration and invasion in oral squamous cancer cells (osccs) resulting in a reduced metastasis in vivo. it is followed that galectin-1 downregulation reduces the production of monocyte chemotactic protein-1 (mcp-1/ccl2) which promotes the migration of osccs by binding to ccr2 receptor. blocking the interaction between mcp-1 and ccr2 abolishes migration. moreover, kim. have proposed to target myofibroblasts overexpressing laminin-332 which caused the formation of the dense fibrosis via desmoplastic reaction during epithelial to mesenchymal transition (emt). this alteration of the tumor microenvironment preceded tumor invasion and was found in invasive ductal carcinoma. targeting of laminin-332 overexpressing myofibroblasts was supposed to prevent the formation of the dense fibrosis, thus inhibiting the invasion - friendly stromal alteration. it is important to note that angiotensin-(17), an endogenous 7-amino acid peptide hormone of the renin - angiotensin system, has been shown to target the tumor microenvironment to inhibit caf growth and tumor fibrosis. additionally, liu. proposed the targeting of the coagulation cascade that is activated in the tumor microenvironment and presented preclinical data targeting tissue factor (tf), an enzyme cofactor in activating coagulation that plays a critical role in tumor growth. coenegrachts. demonstrated that the selective neutralization of host - derived bone - derived placental growth factor (plgf) by anti - mouse alphaplgf reduced the engraftment of tumor cells in the bone, inhibited their interaction with matrix components, reduced the incidence, number, and size of bone metastases, and preserved bone therefore inhibiting both the progression of metastasis and the settlement of tumor in the bone. have shown the role that eph receptor tyrosine kinase ephb6 plays in suppressing cancer invasiveness through c - cbl - dependent signaling, morphologic changes, and cell attachment and that its targeting might enable the regulation of both cell attachment and migration. these stroma - targeted therapies combined with antitumor approaches will be translated into a double - edged sword that cancerous cells will not easily survive. these therapeutic approaches require a full understanding of the cellular and molecular mechanisms governing the tumor - host interactions, accompanied with the development of new mouse models and intravital imaging techniques. | metastasis is the major cause of death for breast cancer patients. tumors are heterogenous cellular entities composed of cancer cells and cells of the microenvironment in which they reside. a reciprocal dynamic interaction occurs between the tumor cells and their surrounding stroma under physiological and pathological conditions. this tumor - host communication interface mediates the escape of tumor cells at the primary site, survival of circulating cancer cells in the vasculature, and growth of metastatic cancer at secondary site. each step of the metastatic process is accompanied by recruitment of stromal cells from the microenvironment and production of unique array of growth factors and chemokines. stromal microenvironment may play active roles in breast cancer metastasis. elucidating the types of cells recruited and signal pathways involved in the crosstalk between tumor cells and stromal cells will help identify novel strategies for cotargeting cancer cells and tumor stromal cells to suppress metastasis and improve patient outcome. |
since the introduction of the dsm - iii (diagnostic and statistical manual of mental disorders, third edition) in the early 1980s, major depressive disorder has been conceptualized as a unitary entity.1,2 however, there have been a growing number of studies that support revising this model to position melancholia as a distinct depressive subtype.1,3 a number of studies have reported clinical48 and biological7,911 differences between patients with melancholic and nonmelancholic depression, and argued for differing underlying pathophysiological processes.12 however, while there is increasing appreciation of melancholia as a distinct entity,13 its validity involves more precise clinical and biological definition. while the melancholic subtype (also classically known as endogenous, autonomous, or type a depression) has been historically used to name a clinical presentation of depression characterized by specific signs and symptoms (eg, nonreactivity of mood, anhedonia, and psychomotor disturbance), its putative pathophysiology has imputed a large number of potential genetic and other biological determinants.14 for example, several studies have implicated an abnormal dexamethasone suppression test,15,16 changes in plasma levels of neuroactive amino acids and nitric oxide,17 hypercortisolism,11,18 and selective responsivity to physical treatments, such as antidepressant drugs19 and electroconvulsive therapy.20,21 however, other biological markers have been less studied or generated less consistent findings. serum brain - derived neurotrophic factor (bdnf) has been reported consistently as being low in depressive patients,22 but very few studies have compared bdnf in depressive subtypes. while bus found no differences between depressive groups,23 patas reported lower levels in nonmelancholic depressive patients compared with a control group, but no difference between melancholic and nonmelancholic depressive subtypes. there has also been a lack of studies investigating differences in oxidative stress markers between melancholic and nonmelancholic depression, although there is a growing body of evidence considering depression as an inflammatory disorder.25 another growing line of research has involved investigating immune parameters across melancholic and nonmelancholic subtypes. however, while several studies have found higher proinflammatory immune activation in nonmelancholic depression,10,26,27 contradictory findings have been reported in relation to inflammatory markers. while interleukin (il)-6 has been reported to be increased11,27,28 or normal29 in melancholic patients, other markers of t helper lymphocyte 1 (th1) proinflammatory response, such as tumor necrosis factor - alpha (tnf-) and interferon - gamma (ifn-), the same variability in findings is evident for t helper lymphocyte 2 (th2) anti - inflammatory markers, such as il-10,10,31,33 as well as other th1 cytokines such as il-19,2931,34 and il-2.10,35 one possible cause of the contradictory results may be the limited discriminatory power of the symptom - based dsm diagnosis of melancholia, being used in almost all reported studies, and which remains practically unchanged in dsm-5.36 in its classical definition, melancholia has as a core feature observable psychomotor disturbance (pmd, ie, a sign rather than a symptom).37 in several studies, parker demonstrated a sign - based approach as being more precise in diagnosing melancholia than symptom - based criteria sets,38 and suggested that the latter may confound interpretation of investigatory neurobiological studies.3 they developed and validated the core measure,38 now one of the most widely used measures of pmd in melancholia.4,5,3941 however, few studies involving the core measure have evaluated biological parameters. joyce reported that patients with core - defined melancholia, but not if subtyped by dsm - iv criteria, had greater neuroendocrine dysfunction,40 with a blunted thyroid - stimulating hormone response after infusion of thyrotrophin - releasing hormone and higher afternoon cortisol levels. mitchell42 reported a higher rate of nonsuppression on the dexamethasone suppression test in melancholic patients defined by the core measure, but not by dsm - iii criteria. while many studies link depression with immune dysregulation4345 via an imbalanced production of proinflammatory / anti - inflammatory cytokines, neurotrophin changes,46,47 and increased oxidative stress,48,49 there are no studies examining these biological markers in relation to the core measure. in summary, despite evidence supporting melancholia as a distinctive depressive subtype therefore, the aim of this study was to compare potential neurobiological parameters of melancholic and nonmelancholic depressed patients subtyped by the core measure and also as compared with controls. the study compares data for three neurobiological parameters not previously studied by the core measure : examining neurotrophins (bdnf), oxidative stress (protein carbonyl content [pcc ] and thiobarbituric acid reactive substances [tbars ]), and immune system (il-2, il-4, il-6, il-10, il-17, tnf-, and ifn-) variables. depressive patients were recruited at the mood disorders program, an outpatient facility based at the hospital de clnicas de porto alegre (hcpa), brazil. a diagnosis of unipolar depression was accorded by dsm - iv criteria and rated by the mini international neuropsychiatric interview, brazilian version (mini - plus).50 all depressed patients were classified as melancholic or nonmelancholic according to the core measure that evaluates 18 observable features of melancholia on a four - point scale, measuring its absence (0) or presence (three levels of severity, from 1 to 3).38 examples of items include facial nonreactivity, facial apprehension, and delay in motor activity. three authors (ls, mac, eav) were trained by video and printed guidelines provided by the black dog institute.51 a core score 8 was adopted in this study as defining melancholia, according to core development studies.52 depressive symptoms were evaluated using the 17-item hamilton depression rating scale (hdrs-17).53 a hdrs-17 18 indicating moderate to severe depression was considered as an inclusion criterion for this study.54 exclusion criteria included any history of autoimmune disease, chronic infection, or an inflammatory disorder, as well as any severe systemic disease or use of immunosuppressive therapy. we also recruited a control group consisted of 54 healthy volunteers attending the hcpa blood donor center, who had no current or previous history as well as no first - degree family history of a major psychiatric disorder, including dementia or mental retardation, assessed by the nonpatient version of the structured clinical interview for dsm - iv. the investigation was approved by the medical ethics committee at hcpa, and all participants provided written informed consent. ten milliliters of blood were collected from each patient and control by venipuncture into a free - anticoagulant vacuum tube. immediately after withdrawal, the blood was centrifuged at 4,000 rpm for 10 minutes and serum was aliquoted, labeled, and stored at 80c until assayed. serum cytokine concentrations were determined by flow cytometry using the bd cytometric bead array th1/th2/th17 human cytokine kit (bd biosciences, san diego, ca, usa). the cytometric bead array kit employed allows discrimination of the following cytokines : il-2, il-4, il-6, il-10, tnf-, ifn-, and il-17a. sample processing and data analyses were performed following the manufacturer s guidelines using a facscalibur flow cytometer (bd biosciences). results were generated in graphical and tabular format using fcap array cytometric bead array analysis software (bd biosciences). serum concentrations of bdnf were measured by sandwich enzyme - linked immunosorbent assay using monoclonal antibodies specific for bdnf from r&d systems (minneapolis, mn, usa). human bdnf monoclonal antibody (clone 37129), a mouse immunoglobulin g2a, was used as the capture antibody, and the human bdnf biotinylated monoclonal antibody (clone 37141), a mouse immunoglobulin g2a, was used as the detection antibody. the amount of bdnf was determined by absorbance at 450 nm with a correction at 540 nm, and the standard curve demonstrated a direct relationship between optical density and bdnf concentration. the levels of lipid peroxidation were measured using the tbars assay kit (cayman chemical company, ann arbor, mi, usa), according to the manufacturer s instructions. the results are expressed in m of malondialdehyde. oxidative damage to proteins was analyzed by determination of carbonyl groups (pcc method for carbonyl content in proteins), as previously described by levine.55 analyses were performed in serum samples and the values expressed in nmol / mg of protein. all analyses were performed using statistical package for the social sciences version 18 software (spss inc, chicago, il, usa). wilk test.56 the chi - square or fisher s exact test was used to analyze the association between dichotomous categorical variables. the student s t - test was used to compare means between unpaired groups. the mann one - way analysis of variance followed by the tukey post hoc test was used for comparison of parametric variables among three or more groups, and the kruskal quantitative variables were expressed as the mean standard deviation or median and interquartile range, according to the sample distribution. in all experiments, p - values less than 0.05 depressive patients were recruited at the mood disorders program, an outpatient facility based at the hospital de clnicas de porto alegre (hcpa), brazil. a diagnosis of unipolar depression was accorded by dsm - iv criteria and rated by the mini international neuropsychiatric interview, brazilian version (mini - plus).50 all depressed patients were classified as melancholic or nonmelancholic according to the core measure that evaluates 18 observable features of melancholia on a four - point scale, measuring its absence (0) or presence (three levels of severity, from 1 to 3).38 examples of items include facial nonreactivity, facial apprehension, and delay in motor activity. three authors (ls, mac, eav) were trained by video and printed guidelines provided by the black dog institute.51 a core score 8 was adopted in this study as defining melancholia, according to core development studies.52 depressive symptoms were evaluated using the 17-item hamilton depression rating scale (hdrs-17).53 a hdrs-17 18 indicating moderate to severe depression was considered as an inclusion criterion for this study.54 exclusion criteria included any history of autoimmune disease, chronic infection, or an inflammatory disorder, as well as any severe systemic disease or use of immunosuppressive therapy. we also recruited a control group consisted of 54 healthy volunteers attending the hcpa blood donor center, who had no current or previous history as well as no first - degree family history of a major psychiatric disorder, including dementia or mental retardation, assessed by the nonpatient version of the structured clinical interview for dsm - iv. the investigation was approved by the medical ethics committee at hcpa, and all participants provided written informed consent. ten milliliters of blood were collected from each patient and control by venipuncture into a free - anticoagulant vacuum tube. immediately after withdrawal, the blood was centrifuged at 4,000 rpm for 10 minutes and serum was aliquoted, labeled, and stored at 80c until assayed. serum cytokine concentrations were determined by flow cytometry using the bd cytometric bead array th1/th2/th17 human cytokine kit (bd biosciences, san diego, ca, usa). the cytometric bead array kit employed allows discrimination of the following cytokines : il-2, il-4, il-6, il-10, tnf-, ifn-, and il-17a. sample processing and data analyses were performed following the manufacturer s guidelines using a facscalibur flow cytometer (bd biosciences). results were generated in graphical and tabular format using fcap array cytometric bead array analysis software (bd biosciences). serum concentrations of bdnf were measured by sandwich enzyme - linked immunosorbent assay using monoclonal antibodies specific for bdnf from r&d systems (minneapolis, mn, usa). human bdnf monoclonal antibody (clone 37129), a mouse immunoglobulin g2a, was used as the capture antibody, and the human bdnf biotinylated monoclonal antibody (clone 37141), a mouse immunoglobulin g2a, was used as the detection antibody. the amount of bdnf was determined by absorbance at 450 nm with a correction at 540 nm, and the standard curve demonstrated a direct relationship between optical density and bdnf concentration. the levels of lipid peroxidation were measured using the tbars assay kit (cayman chemical company, ann arbor, mi, usa), according to the manufacturer s instructions. the results are expressed in m of malondialdehyde. oxidative damage to proteins was analyzed by determination of carbonyl groups (pcc method for carbonyl content in proteins), as previously described by levine.55 analyses were performed in serum samples and the values expressed in nmol / mg of protein. serum cytokine concentrations were determined by flow cytometry using the bd cytometric bead array th1/th2/th17 human cytokine kit (bd biosciences, san diego, ca, usa). the cytometric bead array kit employed allows discrimination of the following cytokines : il-2, il-4, il-6, il-10, tnf-, ifn-, and il-17a. sample processing and data analyses were performed following the manufacturer s guidelines using a facscalibur flow cytometer (bd biosciences). results were generated in graphical and tabular format using fcap array cytometric bead array analysis software (bd biosciences). serum concentrations of bdnf were measured by sandwich enzyme - linked immunosorbent assay using monoclonal antibodies specific for bdnf from r&d systems (minneapolis, mn, usa). human bdnf monoclonal antibody (clone 37129), a mouse immunoglobulin g2a, was used as the capture antibody, and the human bdnf biotinylated monoclonal antibody (clone 37141), a mouse immunoglobulin g2a, was used as the detection antibody. the amount of bdnf was determined by absorbance at 450 nm with a correction at 540 nm, and the standard curve demonstrated a direct relationship between optical density and bdnf concentration. the levels of lipid peroxidation were measured using the tbars assay kit (cayman chemical company, ann arbor, mi, usa), according to the manufacturer s instructions. the results are expressed in m of malondialdehyde. oxidative damage to proteins was analyzed by determination of carbonyl groups (pcc method for carbonyl content in proteins), as previously described by levine.55 analyses were performed in serum samples and the values expressed in nmol / mg of protein. all analyses were performed using statistical package for the social sciences version 18 software (spss inc, chicago, il, usa). wilk test.56 the chi - square or fisher s exact test was used to analyze the association between dichotomous categorical variables. the student s t - test was used to compare means between unpaired groups. the mann one - way analysis of variance followed by the tukey post hoc test was used for comparison of parametric variables among three or more groups, and the kruskal quantitative variables were expressed as the mean standard deviation or median and interquartile range, according to the sample distribution. in all experiments, p - values less than 0.05 data from a total of 33 depressed patients were analyzed after application of inclusion and exclusion criteria. according to the core measure, 13 (39.3%) there were no significant differences between patient groups with regard to age, female sex, hrsd-17, lifetime tobacco use, body mass index, time of current episode, number of previous episodes, and medication use. while core - defined melancholic patients had more psychotic episodes than nonmelancholic patients (30.8% versus 10%, respectively), this difference was not significant. our first strategy was to compare the biological parameters between depressed patients (irrespective of depressive subset) and healthy controls. table 2 data quantify that depressed patients had significantly higher levels than controls on the pcc, il-4, and il-6 variables. nonmelancholic patients and controls returned higher inf- values than melancholic patients, while the controls and nonmelancholic patients did not differ from each other. melancholic patients generated lower tbars values than nonmelancholic patients, but there was no difference between the depressive subset and control groups. both depressive groups showed significantly higher pcc values than controls but did not differ between melancholic and nonmelancholic groups. there was no statistically significant difference in bdnf, il-2, il-4, il-10, tnf-, and il-17 levels between melancholic and nonmelancholic groups. we analyzed biological markers in relation to melancholic and nonmelancholic depression subtyped by a sign - based measure of melancholia. to our knowledge, this is the first study evaluating inflammatory markers, neurotrophins, and oxidative stress in melancholic depression using a sign - based diagnostic tool, ie, the core measure. this is also the first study comparing oxidative stress parameters (tbars and pcc) irrespective of diagnostic system between melancholic and nonmelancholic groups. first, we compared all depressed patients (irrespective of any subdivision) with controls in relation to potential neurobiological markers. we found that pcc, il-4, and il-6 values were higher in the depressive group, replicating previous studies.43,5759 other measures (bdnf, il-2, il-10, ifn-, tnf-, and il-1 levels) failed to indicate any differences across groups. the increase in il-6 is one of the most consistently replicated findings in depressive patients, confirmed in a meta - analysis by dowlati which also found increased tnf- in depressed patients, albeit not replicated in the current study. the higher levels of il-4 (a th2 anti - inflammatory marker) in depressed patients compared with controls is a less consistent finding, with only a few studies reporting this58,59 and a meta - analysis finding no differences.43 in relation to oxidative stress, the increase in carbonyl products (pcc) in our depressive patients is consistent with a study by magalhaes in unipolar depressed subjects from a population - based sample.57 whereas pcc has been found to be increased in those with a bipolar disorder,57,60 more studies are needed to examine this in patients with unipolar depression. our findings did not replicate the higher tbars49 and lower bdnf61 values in depression reported by previous studies, and may reflect some limitations to our study (discussed below). in addition to the differences in neurobiological parameters between patients with depression and controls described above, we also found biological differences between subsets of depressive patients (melancholic and nonmelancholic) using the core subtyping measure. while differences in pcc between depressive patients and controls were evident in both depressive subgroups (with no differences between those subsets), differences in the inflammatory marker il-6 moreover, the statistical difference between depressive patients and controls in il-4 disappears, whereas differences emerge between depressive subsets in ifn- and tbars, with melancholic participants achieving lower ifn- (than both nonmelancholic patients and controls) and tbars (than nonmelancholic) values. while there were no differences in the th1 proinflammatory marker ifn- between depressive patients and controls in the initial analysis, we found lower values in melancholic patients when the depressive groups were split according to clinical signs of pmd. this finding is consistent with a study by rothermundt who found lower ifn- and il-2 levels in melancholic patients, using a symptom - based definition of melancholia. they suggested a decrease in cell - mediated immunity in patients with melancholic depression, possibly induced by hypercortisolemia. thereby, while melancholic depression is classically associated with hypercortisolemia62 and an increase in plasma adrenocorticotropic hormone,18 this increase added to an increase in catecholamines (the major stress hormones) may systematically inhibit the th1 proinflammatory response (via ifn-), while upregulating th2 anti - inflammatory production (via il-4).63 a possible explanation for our results is that melancholic depression may be represented by a specific dysregulation of the proinflammatory / anti - inflammatory and th1/th2 cytokine balance, with a sustained increase in hypercortisolemia and catecholamines that then upregulate il-6 levels. although the initial higher il-4 value was not statistically significant when we split the depressive group into subsets, this change may reflect our small sample size and limited statistical power. similarly, the significance of the higher il-6 value was limited to melancholic patients when compared with controls, with no differences between depressive subtypes when directly compared. although increased il-6 is a well established change in depression43 and is more distinctive in melancholic than nonmelancholic patients,27 our small sample subsets may have compromised our ability to demonstrate any such difference. turning to oxidative stress markers, the difference in pcc was sustained in both depressive subsets when compared with controls, with no difference between subsets. the lack of ability of pcc to discriminate depressive subgroups may be more a reflection of depression than of depressive subtype. in addition, pcc is not only a sign of oxidative stress, but also of protein dysfunction. carbonyl products are altered in a number of systemic diseases, including alzheimer s disease, inflammation, and diabetes,64 are increased consistently in bipolar disorder,57 and may simply be a nonspecific marker of disease. therefore, pcc may be seen as a sign of activity in mood disorders, rather than a marker of a specific subcategory. on the other hand, we found an initially counterintuitive decrease in tbars, a measure of lipid oxidative stress, in melancholic patients in comparison with the nonmelancholic group. most studies have found no changes57 or reported an increase48,65,66 in tbars in depression. to our knowledge, no previous studies have investigated tbars in melancholic depression, and this result clearly warrants replication as it may represent an indirect peripheral metabolic difference in depressive subsets. tbars is a marker of lipid oxidation, and is altered according to metabolic status.67 however, our sample subtyped by pmd did not differ regarding body mass index. previous studies have used symptoms to subtype depressive patients (where nonmelancholic subsets may include atypical depression) and differentiate them according to patterns of changes in weight and appetite (increased in atypical depression and decreased in melancholia). for instance, lamers found minimal or no difference between melancholic patients and healthy controls across several metabolic parameters, although their melancholic patients had lower values for some variables (waist circumference and body mass index). on the other hand, atypical (nonmelancholic) depression was associated with much greater metabolic dysregulation compared with both melancholic patients and controls. in that study, lamers used a modified method to rate depressive subsets, with symptoms of weight and appetite distinctly differentiating the subgroups of depression. although we did not evaluate atypical depression or other metabolic markers in our study, and body mass index values were no different between subsets, other metabolic differences between depressive subsets might be a putative explanation for this finding. most studies examining biological differences (including inflammatory markers) in melancholic patients have made comparisons with symptom - defined groups with nonmelancholic depression, atypical depression, or nondepressive controls.911,18,27,2932,34,35,68 such studies have generally reported hypercortisolemia and other hypothalamic - pituitary - adrenal axis changes in melancholic patients11,18,32,34,69 but contradictory or less robust results in relation to inflammatory markers. these contradictory results may reflect limitations of symptom - based criteria in accurately discriminating the melancholic depressive subtype.70 our results bring a contribution, demonstrating that a sign - based criteria set might help to refine the diagnosis of melancholia, advancing the search for biological determinants of depressive disorder, with potential therapeutic implications. although no differences were found in medication classes among the groups, antidepressants and neuroleptic medications may change inflammatory biomarkers,10,71 levels of bdnf,46 and oxidative stress parameters.49,66,72 third, other potential variables associated with changes in biomarkers were not evaluated, including nutritional status73 and menstrual cycle.74 fourth, although observational rating of pmd is necessary when using the core measure, observable pmd has been shown to be less distinctive in younger patients with seemingly true melancholic depression, while a valid rating of pmd requires observing patients at or near the nadir of their depressive episode.6 while auxiliary tools are being developed to complement the assessment, such as the sydney melancholia prototype index,6 only the core measure was used in the current study. diagnoses such as major depression comprise subsets of depressive conditions, and there is evidence that each may have differing causes, illness trajectories, and treatment responses. valid identification of one historically weighted subset condition (ie, melancholia) may be clinically helpful, and our results add to the body of evidence suggesting that this differentiation may contribute to understanding the underlying neurobiology. although psychiatry disorders are generally defined by symptoms, clinically observable signs can lead to a more accurate diagnosis of melancholia and should be considered in future research studies. depressive patients differed from healthy controls on three biological parameters, returning higher pcc, il-4, and il-6 values. melancholic patients classified by a sign - based measure (core) scored lower across lipid oxidative stress markers (versus nonmelancholic patients) and immunological markers (versus both controls and nonmelancholic patients). a sign - based measure (core) used to subtype depressive patients demonstrated biological differences across depressive subsets and might enhance accuracy of the clinical diagnosis of melancholia. the limitations of our study were that : its small sample size may have limited its analytic power ; all patients were on medication, a potential confounding variable in relation to measuring biological data ; and the fact that younger patients may show less pmd, limiting the power of the core measure in this population. | backgroundthe purpose of this study was to compare melancholic patients rated by the core measure of observable psychomotor disturbance with nonmelancholic and control subjects across a set of biomarkers.methodsdepressed patients were classified as melancholic or nonmelancholic by using the core measure. both groups of patients, as well as control subjects, were compared for a set of clinical and laboratory measures. serum levels of brain - derived neurotrophic factor, of two markers of oxidative stress (protein carbonyl content [pcc ] and thiobarbituric acid reactive substances [tbars ]), and of several immunity markers (interleukin [il]-2, il-4, il-6, il-10, il-17, tumor necrosis factor - alpha, and interferon - gamma) were analyzed.resultsthirty-three depressed patients and 54 healthy controls were studied. depressive patients showed higher il-4, il-6, and pcc values than healthy controls. thirteen (39%) of the depressed patients were assigned as melancholic by the core measure. they generated lower interferon - gamma (compared with nonmelancholic depressed patients) and tbars (compared with both the nonmelancholic subset and controls) and returned higher il-6 levels than controls. both depressive groups generated higher pcc scores than controls, with no difference between melancholic and nonmelancholic subsets.conclusiona sign - based measure to rate melancholia was able to replicate and extend biological findings discriminating melancholic depression. signs of psychomotor disturbance may be a useful diagnostic measure of melancholia. |
epitheloid hemangioma, a benign vascular tumor that arises in skin and soft tissues can also involve the skeletal system. authors report a case of epitheloid hemangioma of the middle phalanx in a young girl without any cutaneous manifestations. the lesion presented as a swollen middle finger, and plain radiographs showed a geographic area of destruction with cortical thinning and intra lesional calcifications. epitheloid hemangioma should be considered in the differential diagnosis of hand masses with expansile lytic lesions with cortical thinning. epitheloid hemangioma [eh ] is a benign vascular tumor that arises in skin, soft tissue, lungs, and bone[1 - 3 ]. eh is a tumor predominantly of the long bones with lesions reported from the femur, humerus, tibia, scapula and the vertebrae[4 - 9 ]. radiologically they present as expansile lytic area of geographic destruction with thinning of the cortex, and definitive diagnosis is by histopathology. immunohistochemistry for vascular markers shows cd 31 positive in cords of cells lining the blood vessels. 16-year - old girl presented in the orthopedic clinic with a swollen right middle finger associated with occasional pain of one month duration. plain films of the right middle finger showed an expansile lesion with thinning of the cortex and intralesional calcifications. [fig 1 ] plain radiographs showing expansile lesion of the middle phalanx with calcifications. computerized tomographic scan[ct scan ] showed a central oval round area of geographic destruction in the medullary canal at the metaphyseal region of the middle phalanx of the right middle finger. [fig 2 ] ct scan showing geographic destruction, with bulbous expansion and calcifications. with a provisional diagnosis of enchondroma the defect in the bone was packed with cancellous grafts harvested from the proximal tibia. histopathology showed grey and dark brown tissue, with sections showing fragments of tissue composed of many perforating capillary sized blood vessels that are lined by plump endothelial cells with abundant cytoplasm and vesicular nuclei. immunohistochemistry for vascular markers showed cd31 positive in cords of cells and cells lining blood vessels, many cd 68 positive cells were also seen. hematoxylin and eosin stain showing blood vessels lined by plump eitheloid cells and eosinophils in the stroma x 20. at one - year [fig 5 & 6 ]. clinical photograph showing the range of movements of the finger. radiographs antero - posterior [a ] and lateral [b ] showing no evidence of recurrence. the differential diagnosis of expansile multi- loculated phalangeal lesions includes enchodroma, giant cell tumor of bone [gct ], aneurysmal bone cyst [abc ], and chondromyxoid fibroma. enchondromas are the most common primary tumor of the hand, presenting as expansile lesion usually of the proximal phalanx. aneurysmal cysts are occasionally reported in the phalanx, and when it occurs in the phalanx it is practically impossible to differentiate it from gct or enchondroma. in gct of the phalanges, there is bone destruction and diaphyseal and soft tissue extension. in our case because of her age and the clinical and radiological findings, a diagnosis of enchodroma was made, as enchondromas are the second commonest benign cartilaginous tumor in her age group, second only to osteochondroma. in small tubular bone tumors like gct and enchondroma there is significant amount of bone loss due to matrix resorption by osteoclasts. presentation of eh is pain in the involved bone ; however it may be seen as an incidental finding. eh has been reported from the femur, humerus, tibia, scapula, and vertebrae, which are the usual sites [4 - 9 ]. it is a tumor predominantly of the long bones ; however lesions in the small tubular bones have been reported. in an analysis of 50 cases of eh, 8% of cases were with lesions of the small bones of the hand. lesions in the hand are expansile, with endosteal scalloping and thinning of the cortex in the meta - diaphyseal region, and rarely with matrix mineralization endosteal scalloping was seen in 100% of cases in a report by bierry ; however none of their cases showed matrix mineralization. there are reports of eh presenting as well defined lytic lesions of bone with sclerotic margins and intralesional calcifications, giving the appearance of soap bubble. eh is known to be associated with skin lesions, and the absence of skin lesions made us think of enchondroma as the only diagnosis. histologically there are large polyhedral cells in sheets or cords or lining numerous well defined vascular spaces. there is a lobular growth pattern, with cellular lobules separated by loose connective tissue containing well formed vessels which are of capillary size. the sheets of epitheloid cells lining the vascular structures have tomb stone like arrangement. spontaneous remission of the tumor has also been reported suggesting a benign nature of the tumor, however a case of recurrence was reported by nielson.[3, 5 ]. authors report a case of epitheloid hemangioma in a 16-year- old girl, which was clinically and radiologically diagnosed as enchondroma. this is a rare presentation of phalangeal tumor, and the diagnosis can be confirmed by immunohistochemistry for vascular markers. | introduction : epitheloid hemangioma, a benign vascular tumor that arises in skin and soft tissues can also involve the skeletal system. occasionally this has been reported from small tubular bones of the hand.case report : authors report a case of epitheloid hemangioma of the middle phalanx in a young girl without any cutaneous manifestations. the lesion presented as a swollen middle finger, and plain radiographs showed a geographic area of destruction with cortical thinning and intra lesional calcifications. the case was managed by curettage and bone grafting. histology confirmed this as a case of epitheloid hemangioma.conclusion:epitheloid hemangioma should be considered in the differential diagnosis of hand masses with expansile lytic lesions with cortical thinning. |
every year about 14 million new cancer cases are detected and 8 million people die of cancer. however, there is a marked difference in the distribution of cancer sites across different regions of the world. in contrast to developed countries, cervical cancer is a public health problem in developing countries like india, so much so that india alone accounts for one - quarter of the worldwide burden of cervical cancers. it is the one of the leading cause of cancer mortality, accounting for 17% of all cancer deaths among women aged between 30 and 69 years. it is estimated that cervical cancer will occur in approximately 1 in 53 indian women during their lifetime compared with 1 in 100 women in more developed regions of the world. however, there are two prerequisites for screening to reduce the rate of death from cancer. first, screening must advance the time of diagnosis of cancers that are destined to cause death. second, early treatment of these cancers must confer some advantage over treatment at clinical presentation. unlike other cancer sites, cervix can be subjected to screening for early diagnosis and treatment. however, despite availability of various cervical cancer screening methods, as well as large burden of disease in india, there is no countrywide government - sponsored public health policy on prevention of cervical cancer by either screening or vaccination or both. therefore, this study was carried out to understand and present burden of cervical cancer in india, as well as to appraise the various cervical cancer screening methods and studies conducted for evaluating screening test for the detection of cervical carcinoma. however, since india is culturally, economically, and sociodemographically dissimilar from other western countries, we limited the scope of our study to screening trials conducted in indian population, so as to provide locally relevant evidence - based recommendations for cervical cancer screening in indian population. this paper is based on information gathered from a review of peer - reviewed publications on cervical cancer screening and prevention in india. medline (http://www.pubmed.com) and web of science electronic database were searched from january 1990 to december 2015 using the keywords such as cervical cancer, screening, early detection, human papillomavirus (hpv), cervical cytology and visual inspection, and their corresponding mesh terms were also used in combination with boolean operators or, and. we also examined bibliographies of included articles to identify additional references. summary of evidence search and selection study designs eligible for inclusion in our study were randomized controlled trials, nonrandomized controlled trials, cohort studies, and cross - sectional studies conducted to evaluate the performance of the screening tests for detection of cervical cancerstudies conducted in india only were included in the study. study designs eligible for inclusion in our study were randomized controlled trials, nonrandomized controlled trials, cohort studies, and cross - sectional studies conducted to evaluate the performance of the screening tests for detection of cervical cancer studies conducted in india only were included in the study. studies not providing data on sensitivity and specificity of screening test were evaluated and excluded from the study. studies not providing data on sensitivity and specificity of screening test were evaluated and excluded from the study. the title and abstract of each citation were screened first, and full report was screened second if necessary to select the relevant articles according to selection criteria. full texts of these selected studies were retrieved, reviewed, and extracted for relevant data by authors independently. a total of 11 studies were included in the review, and their findings have been presented. the descriptive statistical data for cervical cancer (international classification of diseases, tenth edition, c53) were mainly obtained from publications of the indian cancer registry, union for international cancer control, international agency for research on cancer, and who (globocan-2012). this paper is based on information gathered from a review of peer - reviewed publications on cervical cancer screening and prevention in india. medline (http://www.pubmed.com) and web of science electronic database were searched from january 1990 to december 2015 using the keywords such as cervical cancer, screening, early detection, human papillomavirus (hpv), cervical cytology and visual inspection, and their corresponding mesh terms were also used in combination with boolean operators or, and. we also examined bibliographies of included articles to identify additional references. study designs eligible for inclusion in our study were randomized controlled trials, nonrandomized controlled trials, cohort studies, and cross - sectional studies conducted to evaluate the performance of the screening tests for detection of cervical cancerstudies conducted in india only were included in the study. study designs eligible for inclusion in our study were randomized controlled trials, nonrandomized controlled trials, cohort studies, and cross - sectional studies conducted to evaluate the performance of the screening tests for detection of cervical cancer studies conducted in india only were included in the study. studies not providing data on sensitivity and specificity of screening test were evaluated and excluded from the study. studies not providing data on sensitivity and specificity of screening test were evaluated and excluded from the study. study designs eligible for inclusion in our study were randomized controlled trials, nonrandomized controlled trials, cohort studies, and cross - sectional studies conducted to evaluate the performance of the screening tests for detection of cervical cancerstudies conducted in india only were included in the study. study designs eligible for inclusion in our study were randomized controlled trials, nonrandomized controlled trials, cohort studies, and cross - sectional studies conducted to evaluate the performance of the screening tests for detection of cervical cancer studies conducted in india only were included in the study. studies not providing data on sensitivity and specificity of screening test were evaluated and excluded from the study. studies not providing data on sensitivity and specificity of screening test were evaluated and excluded from the study. the title and abstract of each citation were screened first, and full report was screened second if necessary to select the relevant articles according to selection criteria. full texts of these selected studies were retrieved, reviewed, and extracted for relevant data by authors independently. a total of 11 studies were included in the review, and their findings have been presented. the descriptive statistical data for cervical cancer (international classification of diseases, tenth edition, c53) were mainly obtained from publications of the indian cancer registry, union for international cancer control, international agency for research on cancer, and who (globocan-2012). the national cancer registry programme (ncrp) has been in existence since 1982, and permanent institute (national centre for disease informatics and research) of the indian council of medical research is the crucial repository of data from the collaborating cancer registries located in medical colleges / institutions and hospitals throughout india. as of march 2016, there are 29 hospital - based cancer registries (including all regional cancer centres) and 29 population - based cancer registries (pbcrs) under ncrp. the data obtained from these indian cancer registries indicate that cervical cancer contributes to approximately 629% of all cancer in females. the age - adjusted incidence rate of cancer cervix was found to vary widely among registries, highest being 23.07/100,000 in mizoram state, followed by 22.54/100,000 in pasighat and the lowest being 4.91/100,000 in dibrugarh district. the older pbcrs such as bengaluru, bhopal, chennai, delhi, and barshi rural reported an age - adjusted incidence rate between 13 and 16/100,000 [table 1 ]. all the major registries from 1998 to 2014 reported a statistically significant decreasing trend (negative annual percentage change) of cervical cancer [table 2 ]. the maximum numbers of cases were reported in 5059 years of age group amounting to 27.37% of all cervical carcinoma cases [table 3 ]. age - adjusted (world) incidence rates of cervix uteri cancer and its relative proportion in indian population - based cancer registries the age - adjusted (world) incidence rates and annual percentage change for cervix uteri cancer in indian population - based cancer registry during the years 1988 - 2014 age - wise distribution of cervix uteri cancer the 5-year relative survival rate for cancer cervix in india has been reported to be approximately 46% (3460), which much lesser than survival rates reported from other asian countries such as china, south korea, singapore, and thailand [table 4 ]. in addition, cervical cancer survival rates in india also shows a wide variation ranging from 59.6% in chennai to 34.5% in bhopal. the 5-year age - standardized relative survival of cervical cancer for different indian registries and its comparison with other asian registries are shown in table 4. five - year age - standardized (074 years) relative survival for cervix uteri cancer in selected indian populations and selected asian countries cervix is amenable to screening by a number of methods which include visual inspection with acetic acid (via), magnified via (viam) visual inspection with lugol 's iodine (vili), the papanicolaou test, and hpv dna testing. a brief overview including strengths and weaknesses of each screening modality is presented in table 5. salient findings of indian studies on screening test performance are summarized in table 6. the pooled sensitivity and specificity for via was found to be 67.65% and 84.32%, for viam, the pooled sensitivity and specificity were 65.36% and 85.76% and for vili, the pooled sensitivity and specificity were 78.27% and 87.10%, respectively. the pooled sensitivity and specificity for cytology positivity at low - grade squamous intraepithelial neoplasia threshold were 62.11% and 93.51% and for hpv, the pooled sensitivity and specificity were 77.81% and 91.54%, respectively. overview of primary screening tools for cervical cancer indian studies providing accuracy of cervical screening tests in detecting cervical intraepithelial neoplasia the national cancer registry programme (ncrp) has been in existence since 1982, and permanent institute (national centre for disease informatics and research) of the indian council of medical research is the crucial repository of data from the collaborating cancer registries located in medical colleges / institutions and hospitals throughout india. as of march 2016, there are 29 hospital - based cancer registries (including all regional cancer centres) and 29 population - based cancer registries (pbcrs) under ncrp. the data obtained from these indian cancer registries indicate that cervical cancer contributes to approximately 629% of all cancer in females. the age - adjusted incidence rate of cancer cervix was found to vary widely among registries, highest being 23.07/100,000 in mizoram state, followed by 22.54/100,000 in pasighat and the lowest being 4.91/100,000 in dibrugarh district. the older pbcrs such as bengaluru, bhopal, chennai, delhi, and barshi rural reported an age - adjusted incidence rate between 13 and 16/100,000 [table 1 ]. all the major registries from 1998 to 2014 reported a statistically significant decreasing trend (negative annual percentage change) of cervical cancer [table 2 ]. the maximum numbers of cases were reported in 5059 years of age group amounting to 27.37% of all cervical carcinoma cases [table 3 ]. age - adjusted (world) incidence rates of cervix uteri cancer and its relative proportion in indian population - based cancer registries the age - adjusted (world) incidence rates and annual percentage change for cervix uteri cancer in indian population - based cancer registry during the years 1988 - 2014 age - wise distribution of cervix uteri cancer the 5-year relative survival rate for cancer cervix in india has been reported to be approximately 46% (3460), which much lesser than survival rates reported from other asian countries such as china, south korea, singapore, and thailand [table 4 ]. in addition, cervical cancer survival rates in india also shows a wide variation ranging from 59.6% in chennai to 34.5% in bhopal. the 5-year age - standardized relative survival of cervical cancer for different indian registries and its comparison with other asian registries are shown in table 4. five - year age - standardized (074 years) relative survival for cervix uteri cancer in selected indian populations and selected asian countries cervix is amenable to screening by a number of methods which include visual inspection with acetic acid (via), magnified via (viam) visual inspection with lugol 's iodine (vili), the papanicolaou test, and hpv dna testing. a brief overview including strengths and weaknesses of each screening modality is presented in table 5. salient findings of indian studies on screening test performance are summarized in table 6. the pooled sensitivity and specificity for via was found to be 67.65% and 84.32%, for viam, the pooled sensitivity and specificity were 65.36% and 85.76% and for vili, the pooled sensitivity and specificity were 78.27% and 87.10%, respectively. the pooled sensitivity and specificity for cytology positivity at low - grade squamous intraepithelial neoplasia threshold were 62.11% and 93.51% and for hpv, the pooled sensitivity and specificity were 77.81% and 91.54%, respectively. overview of primary screening tools for cervical cancer indian studies providing accuracy of cervical screening tests in detecting cervical intraepithelial neoplasia in today 's era, in spite of the availability of hpv vaccines and affordable and effective methods for early detection and treatment of cervical cancer precursor lesions, cervical cancer still continues to be a public health problem in india. the age - adjusted incidence rates of cancer cervix reported by majority of indian cancer registries are much higher than the world age - adjusted incidence rate of 7.9/100,000 population but is lower or similar to cervical cancer incidence rates of 19.2/100,000 population seen in the south - east asian region. the high burden of cervical cancer in india and southeast asian countries is due to poor to moderate living standards, a high prevalence of hpv (more than 10% in women aged more than 30 years) and due to lack of screening. there is a wide variation (range : 4.9123.07/100,000) in the incidence rates reported by individual pbcrs [table 1 ], more so these extreme rates have been reported by newer registries. this is because some pbcrs are reporting the data for the first time and hence, it needs to be viewed with some caution as in the initial years of registry operation there could be some degree of over / under reporting. most of the major registries have shown a decreasing trend of cervical cancer, however, the decrease was very small. the mean annual percentage decrease in the average age - adjusted rate ranged from 1.81% to 3.48% among various registries. it should be noted that in india most of the cervical cancer cases are detected with regional spread of the disease, and a very small proportion are diagnosed at a localized stage. in india, an organized mass - screening program for early detection of cervical cancer is not in practice ; hence, this decrease can not be attributed to screening. moreover, it is also highly probable that cervical cancer incidence rates are an underestimate for india possibly due to underdiagnosis of cervical cancer cases in rural areas and among most impoverished women, as well as due to noninclusion of subclinical cervical cancers in routine hysterectomy specimens not subjected to histopathology, which is a common practice in many regions of india. on the other hand, it has also been suggested that probably an improvement in the living standard of women may have resulted in a reduction in the incidence of cervical cancer, but the same has not been substantiated. the 5-year age - standardized (074 years) relative survival for cervical cancer in india is much lower as compared to certain south east asian countries [table 4 ]. this can be attributed to the absence of a nationwide screening program as survival is determined by age and the extent of disease, with younger women having longer survival. this could be because indian registries suffer with the problem of under - reporting of death, which is more pronounced in the rural area. in addition, the lack of health infrastructure in rural india results in lower survival rates as compared to urban areas. in india, despite such alarming statistics there has been no synchronized initiative from public health authorities for prevention and control cervical cancer. in developed countries, conventional cytology screening programs have shown a marked decline in the incidence of cervical cancer. however, its successful implementation requires a variety of requirements to be fulfilled, which are not feasible in many low - resource settings where a high risk of cervical cancer is experienced. for instance, cytology screening requires a laboratory infrastructure, microscopes, several resource personnel (smear collectors, cytotechnicians, and pathologists), consumables (slides, fixative, pap stain containing five dyes, and three solutions), and several steps with inbuilt quality assurance procedures. moreover, cytology must be repeated at frequent 35-year intervals to ensure satisfactory sensitivity and optimum detection of cervical cancer precursor lesions and repeat visits are necessary after a positive cytology for diagnosis and treatment, which may lead to drop outs. several years of cytology screening in low- and middle - income countries have not led to significant reductions in cervical cancer in these countries, possibly due to the difficulties in offering high - quality cytology, programmatic deficiencies in follow - up and treatment of screen - positive women, and also due to the considerable financial, technical, and logistic inputs necessary for effective cytology programs. hence, these challenges have prompted the search for programs based on alternative cervical screening tests, which are implementable in resource - poor settings. via is one of the alternative methods for cervical cancer screening that has been widely investigated. in our review of indian studies, the sensitivity of via has been found to be better than cytology, but its specificity is lower [table 4 ]. however, the biggest advantage of visual tests is that it can be implemented through primary health - care workers, it does not require a laboratory infrastructure, and the results are obtained immediately following testing, allowing diagnosis and treatment to be instituted during the same visit. it has been well established that cervical neoplasia is caused by persistent infection with certain oncogenic types of hpv. indian studies of testing for hpv dna indicate higher sensitivity and comparable specificity as compared to visual inspection and cytology. however, the requirement of sophisticated laboratory infrastructure and high cost make it impracticable to be implementable in resource - poor, low - income countries. the efficacy of screening by via in reducing the incidence of and mortality from cervical cancer has been investigated through cluster randomized controlled trial in dindigul (india), a single round of via - based screening led to a 25% reduction in cervical cancer incidence and a 35% reduction in mortality over 7 years of follow - up. similarly, a randomized controlled trial carried out in maharashtra showed a 31% reduction in cervical cancer mortality in the screening group (mortality rate ratio relative risk = 0.69 ; 95% confidence interval : 0.54 - 0.88 ; p = 0.003) as compared to the control group. the studies conducted in india provide sufficient evidence that cervical cancer screening through simple test like via / vili is affordable, feasible, and an accurate tool for implementation in all health - care settings. in addition, via / vili also provides an opportunity to adopt see and treat approach, which is very useful in resource - poor countries where follow - up is poor. these tests can also be easily taught to grass root health workers, who can help in conducting the screening program in remote areas. however, for any cervical screening program to be successful in addition to the use of a reliable and accurate screening test, high rates of coverage and the ability to effectively provide treatment to test positive women are very important. hence, the development of health services and generation of community involvement are keys to the initiative in reducing the burden of cervical cancer. our study highlights the success of visual screening tool in early detection and mortality reduction of cervical cancer in a resource - poor setting and thus, provides a unique opportunity for developing countries to integrate screening of cervical neoplasia in primary health - care settings. | background : cervical cancer is a major cause of cancer mortality in women and more than a quarter of its global burden is contributed by developing countries. in india, in spite of alarmingly high figures, there is no nationwide government - sponsored screening program. this study was conducted to assess the burden of cervical cancer in india and review the performance characteristics of available cervical cancer screening tools, so as to provide evidence - based recommendations for application of most practically suited screening test to be used in resource - poor field settings.materials and methods : medline and web of science electronic database were searched from january 1990 to december 2015, using the keywords such as cervical cancer, screening, early detection, cervical cytology and visual inspection, and their corresponding mesh terms in combination with boolean operators or, and. two authors independently selected studies that are published in english and conducted in india. a total of 11 studies were found to be relevant and eligible to be included in the present study.results:in india, cervical cancer contributes to approximately 629% of all cancers in women. the age - adjusted incidence rate of cervical cancer varies widely among registries ; highest is 23.07/100,000 in mizoram state and the lowest is 4.91/100,000 in dibrugarh district. the pooled estimates of sensitivity and specificity of visual inspection with acetic acid (via), magnified via, visual inspection with lugol 's iodine (vili), cytology (pap smear), and human papillomavirus dna were found to be 67.65% and 84.32%, 65.36% and 85.76%, 78.27% and 87.10%, 62.11% and 93.51%, and 77.81% and 91.54%, respectively.conclusions:in developing countries because of lack of necessary infrastructure and quality control, high - quality cytology screening may not be feasible for wide - scale implementation. hence, cervical cancer screening program based on visual screening test such as via / vili should be adopted as an integral part of primary health - care setup in resource - poor countries like india. |
intramuscular hemangioma (imh) is a rare vascular disease involving skeletal muscle, comprising only 0.8% of all hemangiomas1. about 10% to 15% of imhs occur in the head and neck region, mostly involving the masseter muscle23. imh of the head and neck may be localized mainly in the masseter muscle (36%), trapezius (24%), periorbital muscles (12%), sternocleidomastoid muscle (10%), temporalis muscle, or orbicularis oris muscle4. however, it often is not found until unexpected enlargement, pain, or cosmetic asymmetry occurs in adulthood. several non - surgical treatments including cryotherapy, sclerosant injection, and arterial ligature have been described, but complete surgical resection is the curative intervention78. in the case of a large feeding vessel, embolization of the feeding vessels one imh case in the masseter muscle feeding from the transverse facial artery (tfa) and a second imh case in the orbicularis oris muscle feeding from the superior labial artery. written informed consent was obtained from the patients for publication of this case report and any accompanying images. in august 2014, a 48-year - old male visited our clinic with the chief complaint of swelling of the left buccal area.(fig. 1) on palpation, a painless and compressible mass was found near the left masseter muscle. there was no wattle sign or beating, and only bloody discharge was observed in fine needle aspiration. immediately following fine needle aspiration, initial diagnosis was a vascular lesion, such as hemangioma or vascular malformation. on magnetic resonance imaging (mri), a mass about 2.23.33.4 cm at the left masseter muscle was strongly enhanced with heterogeneous t2 hyperintensity.(fig. external carotid angiography (eca) was performed for further evaluation. a vascular mass feeding from the left tfa and the maxillary artery was identified.(fig. 3, 4) in the lateral view of angiography, the prosthesis of a dental implant on the upper left first molar overlapped the mass. the mass became blushed and then disappeared over time as contrast agent diffused through the blood vessels. based on clinical and radiographic examinations, the patient was provisionally diagnosed with imh of the left masseter muscle feeding from the left tfa (main) and maxillary artery. to reduce the risk of perioperative and postoperative bleeding, embolization of the distal tfa branch was performed.(fig. 5) a guiding catheter (diameter of 5 fr, enboy ; depuy synthes, west chester, pa, usa) was inserted in the left eca, then selection of the distal branch of the tfa was performed by prowler select plus (cordis, miami, fl, usa). a) an embolizing agent, polyvinyl alcohol (pva) particle (contour of 150 - 200 nm), was injected at slow speed into the selected site. pva particles are invisible in angiography, so the contrast agent was injected locally to the selected site to verify embolization. because the pathway was blocked at the selected site, back pressure was observed intensively at the backside of the selected site instead of the mass.(fig. b) to confirm effective embolization, contrast agent was injected into the eca again. blood supply from the maxillary artery was not significant, so embolization of the vessel was not performed. the following day, there was no change in the strongly enhancing mass at the left masseter muscle on post - embolization mri.(fig. two days after embolization, the buccal mass was excised via intraoral approach under general anesthesia. after insertion of a fine tube to stensen 's duct for position marking, mucosal incision anterior to stensen 's duct was performed. at the eight month check up, the patient reported no symptoms and was thereafter lost to follow - up. in april 2015, 9) in another dental clinic, she had undergone laser therapy at that area more than 10 years prior for the same symptom, but the mass had progressively enlarged. a 2.7 cm ovoid submucosal mass was well defined on mri stir (short - t1 inversion recovery) and t2 images (fig. 10), and the mass was feeding from small branches of the superior labial artery on angiography.(fig. 11) the mass was excised under general anesthesia without embolization. via the intraoral approach, mucosal incision on the buccal mucosa and undermining of the mass were conducted under local bleeding control. in august 2014, a 48-year - old male visited our clinic with the chief complaint of swelling of the left buccal area.(fig. 1) on palpation, a painless and compressible mass was found near the left masseter muscle. there was no wattle sign or beating, and only bloody discharge was observed in fine needle aspiration. immediately following fine needle aspiration, initial diagnosis was a vascular lesion, such as hemangioma or vascular malformation. on magnetic resonance imaging (mri), a mass about 2.23.33.4 cm at the left masseter muscle was strongly enhanced with heterogeneous t2 hyperintensity.(fig. a vascular mass feeding from the left tfa and the maxillary artery was identified.(fig. 3, 4) in the lateral view of angiography, the prosthesis of a dental implant on the upper left first molar overlapped the mass. the mass became blushed and then disappeared over time as contrast agent diffused through the blood vessels. based on clinical and radiographic examinations, the patient was provisionally diagnosed with imh of the left masseter muscle feeding from the left tfa (main) and maxillary artery. to reduce the risk of perioperative and postoperative bleeding, embolization of the distal tfa branch was performed.(fig. 5) a guiding catheter (diameter of 5 fr, enboy ; depuy synthes, west chester, pa, usa) was inserted in the left eca, then selection of the distal branch of the tfa was performed by prowler select plus (cordis, miami, fl, usa). a) an embolizing agent, polyvinyl alcohol (pva) particle (contour of 150 - 200 nm), was injected at slow speed into the selected site. pva particles are invisible in angiography, so the contrast agent was injected locally to the selected site to verify embolization. because the pathway was blocked at the selected site, back pressure was observed intensively at the backside of the selected site instead of the mass.(fig. b) to confirm effective embolization, contrast agent was injected into the eca again. blood supply from the maxillary artery was not significant, so embolization of the vessel was not performed. the following day, there was no change in the strongly enhancing mass at the left masseter muscle on post - embolization mri.(fig. two days after embolization, the buccal mass was excised via intraoral approach under general anesthesia. after insertion of a fine tube to stensen 's duct for position marking, mucosal incision anterior to stensen 's duct was performed. local control of bleeding and direct resection of the mass at the eight month check up, the patient reported no symptoms and was thereafter lost to follow - up. 9) in another dental clinic, she had undergone laser therapy at that area more than 10 years prior for the same symptom, but the mass had progressively enlarged. a 2.7 cm ovoid submucosal mass was well defined on mri stir (short - t1 inversion recovery) and t2 images (fig. 10), and the mass was feeding from small branches of the superior labial artery on angiography.(fig. 11) the mass was excised under general anesthesia without embolization. via the intraoral approach, mucosal incision on the buccal mucosa and undermining of the mass were conducted under local bleeding control. the term hemangioma has been applied to a wide variety of vascular lesions and continues to be used to describe vascular malformations. several authors have recently suggested that vascular malformations should be distinguished from hemangiomas10111213. in 1982, the international society for the study of vascular anomalies adopted mulliken and glowacki 's classification system and modified the classification of vascular malformations versus vascular tumors10. vascular malformations are present at birth and enlarge in proportion to the growth of the child11. they are subcategorized as slow - flow vascular malformations (capillary malformation, venous malformation, and lymphatic malformation), fast - flow vascular malformations (including arteriovenous malformation), and complex - combined vascular malformations. phlebolith (especially in venous malformation on computed tomography [ct ]) and signal void (especially in arteriovenous malformation on mri) are common features. there is often a wattle sign, beating, and thrilling in vascular malformations. on the other hand, vascular tumors are true neoplasms with cellular hyperplasia and include infantile hemangioma, congenital hemangioma, and hemangioendothelioma. on radiographic images, they show a well - defined mass in the majority of patients13. arteriovenous shunting and phlebolith are unusual in vascular tumors. histologically, they grow by endothelial proliferation, and tumors can grow, regress, or persist. adipose tissue can be observed, because regressed tumors are replaced by adipose tissue1014. imh is a special form of hemangioma involving skeletal muscle and comprises only 0.8% of all hemangiomas1. about 10% to 15% of imhs occur in the head and neck region, mostly involving the masseter muscle23. they therefore have somewhat different characters from other types of hemangiomas. due to the rarity of these tumors and their deep location and unfamiliar presentation, inaccurate diagnosis, inappropriate excision, and unnecessary risk to the facial nerve occur when imh is present in the face4. over 90% of all imhs differential diagnosis of imh includes lymphangioma, lymphomas, rhabdomyosarcoma, and schwannomas9. in particular, imh arising from the masseter muscle is frequently confused with parotid tumor715. however, it is often not found until unexpected enlargement, pain, or cosmetic asymmetry occurs in adulthood. allen and enzinger16 retrospectively reviewed the histopathological findings of a large group of patients described as having imh. they classified these patients into three categories based on vessel size and histological features : small - vessel type (capillary type, less than 140 m), large - vessel type (cavernous type, more than 140 m), and mixed - vessel type. however, yilmaz.13 argued that the description of the small - vessel type was identical to that of intramuscular capillary - type hemangioma, while the description of large - vessel type was more consistent with what is now designated as venous malformation. capillary - type imh accounts for 50% of all imh, and 68% of these tumors are in the head and neck4. to diagnosis imh, mri is considered the gold standard 10111215 and typically shows a well - marginated soft tissue mass. on t2 and stir images, lesions appear homogeneous and moderately hyperintense during the proliferative phase and increased heterogeneity during the involution phase owing to areas of fat replacement. flow voids may be seen at the periphery of the lesion on spin - echo sequences, reflecting high - flow arterial feeders10. angiography is useful for diagnosis of arteriovenous malformation and is not routinely performed for diagnosis of hemangioma. non - surgical treatments such as cryotherapy, sclerosant injection, embolization, and arterial ligature have been described, but there is dispute about the results of these treatments. therefore, non - surgical treatments are currently recommended only when surgery is contraindicated or refused7. complete surgical resection is the curative intervention. for intramasseteric cases, the intraoral approach for surgical resection may be useful as it avoids any visible scar or facial nerve dissection78. localized recurrence rate ranges from 9% to 28%, despite wide resection of the tumor due to the absence of capsule surrounding the tumor2. minor feeding vessels and residual tumor may be responsible for the recurrence rate16. in case 1 a mass with well - defined border, strongly enhanced with heterogeneous t2 hyperintensity was found on mri.(fig. 2) on angiography, a vascular mass feeding from the left tfa (main) and the maxillary artery was found.(fig. 3) tfa is a branch arising from the superficial temporal artery, and it anastomoses with the facial artery, mesenteric artery, infraorbital artery, and others17. 5), and the mass was surgical excised by intraoral approach two days later.(fig. 10, 11), and there was wattle sign or beating. on microscopic exam of case 1, well developed vessels with proliferation of endothelial cells between skeletal muscle tissue were found.(fig b) a large amount of adipose tissue was observed because regressed tumors are replaced by adipose tissue.(fig. c) larger vessels (possibly from feeding vessels) were also observed within the mass, but there was no obvious arteriovenous shunting.(fig. 8. d) this tumor was therefore considered mixed - type imh. in case 2, large vessels (possibly from feeding vessels) were observed in the lateral side of the mass (fig. b) however, there was only a small amount of adipose tissue in this case. 13), an antigen protein that shows expression on immature hematopoietic cells and endothelial cells. preoperative embolization and surgical excision are recommended. in these two rare cases of imh in masseter and orbicularis oris muscle, the intraoral approach provided a direct approach and allowed removal of the tumor without any visible scar or facial nerve dissection. | intramuscular hemangioma (imh) is a rare vascular disease involving skeletal muscle, comprising only 0.8% of hemangiomas. about 10% to 15% of imhs occur in the head and neck region, mostly involving the masseter muscle. imh occurs mostly in childhood, but is often not found until unexpected enlargement, pain, or cosmetic asymmetry occurs in adulthood. several non - surgical treatments including cryotherapy, sclerosant injection, and arterial ligature have been described, but complete surgical resection is the curative intervention. in this report, we present two rare cases of imh. one imh case in a 48-year - old male occurred in the masseter muscle feeding from the transverse facial artery. embolization of the distal branch of the facial artery was first conducted, and then the buccal mass was removed surgically via the intraoral approach. a second imh case in a 58-year - old female occurred in the orbicularis oris muscle feeding from the superior labial artery, and the mass was excised surgically without embolization. |
over the past few years, there has been recognition of a distinctive group of renal tumors composed grossly of cystic and solid portion and microscopically of stromal and epithelial proliferation. while it is not clear whether they represent a heterogenous group or a single disease entitiy, these tumors have been reported under various names such as adult mesoblastic nephroma, cystic nephroma, mixed epithelial and stromal tumor and some others. we believe that the descriptive and unifying term of mixed epithelial and stromal tumor is appropriate for this group of tumors until further characterization is made and report a case of renal tumor which appears to represent a good example of this entity. a 47-yr - old woman was detected to harbor a right renal mass by ultrasonography on routine examination. microscopic hematuria was also present at that time but there were no other abnormal clinical or laboratory findings or past history of medical problem. on computed tomography, the mass measured 7 cm in the largest dimension and was multiseptated with irregularly enhancing solid component (fig. the mass was occupying mid to lower pole of the right kidney close to renal sinus and partially protruding into renal pelvis. it measured 86 cm and was largely cystic with a whitish yellow solid mural nodule of 33 cm (fig. microcystic lesions were present with regional sponge - like appearance and focal calcification was also found. microscopically, solid portion was composed of irregularly arranged bundles of cigar - shaped spindle cells sprinkled with a few inflammatory cells in the fibrillar background (fig. the nodule of spindle cells displayed hypocelluar areas at the periphery entrapping several tubular structures which occasionally exhibited cyst - like dilatation. epithelial proliferation was exuberant around the sponge - like areas and occupied an area measuring approximately 1.5 cm in largest dimension. eosinophilic columnar epithelial cells containing occasional single unconspicuous nucleolus constituted papillary structures with fibrovascular core and intervening foamy histiocytes (fig. immunohistochemically, the spindle cells were positive for vimentin, smooth muscle actin, but negative for cd34, desmin, hmb45, estrogen receptor and progesterone receptor while epithelial cells were reactive for epithelial membrane antigen, high molecular weight cytokeratin and pancytokeratin. on electron microscopic examination, the spindle cells had many subplasmalemmal pinocytotic vesicles and intracytoplasmic filaments occasionally forming dense bodies, thus suggesting myofibroblastic differentiation (fig. 5). michal and syrucek first proposed the term of mixed epithelial and stromal tumor of the kidney in 1998 (1) and later adsay. reported a group of 12 patients under the same name and regarded the diagnosis as an appropriate preliminary title for that category of tumor which is characterized grossly by a mixture of solid and cystic areas and various diagnoses have been rendered to tumors with similar morphologic findings such as adult mesoblastic nephroma (3 - 6), cystic hamartoma of pelvis (7), cystic nephroma (8) or mature nephroblastic tumor and cystic partially differentiated nephroblastoma (9). it remains to be determined whether these tumors are comprised of a heterogenous group of tumors or represent one distinct entity. however, these tumors basically share similar clinicopathologic characteristics and therefore it appears reasonable, at least for the present, to regard them as a single entity. approximately 50 cases of such tumors have been reported in the literature (2 - 5) and three cases have been documented in korean literature under the name of adult mesoblastic nephroma (10 - 12). common clinical presentations were those of usual renal mass lesions such as flank pain, hematuria, symptoms of infection or no attributable symptoms. mean tumor size is approximately 6 cm and the tumor shows a female preponderance centered around perimenopausal age and frequent reactivity for estrogen or progesterone receptors. and in rare reported cases of male patients these facts led to the suggestion of hormonal mechanism in pathogenesis and the estrogen- and progesterone - receptor positivity are sometimes deemed sufficient characteristics to warrant its utility as an adjunct diagnostic criteria for this tumor. however, our case lacked evidence of hormonal receptor expression and therefore it is reasonable to conclude that not all cases of mixed epithelial and stromal tumor implicate hormonal mechanism in pathogenesis and it also can be speculated that this presumed difference in pathogenesis might eventually result in subclassification of this tumor after accumulation of further data. the stromal component of mixed epithelial and stromal tumor varies in cellularity and can assume various histologic appearance reminiscent of leiomyoma, solitary fibrous tumor or ovarian stroma. ovarian stroma, frequently found in mixed epithelial and stromal tumor, is absent in this case, which might be related to estrogen and progesterone receptor negativity of this case. epithelial proliferation in this case is exuberant and spans an area over 1 cm in diameter. this feature is worrisome especially when we consider the newly accepted criteria of 0.5 cm as a cut - off point between papillary adenoma and carcinoma (13). however, malignancy of epithelial component is not yet documented in mixed epithelial and stromal tumor whereas there is a report of stromal malignancy (14). therefore, while it appears reasonable to accept, at least for the present, that benign epithelial proliferation can be florid, the possibility can not be completely ruled out that there might develop epithelial malignancy. adult mesoblastic nephroma was first reported in 1973 (15) and was the primary diagnosis in a majority of this group of tumors (3 - 6). while it is clear that this tumor shares a few morphologic characteristics of congenital mesoblastic nephroma, it also appears evident that there are a few unmistakable differences. firstly and most importantly, the epithelial proliferation seems an integral neoplastic component in this tumor (2) whereas in congenital mesoblastic nephroma, it is more likely entrapped epithelial portion with occasional metaplastic changes and focal hyperplasia. furthermore, genetic alterations typical of congential mesoblastic nephroma were not found in this tumor (16). and frequent infiltration into surrounding renal parenchyma found in congenital mesoblastic nephroma is rarely encountered (4). therefore this tumor is more likely unrelated to mesoblastic nephroma and it appears more reasonable to apply the descriptive term of mixed epithelial and stromal tumor at least for the present under which name it is included in the current who classification (13). when we accept the entity of mixed epithelial and stromal tumor, the differential diagnosis is limited. the spindle cell component of mixed epithelial and stromal tumor can be found in solitary fibrous tumor (17) or angiomyolipoma (18), both of which can be excluded with immunostaining and by lack of epithelial proliferation. biphasic renal lesions may encompass tumors such as nephroblastoma, cystic partially differentiated nephromablastoma and cystic embryonal sarcoma (19), all of which are rare in adulthood or contain malignant mesenchymal component. rare benign biphasic renal tumor such as nephrogenic adenofibroma (20) can be differentiated on the basis of negative immunoreactivity to actin or desmin and the lack of cystic component. in summary, we report a case of renal tumor showing unique histologic features of biphasic epithelial and stromal component and briefly discuss the differential diagnosis and its relationship with mesoblastic nephroma. | the descriptive term " mixed epithelial and stromal tumor of the kidney " was recently proposed for a group of renal tumors characterized histologically by a mixture of stromal and epithelial proliferation. it is a rare benign neoplasm of the kidney which has been reported under various names such as adult type mesoblastic nephroma or others. we report a case of mixed epithelial and stromal tumor in a 47 yr old female patient presenting as a partly cystic and partly solid renal mass. microscopically, the tumor exhibited spindle cell component in solid portion and epithelial proliferation around microcystic areas. immunoreactive profiles and ultrastructural examination suggested myofibroblastic nature of the stromal cells. we believe this case exemplifies a unique adult renal tumor displaying both epithelial and stromal neoplastic component and has a few unusual features worthy of attention. |
recurrent microdeletions and microduplications of a 600 kb genomic region of chromosome 16p11.2 have been implicated in childhood - onset developmental disorders1 - 3. here we report the strong association of 16p11.2 microduplications with schizophrenia in two large cohorts. in the primary sample, the microduplication was detected in 12/1906 (0.63%) cases and 1/3971 (0.03%) controls (p=1.210 - 5, or=25.8). in the replication sample, the microduplication was detected in 9/2645 (0.34%) cases and 1/2420 (0.04%) controls (p=0.022, or=8.3). for the series combined, microduplication of 16p11.2 was associated with 14.5-fold increased risk of schizophrenia (95% c.i. [3.3, 62 ]). a meta - analysis of multiple psychiatric disorders showed a significant association of the microduplication with schizophrenia, bipolar disorder and autism. the reciprocal microdeletion was associated only with autism and developmental disorders. analysis of patient clinical data showed that head circumference was significantly larger in patients with the microdeletion compared with patients with the microduplication (p = 0.0007). our results suggest that the microduplication of 16p11.2 confers substantial risk for schizophrenia and other psychiatric disorders, whereas the reciprocal microdeletion is associated with contrasting clinical features. |
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diverticular disease primarily involves the sigmoid colon, followed by the ascending colon and cecum. rectal involvement is extremely rare, with only a few reports in the literature since 1911. surgical intervention is necessary when the patient suffers from a diverticular complication. in this case, an emergent laparotomy following an abdominal perineal resection (apr) and permanent colostomy was performed because of an impending bowel perforation caused by a mucinous adenocarcinoma within the rectal diverticulum. a 77-year - old man with a 10-day history of chronic constipation and abdominal distension was seen at the emergency room. the patient 's medical history included right hemiparesis due to left basal ganglia infarction 8 years prior and systemic hypertension. on physical examination, he had abdominal distension with mild tenderness ; however, he did not have rebound tenderness and his bowel sounds were increased. on digital rectal examination, there was an edematous soft mass - like lesion at a one - finger depth from the anal verge which was not tinged with blood. laboratory studies revealed that the white blood cell count, hemoglobin, electrolytes, and liver function tests were within the normal range. the level of carcinoembryonal antigen was 17.0 ng / ml (normal range, 0 to 6 ng / ml), and that of carbohydrate antigen 19 - 9 was 99.3 u / ml (normal range, 0 to 37 u / ml). a contrast enhanced computed tomography scan was suggestive of rectal cancer with a diffuse wall thickening that was 7 cm in length and was located 5 cm above the anal verge. the small bowel and colon were dilated with fecal retention due to a mechanical obstruction proximal to the mass lesion (fig. endoscopic biopsy showed only an inflammatory change ; however, we clinically suspected rectal cancer, so additional pelvic magnetic resonance imaging (mri) was performed. the pelvic mri revealed a submucosal infiltrating enhanced lesion involving the rectum and containing round and tubular cystic lesions with perirectal fat infiltration (fig. a positron emission tomography scan showed rectal cancer because of increased fluorine-18 2-fluoro-2-deoxy - d - glucose uptake (max standardized uptake value, 7.7). because of negative biopsy results and radiologic findings that showed a submucosal infiltrating enhanced lesion, we suspected that the tumor was most likely a primary rectal linitis plastica and recommended surgical treatment for the patient. however, the patient declined surgery without pathologic confirmation and chose close observation and symptomatic treatment. one month later, he returned to the emergency room with recurrent abdominal distension and severe constipation. an emergent apr and permanent colostomy was performed due to signs of impending bowel perforation. during surgery which lasted for 5 hours, there were no special events, but pelvic dissection was difficult due to the infiltrative tumor mass and neighboring fibrosis. pathologic examination of the resected tissue revealed a mucinous adenocarcinoma in the rectal diverticulum involving the rectum (4.2 2 1.5 cm) with extension to perirectal soft tissue ; however, no lymphovascular perineural invasion or distant metastasis was detected (fig. the cancer was located within a diverticulum, but the entrance of the diverticulum was not seen during endoscopy (fig. the negative endoscopic biopsy result was due to the fact that the mucous membrane was unaffected by the adenocarcinoma within the rectal diverticulum. after surgery, the patient received adjuvant radiation therapy, and no recurrence has been detected for 12 months. involvement of the entire colon occurs in up to 10% of the cases, with between 2 to 10% being limited to the transverse or ascending colon. however, rectal involvement is extremely rare. first, the muscle fibers of the taenia coli spread outward, thus surrounding the rectum and protecting it against intraluminal pressures. secondly, less constant internal pressure is exerted on the rectum by accumulated feces and by a lower peristaltic activity as compared with the sigmoid colon. the causes of rectal diverticula remain unknown ; possible predisposing factors include congenital anomalies (e.g., weakness in the circumferential muscle that surrounds the rectum), primary muscle atrophy, or the absence of supporting structures (e.g., the coccyx). in this case, most patients that are symptomatic have a concurrent rectal diverticulum, as in the present case. rectal diverticula may be inflamed with impacted feces and can progress to abscess formation and perforation. other reported complications include rectal strictures, fistula development, and rarely rectal prolapse because of a large inverted diverticulum. although colonic diverticulosis and cancer are common diseases in patients over 60 years of age, cancer arising within a diverticulum is rare. the possibility of a casual relationship between diverticulitis and cancer of the colon has been suggested. some investigators have attempted to show an increased incidence of colonic carcinomas in patients with diverticulitis, suggesting that chronic inflammation leads to metaplasia and neoplastic changes. however, others have suggested that no cause - effect relationship exists between an adenocarcinoma and diverticular disease of the colon based on the following evidence : 1) the rarity of adenocarcinomas arising within the diverticular mucosa, 2) the absence of occasional dysplasia in the diverticular mucosa, and 3) the often documented occurrence of adenocarcinomas in areas with no diverticula. to the best of our knowledge, only one case of an adenocarcinoma in a rectal diverticulum has been reported in japan. this report was the first case of a symptomatic mucinous adenocarcinoma within a rectal diverticulum, which caused a mechanical obstruction. during colonoscopic examination, the rectal diverticulum was not visible because the orifice of the diverticulum was narrow and collapsed. in this case, the diagnosis was difficult because radiographic features and endoscopic findings of the lesion were similar to those of a primary rectal linitis plastica ; however, the cancer was diagnosed at a relatively early stage because the mucinous adenocarcinoma secreted a thick mucinous material into the submucosal layer, which caused a colonic obstruction before perforating the diverticular wall. | the occurrence of an adenocarcinoma arising from a rectal diverticulum that causes mechanical ileus is very rare. recently, we diagnosed a case of a mucinous adenocarcinoma in a rectal diverticulum after an emergent abdominal perineal resection and permanent colostomy by laparotomy. here, we present a case report and a review of the literature. |
the study adhered to the tenets of the declaration of helsinki and followed the guidelines, which the ethics committee of our institution approved. this study included 169 consecutive patients who were native japanese (73 men and 96 women ; age, mean sd, 59.0 11.1 years) and had type 2 diabetes between april 2001 and march 2005. diabetes was diagnosed based on the criteria of the american diabetes association (8). if the patients were treated with insulin or oral hypoglycemic agents or if the fasting blood glucose value exceeded 126 mg / dl, they were considered to have diabetes. if the blood pressure (bp) exceeded 140/90 mmhg or if they used antihypertensive drugs, the patients were considered to have hypertension (9). dyslipidemia was diagnosed in patients with serum ldl cholesterol levels 140 mg / dl and/or hdl cholesterol levels 300 mg / g creatinine (or dipstick urinalysis showing 2 +, 3 +, or 4 +) (11). the serum creatinine was measured within 4 h of fasting venous blood collection using a hitachi 747 biochemistry analyzer (hitachi high - technologies corporation, tokyo, japan). renal function also was evaluated based on the estimated glomerular filtration rate (egfr), which was calculated using a previously reported formula (12). the following equation originated from the modification of diet in renal disease study group compiled for japanese individuals and was recommended by the japanese society of nephrology : egfr (ml / min/1.73 m) = 194 scr age 0.739 (if female). the ckd stages were based on the national kidney foundation disease outcomes quality initiative clinical practice guidelines (13). the absence of ckd was defined as with no microalbuminuria and an egfr exceeding 90 ml / min/1.73 m. in the current study, we recruited patients with type 2 diabetes, no or minimal diabetic retinopathy, and no or microalbuminuria. in addition, patients with poorly controlled diabetes (hba1c > 10.0%), uncontrolled hypertension (bp > 160/100 mmhg), and severe anemia (hemoglobin 20/20) and intraocular pressure (iop) levels that were within the normal range (< 20 mmhg). after the pupils were dilated with a 0.5% tropicamide eye drop, an ophthalmologist, who was masked to the rbf measurements, assessed the retinopathy. for each eye, photographs were taken with the topcon trc - nw6s (topcon, tokyo, japan), and the maximal grade in any of the seven standard photographic fields was determined for each lesion and used to define the retinopathy levels (14). the severity of retinopathy was categorized as none (level 10), mild nonproliferative (levels 2137), moderate - to - severe nonproliferative (levels 4353), or proliferative (levels 6065) (15). patients with moderate - to - severe nonproliferative diabetic retinopathy and proliferative diabetic retinopathy and clinically relevant macular edema were excluded. one eye of a patient was included in the study if it had worse retinopathy that met the inclusion criteria. if both eyes of a patient had retinopathy of equal severity, one eye was assigned randomly to the study. the ophthalmologic exclusion criteria included a previous intraocular surgery, a history of laser photocoagulation, moderate - to - severe cataract, vitreous hemorrhage, tractional retinal detachment, and moderate - to - high refractive error (greater than 3.0 diopters). the participants had abstained from drinking coffee for at least 12 h before the rbf was measured. we used a retinal ldv system (model clbf 100, laser blood flowmeter ; canon, tokyo, japan) to estimate the blood flow in the superior branch of the first - order major temporal retinal artery. the system noninvasively measures the absolute values of the erythrocytes flowing in the centerline of the vessel, based on the bidirectional ldv (16). the mean retinal blood velocity (vmean) was defined as the v of the averaged maximal speed during one cardiac cycle. the diameter of the retinal artery also was determined automatically by computer analysis of the signal produced by the arterial image on the array sensor using the half height of the transmittance profile to define the vessel edge (16). the rbf was calculated as rbf = vmean area, where the vmean was calculated as vmean = v of the averaged maximal speed 2, and the area was the cross - sectional area of the retinal artery at the laser doppler measurement site (16). the mean arterial bp was determined by the following formula : diastolic bp + (systolic bp diastolic bp) 3 (16). all values are expressed as the mean sd. the assumption of data normality was assessed using the shapiro - wilk test. comparisons between groups were made using one - way anova (for continuous variables) and the test (for categorical variables). one - way anova was followed by a post hoc comparison with tukey - kramer procedure. standardized regression coefficients from multiple regression analysis of the rbf in relation to various factors were analyzed. all analyses were conducted using spss statistical software, version 16 (chicago, il). the participants had abstained from drinking coffee for at least 12 h before the rbf was measured. we used a retinal ldv system (model clbf 100, laser blood flowmeter ; canon, tokyo, japan) to estimate the blood flow in the superior branch of the first - order major temporal retinal artery. the system noninvasively measures the absolute values of the erythrocytes flowing in the centerline of the vessel, based on the bidirectional ldv (16). the mean retinal blood velocity (vmean) was defined as the v of the averaged maximal speed during one cardiac cycle. the diameter of the retinal artery also was determined automatically by computer analysis of the signal produced by the arterial image on the array sensor using the half height of the transmittance profile to define the vessel edge (16). the rbf was calculated as rbf = vmean area, where the vmean was calculated as vmean = v of the averaged maximal speed 2, and the area was the cross - sectional area of the retinal artery at the laser doppler measurement site (16). the mean arterial bp was determined by the following formula : diastolic bp + (systolic bp diastolic bp) 3 (16). all values are expressed as the mean sd. the assumption of data normality was assessed using the shapiro - wilk test. comparisons between groups were made using one - way anova (for continuous variables) and the test (for categorical variables). one - way anova was followed by a post hoc comparison with tukey - kramer procedure. standardized regression coefficients from multiple regression analysis of the rbf in relation to various factors were analyzed. all analyses were conducted using spss statistical software, version 16 (chicago, il). we divided the patients with type 2 diabetes with normoalbuminuria (dn stage 1 ; n = 120) or microalbuminuria (dn stage 2 ; n = 49) into four groups based on the ckd stage (non - ckd, n = 99 ; ckd stage 1, n = 22 ; stage 2, n = 27 ; stage 3, n = 21). no patient had stage 4 or 5 ckd. although the group - averaged values of age and duration of diabetes were significantly higher in stage 2 ckd compared with the other groups, there were no significant differences in the hba1c, systemic bp, heart rate, iop, hemoglobin, total cholesterol, triglyceride, hdl, or ldl among the groups. the serum creatinine and egfr were higher in stage 1 ckd but lower in stages 2 and 3 ckd compared with the non - ckd group (table 1). we found significant decreases in the vessel diameter (p = 0.017) and rbf (p = 0.035) in stage 3 ckd compared with those in the non - ckd group ; however, there were no differences in the retinal arteriolar blood velocity among the groups (table 2). in the current study, 47 (28%) patients had mild retinopathy in at least one eye. there were no significant differences in retinal circulatory parameters (vessel diameter, velocity, and rbf) between the no retinopathy group and the mild nonproliferative diabetic retinopathy group (data not shown). in addition, there also were no significant differences in the retinal circulatory parameters between dn stages 1 and 2 (data not shown). multiple regression analysis was performed to determine whether the ckd stage was an independent variable related to the rbf in patients with type 2 diabetes. table 3 shows the multiple regression analysis of the rbf in relation to the renal stage of ckd, serum ldl, mean arterial bp, and hba1c, based on our previous findings (5). this analysis confirmed that the ckd stage was a significant and independent variable associated with the rbf in these patients (p = 0.02) (table 3) in addition to serum ldl and mean arterial bp. characteristics of patients with type 2 diabetes with early - stage retinopathy retinal circulatory parameters in the study groups multiple regression analysis of rbf in patients with type 2 diabetes although diabetic retinopathy is associated closely with the preclinical morphologic changes of dn (17), about one - third of patients with diabetes have decreased kidney function without either albuminuria or retinopathy (18). these findings suggested that the relation between renal dysfunction and impaired retinal microcirculation remains unclear, especially in early - stage diabetes. we recently reported that the rbf decreases in patients with type 2 diabetes without retinopathy and with mild retinopathy (5). the current study also found that the vessel diameter and rbf in the retinal arterioles decreased in patients with type 2 diabetes with stage 3 ckd and normo / microalbuminuria, suggesting that impaired renal dysfunction might be associated with decreased retinal microcirculation in early - stage type 2 diabetes. taken together, we hypothesized that impaired retinal and renal microcirculation might be involved with the common pathogenesis of diabetic microvascular complications, especially in early - stage type 2 diabetes. (19) reported that higher baseline c - reactive protein (crp) was associated with decreased egfr in patients with hyperlipidemia and a history of myocardial infarction, suggesting that chronic low - grade inflammation might be associated with renal dysfunction. although we did not measure the serum level of crp in patients with diabetes in the current study, we reported previously that the crp impaired retinal endothelial function in isolated porcine retinal arterioles (20). because ckd is associated with low - grade inflammation, endothelial dysfunction, and platelet activation, even among patients with moderate renal impairment (21), chronic inflammation might be associated with the pathogenesis of diabetic microvascular complications, including retinopathy and nephropathy, in patients with type 2 diabetes. previous epidemiologic studies have reported that renal dysfunction might have some effect on ocular disorders in patients without diabetes (6,7). in addition, recent clinical studies have reported that the vessel calibers of retinal arterioles and venules decreased progressively with each ckd stage of renal failure (22,23), suggesting that progression of renal dysfunction might be associated with retinal vessel constriction. although those studies did not include patients with diabetes, the findings seemed to support the current finding that vessel diameter and blood flow in the retinal arterioles were significantly lower in the patients with stage 3 ckd compared with those without ckd in patients with type 2 diabetes and no or minimal retinopathy. the exact mechanism by which vasoconstriction of the retinal arterioles and decreased rbf without any change in blood velocity in patients with ckd stage 3 might be associated with renal dysfunction remains unclear. ckd is associated with low - grade inflammation, platelet activation, and endothelial dysfunction even among patients with moderate renal impairment (21). indeed, the reduced endothelium - dependent dilation in the brachial artery of patients with ckd is related to the severity of the renal failure (24). evidence indicates that oxidative stress occurs early in the course of ckd and is amplified with disease progression (3). in addition, the nitric oxide (no) production decreases in renal disease due to impaired endothelial function and renal no production (25). because we previously reported that no has an important role in vasoregulation in rbf (26), the decreased no production in patients with diabetes with ckd might be involved with constriction of the retinal arterioles and decreased rbf seen in the current study. moreover, the current data do not definitively explain why there were no significant differences in blood velocity among the four diabetic groups. based on our previous findings that the blood velocity in the retinal arterioles decreased in patients with type 2 diabetes with no and/or minimal retinopathy compared with healthy subjects (5), we speculated that the blood velocity in the retinal arterioles decreased in all patients with diabetes independent of the ckd stage without a further decrease according to the ckd stage. in the current study, the multivariable regression model included serum ldl, mean arterial systemic bp, and hba1c, which we selected based on previous findings (5), and suggested that the ckd stage, serum ldl, and systemic bp are independent risk factors for rbf in our patients. because ldls specifically impair endothelium - dependent vasodilation by reducing no synthesis and producing superoxide anion in isolated coronary arterioles (27), the increased serum ldl might be involved with the decreased rbf via the reduced no production in the retinal arterioles, resulting in constricted retinal arterioles. at the onset of diabetes, the kidney enlarges along with glomerular hypertrophy, and the gfr becomes supranormal (28,29). hypertrophy also contributes to cellular oxidative stress, which might precede the reactive oxygen species perturbation (30). this eventually leads to gradual kidney deterioration with reduced gfr and subsequently results in sclerosis and kidney failure. moreover, renal arterial resistance plays an important role in deteriorating renal function in patients with diabetes (31). in the current study, we found that the egfr increased in the group with stage 1 ckd with microalbuminuria compared with the group with no ckd, whereas there was no difference in rbf between the non - ckd and stage 1 ckd groups, suggesting a possible discrepancy in the changes in the retinal and renal microcirculation in early - stage type 2 diabetes with microalbuminuria. because the retinal microvasculature can be visualized directly, noninvasive measurement of the rbf using an ldv system might be a good indicator of general microvascular dysfunction in patients with type 2 diabetes. a prospective study is needed to identify the interaction of the pathogenesis between retinopathy and nephropathy in type 2 diabetes. we also did not examine the gfr directly but estimated it from the serum creatinine. moreover, all patients in the current study were japanese, which might have affected our results, because asian patients with type 2 diabetes have a high prevalence of albuminuria, reportedly 60% (32). moreover, we included only patients with early - stage diabetic retinopathy and nephropathy, and therefore could not comment on the retinal hemodynamics in patients with type 2 diabetes with late - stage diabetic microvascular complications. in addition to the current findings obtained from patients with early - stage renal dysfunction, we previously reported a negative linear correlation between the changes in systemic bp and the rbf induced by hemodialysis in patients with end - stage renal disease (33), suggesting that autoregulation of the rbf might be impaired in these patients. the current finding that the systemic bp is an independent risk factor for rbf in our multivariable regression model (table 3) might be associated with impaired autoregulation of the rbf in patients with diabetes with renal dysfunction. another clinical study is needed to elucidate the relation between renal function and retinal microcirculation in the advanced stages of diabetic microvascular complications. finally, we used only one method to quantify the changes in rbf and had no technique to measure the renal blood flow. although some larger - scale studies have reported that the vessel diameter in the retinal arterioles is reduced when using other techniques to evaluate the vessel diameter, which seems to be in line with current results, further study is warranted to substantiate these changes in blood flow and vessel diameter in the retinal arterioles by using other techniques to evaluate the retinal vessel diameter and retinal blood flow in these patients. in conclusion, our results indicated that the rbf decreases in patients with type 2 diabetes with stage 3 ckd, suggesting that impaired renal function might be associated with decreased rbf in early - phase diabetic retinopathy. because the ldv system enables noninvasive and quantitative evaluation of rbf, evaluation of the retinal microcirculation is likely useful for detecting early changes in the microvasculature in patients with type 2 diabetes. | objectiveto study the relationship between retinal microcirculation and renal function in patients with type 2 diabetes.research design and methodsusing a laser doppler velocimetry system, we obtained the retinal blood flow (rbf) values by simultaneously measuring the retinal vessel diameter and blood velocity. to determine if the rbf is affected in the presence of renal dysfunction, we also evaluated the renal function using the estimated glomerular filtration rate calculated by age and serum creatinine level.resultswe recruited 169 eyes of 169 consecutive japanese patients with type 2 diabetes, no or minimal diabetic retinopathy, and normo / microalbuminuria (mean age sd, 59.0 11.1 years). we divided the patients into four groups based on the stage of chronic kidney disease (ckd) (non - ckd, n = 99 ; ckd stage 1, n = 22 ; stage 2, n = 27 ; stage 3, n = 21). we found significant (p = 0.035) decreases in rbf with decreased vessel diameter (p = 0.017) but no difference in blood velocity (p = 0.54) in stage 3 ckd compared with the non - ckd group. multiple regression analysis showed that the ckd stage was significantly (p = 0.02) and independently associated with decreased rbf.conclusionsour results indicated that the vessel diameter and rbf in the retinal arterioles decrease in patients with type 2 diabetes with stage 3 ckd, suggesting that impaired renal function might be associated with decreased rbf, probably via constriction of the retinal arterioles, in early - phase diabetic retinopathy. |
(2n = 22) is a major grain legume of the arid and semiarid regions of the world. though considered a minor crop, pigeonpea is of considerable importance in areas of south asia (mainly on the indian subcontinent), africa, the caribbean, and latin america, where it is a prominent source of protein in the human diet, as well as wood for fuel and light duty structural applications such as thatch for roofing. pigeonpea has now moved from an orphan legume crop to one of the promising pluses where genomics - assisted breeding approaches for a sustainable crop improvement are routine by pigeonpea genome initiative, an effort of various researchers. the first pigeonpea est dataset provides a transcriptomic resource for gene discovery and development of functional markers associated with biotic stress resistance. root is the major part of water and nutrition uptake in pigeonpea which has a deep and extensive root system that provides access to water stored deep in the soil profile when that in the surface layer is depleted ; this source of water is particularly important for long duration crops. in order to identify the associated genes in pigeonpea root tissues, a normalized cdna library was constructed from pigeonpea root and expression analysis of the identified genes was carried out by reverse transcriptase pcr (rt - pcr) technique. the pigeonpea genotype, namely, grg-295 was selected to construct cdna library and identification of expressed sequence tags (ests). the seeds of grg-295 pigeonpea genotype were grown in petri dish for 15 days and irrigated with water. at the end of the 15th day, total rna was isolated from root tissues by using the trizol reagent (invitrogen, carlsbad, ca, usa), and mrna was further isolated by using the polyatract mrna isolation system (promega, madison, wi, usa). the quantification of rna was verified by absorption ratio of od260/280 and by formaldehyde gel electrophoresis. the first and second strands of cdna were synthesized using clontech smarter pcr cdna synthesis kit. the cdnas were purified by the minelute pcr purification kit (qiagen, valencia, ca, usa) and ligated into a pgem - t easy vector (promega, madison, wi, usa). ligated plasmid dnas were used for transformation into competent e. coli dh5 strain. positive clones were selected on an ampicillin / iptg / x - gal lb plate. plasmid dna from positive clones were isolated by using real 96 plasmid isolation kit (qiagen, netherlands), and purified dna was used for single - pass sanger sequencing by using m13f / r universal sequencing primers on abi sequencing machine 3500xl genetic analyzer. all the ests were processed using vecscreen (http://www.ncbi.nlm.nih.gov/vecscreen/vecscreen.html) to remove vector and cloning oligo sequences and various contaminants to trim a high quality region. based on the qualified sequences, the predicted amino acid sequences were used to search for similar peptide sequences to search for similar protein sequences in public database ncbi (http://www.ncbi.nlm.nih.gov) using the blastx search algorithm by using default parameters of the program. the similarity scores between the cdna clones and known sequences were represented by blastx probability e values. further the ests were classified into different functional categories based on the knowledge of biochemistry, plant physiology, and molecular biology (http://www.metacyc.org/), go (http://www.ebi.ac.uk/goa/), and cog (http://www.ncbi.nlm.nih.gov/cog/) tools and by searching related abstracts in pubmed. total root rnas isolated from pigeonpea root tissues were used for reverse transcription polymerase chain reaction (rt - pcr) analysis. genomic dna contamination was removed by dnase i. first - strand cdna was synthesized from each 2 g of total rna sample using clontech smarter pcr cdna synthesis kit according to the manufacturer 's protocol. the cdnas were purified using a commercial column (qiagen). to determine the expression of candidate genes, pcr was performed with 2 l of the first - strand cdna template and gene - specific primer pairs. gene - specific rt - pcr primers were designed with primer 3.0 according to the est sequences and were synthesized commercially. general pcr was conducted with annealing as required for the specific primer pairs (table 1). rt - pcr experiments were repeated three times, and the pcr products were detected on 1.5% agarose gel. plant root systems serve a number of important functions, including anchoring the plant, absorbing water and nutrients, producing amino acids and hormones, and secreting organic acids, enzymes, and alkaloids. the physiological significance of roots is belied by their relative structural simplicity as compared to other plant organs ; major metabolic pathways such as photosynthesis lacking in root tissues have a stereotypical morphology that is conserved across taxa and throughout the life cycle of individuals. this combination of physiological relevance and structural simplicity has made roots obvious targets for functional genomics analyses. as a major grain legume of semiarid tropics and a deep and extensive root system of pigeonpea represents an excellent source of identification of ests associated with their root tissues. so, the present work was focused on the study of genes associated with pigeonpea root tissues. in the present investigation, the cdna library was constructed in order to identify ests associated with pigeonpea roots and their functional analysis was carried out. the total rna from the pigeonpea root tissue was isolated and the first and second cdna strand was synthesized. the presence of the gene in plasmid construct of colonies was confirmed by colony pcr. the colony pcr showed that the size of these inserts ranged from 200 to 800 bp. out of 400 bacterial clones, the plasmid construct of 250 positive recombinant clones was sequenced in single passed sequencing reaction from 3 end using m13 forward / reverse primer and the sequence data was subjected to blast analysis. the leading sequences, tailing of the sequence, and poor quality sequences were excluded firstly. finally, 105 high quality ests were retained which were clustered into 72 unigenes comprising 25 contigs and 47 singlets (table 2) and were compared with ncbi nonredundant protein database using blastx algorithm and default parameters. in blastx analysis, it was shown that most of the sequences were having a significant homology with known proteins. sequences that had no significant homology with protein database were compared to nucleotide blast using default parameters. the ests were deposited to ncbi dbest with the accession number of jk973637 to jk973741 (table 3). the ests were categorized into 8 diverse functional group consisting of 10% transporter genes, 6% signal transduction genes, 20% cell growth and transcriptional regulator genes, and 26% metabolism genes. in other genes, 8% genes were uncharacterized, 8% hypothetical genes, 10% genes with no significant match, and 14% genes was involved in other functions. there were 8% of the ests found that did not show any match to known proteins in blastx program and that suggest novel nature of those genes (figure 1). in order to validate the ests generated from cdna library, the expression of the four genes which were involved in different metabolic pathways was analysed by rt - pcr. rt - pcr results showed that the expression levels of four candidate genes, namely ; s - adenosylmethionine synthetase, phosphoglycerate kinase, serine carboxypeptidase, and methionine aminopeptidase, were clearly expressed in pigeonpea roots (figure 2). it was concluded that overall, there was a good agreement between the cdna library data and the rt - pcr results. s - adenosylmethionine synthetase (sams) comprises of two cdnas in pinus contorta among which sams1 is expressed in roots and exhibits a specific expression pattern in the meristem at the onset of adventitious root development. sams also catalyses the nucleophilic substitution reaction from between methionine and atp into s - adenosylmethionine which have central role in several biological process in plants, namely, methyl group donor in trimethylation of lignin, dna, and alkaloids as well as donor of aminopropyl moieties in ethylene and polyamine synthesis [8, 9 ]. phosphoglycerate kinase superfamily has a diverse function in numerous metabolic processes like generation of precursor metabolites and energy, carbohydrate metabolism, phosphorous metabolism, glycolysis, kinase activity, atp binding activity, and so forth. the presence of phosphoglycerate kinase transcripts in the cdna library supports its diverse function in pigeonpea root. serine carboxypeptidase differentially expressed in root and other tissues is responsible for the synthesis of sinapoylcholine and sinapoylmalate in arabidopsis which encodes 51 proteins annotated as serine carboxypeptidase - like enzymes and emerged as a new group of acyltransferase enzymes that are able to modify plant natural products [1012 ]. methionine aminopeptidase, a ubiquitous enzyme, differentially expressed in root tissues is one of the central enzymes in protein synthesis that catalyzes n - terminal methionine from proteins. a few important ests, namely, type 2 metallothionein, tbp - associated factor, abc transporter, and phosphatidylcholine transfer protein, were also abundantly found in the cdna library. reddy. found abundant metallothionein genes in normalized library from rice leaves and suggested that they might perform essential functions of plant growth beside metal detoxification. tata - binding protein and tbp - associated factors are transcriptional factors which are predominantly involved in rna polymerase ii mediated transcription process. abc transporters play an important role in organ growth, plant nutrition, plant development, response to abiotic stress, and the interaction of the plant with its environment. phosphatidylcholine is usually the most abundant phospholipids in animals and plants, often amounting to almost 50% of the total, and as such it is obviously the key building block of membrane bilayers making up a very high proportion of the outer leaflet of the plasma membrane. similarly numerous ests like glycine dehydrogenase, signal proteins, root nodule extensions, membrane proteins, and -glucosidase were observed in the cdna library constructed from pigeonpea root tissues, which is thought to play possible roles in plant metabolism, growth, and development. apart from the known ests, 6 est transcripts (jk973668, jk973685, jk973701, jk973714, jk973715, and jk973718) were observed as not having any significant match in ncbi database. along with that uncharacterized and hypothetical proteins were also observed in cdna library which were thought to impart in the cardinal role in root tissues of pigeonpea. our present investigation contains a precise repertoire of transcripts associated with the various metabolic functions in pigeonpea root. these ests appear to be involved in multiple metabolism pathways in the plant 's physiological and biochemical processes. in addition to known genes, some ests were unknown and uncharacterized, whose functional roles remain unclear and require further investigation in future. the root transcriptome characterized in this study markedly these est tags may be useful for functional gene annotation, analysis of splice site variants and intron / exon determination, and evaluation of gene homologies or kegg pathway confirmation. | pigeonpea (cajanus cajan (l) millsp.) is an important food legume crop of rain fed agriculture in the arid and semiarid tropics of the world. it has deep and extensive root system which serves a number of important physiological and metabolic functions in plant development and growth. in order to identify genes associated with pigeonpea root, ests were generated from the root tissues of pigeonpea (grg-295 genotype) by normalized cdna library. a total of 105 high quality ests were generated by sequencing of 250 random clones which resulted in 72 unigenes comprising 25 contigs and 47 singlets. the ests were assigned to 9 functional categories on the basis of their putative function. in order to validate the possible expression of transcripts, four genes, namely, s - adenosylmethionine synthetase, phosphoglycerate kinase, serine carboxypeptidase, and methionine aminopeptidase, were further analyzed by reverse transcriptase pcr. the possible role of the identified transcripts and their functions associated with root will also be a valuable resource for the functional genomics study in legume crop. |
the macrofungi - the mushrooms are eukaryotic, non - photosynthetic, and aerobic organisms that form characteristic fruiting bodies. belonging to the fifth kingdom, all mushrooms are heterotrophic, and they assimilate nutritive substances by absorption of simple molecules as nutrients after complex organic polymers such as celluloses are degraded by extracellular enzymes secreted by them. the mushrooms are taxonomically classified in two different groups : basidiomycetes, which comprise many of the known genera and ascomycetes. edible and medicinal mushrooms, which can be commercially produced, are cultivated on lignocellulosic agricultural residues such as straw, wood chips, and sawdust. mushrooms have been used by humans since thousands of years as food and/or as medicine. more than 14,000 species of mushrooms are recognized, and among them, approximately 2000 are identified as edible. of the 2000 edible mushrooms in 30 genera, 270 species are now considered as potential therapeutic or preventative agents that may ensure wellness of humans. today, the increasing consumption of mushrooms can be attributed not only to the pleasant flavor and aroma of culinary mushrooms but also to the current search for natural products, which are free of pesticides ; food as nutraceuticals or functional food with high vitamins and protein contents as well bioactive activities including antioxidant activities. the culinary mushrooms can be excellent components in our daily diet, because they are rich in proteins, fibers, carbohydrates, vitamins, and minerals. while at the same time, mushrooms are low in calories and fats including cholesterol. in the recent years to unravel the mysteries of mushrooms, especially those used in traditional medicines, mushrooms are being investigated for their many ethnomycological claims of medicinal value. the mushrooms are being developed as nutraceuticals or nutriceuticals to garner the essence of mushrooms and to make consumption easy. further, scientific validation of traditional knowledge bears evidence of the many positive effects of consuming mushrooms fresh or processed on human health. in the words of chang : without leaves, without buds, without flowers ; as a food, as a tonic, as a medicine ; the entire creation is precious. as the recognition of mushrooms as possible food or supplements that can improve health and enhance quality of life increases, numerous products are marketed with claims often based on ethnomycological knowledge. in an attempt to scientifically validate the numerous claims of the health benefits of mushrooms, related research however, at this moment, many of the studies on the efficacy of medicinal mushrooms that are available to the public are based on animal studies (usually in mice) or cultured cells. there is a need to show efficacy in humans via clinical trials. in china, complementary and alternative medicine is permitted as an alternative therapy for life - debilitating diseases, and mushroom nutriceuticals or mushroom functional foods are included in cancer and other life - threatening diseases management. one of the many diseases that threaten humans, neurodegenerative diseases can be very traumatic as one ages. neurohealth is the concern for the predicted silver tsunami to hit humans the aging tsunami is projected to be 80 - 90 million of 65 plus population in 2050. why the concern ? a large percentage of this aging population (projected at about 80%) will have at least one chronic health - related diseases. alzheimer and neurodegenerative diseases are high on the list of chronic diseases of the aged. this disease is characterized biologically by the death of neurons in the forebrain, hippocampus, and cerebral cortex accompanied by the presence of amyloid deposition. the symptoms caused by the death of cholinergic neurons are treated with drugs that enhance neurotransmission at cholinergic synapses. the drugs include aricept[r ] by pfizer, exelon[r ] by novartis, reminyl[r ] by janssen, and cognex[r ] by first horizon. these drugs only delay further deterioration and do not reverse the damage done to cognitive functions. another drug memantine (forest laboratories), which blocks the receptor for the glutamate neurotransmitter which may be responsible for the neurotoxicity in alzheimer 's disease, had temporary beneficial effects, too. once the clinical symptoms are manifested, it will be only possible to arrest or delay further degeneration process. the strategy will be to look for agents that slow down and prevent further damage to the vital cells involved. the search is now for small molecules that can cross the brain - blood and induce the production of nerve growth factor (ngf), a family of proteins responsible for maintenance, survival, and regeneration of neurons during adult life. it has been shown that ngf absence caused an alzheimer's - like symptom in the adult brain of mice.. we may have to turn to traditional knowledge that taps nature for medicine to prevent or reduce the severity of nerve - related diseases as we age. for example, in india, it is noted that alzheimer among the older generation is not in alarming numbers. the regular consumption of spices including turmeric and pepper may be the reason, and currently, this is actively been studied. almost all culinary mushrooms are noted for their polysaccharide components, which play a role in stimulation of immune system and possible applications in cancer management. many edible genera, however, are also noted for their specific activities. among the special activities, a number of mushrooms including sarcodon scabrosus, ganoderma lucidum, grifola frondosa, and hericium erinaceus are reported to have activities related to nerve and brain health. in this paper, the potentials of mushrooms as nutraceutical or nutriceuticals to help in the reduction or even prevention of age - related neurodegenerative diseases are discussed. when rat pheochromocytoma cells (pc12) were treated with scabronines a and g, there was neurite outgrowth. extracts of g. lucidium contained neuroactive compounds that induced neuronal differentiation and prevented ngf - dependent apoptosis of rat pheochromocytoma pc12 neuronal cells. these effects are probably mediated through the ras / erk and camp - response element - binding protein (creb) signaling pathways, because g. lucidum extracts induced phosphorylation of erk 1, erk 2, and creb. the culinary mushroom that has been extensively studied for its neurohealth properties is h. erinaceus (lion 's mane mushroom). the polysaccharides in an aqueous extract of the lion 's mane mushroom could induce neuronal differentiation and promote neuronal survival. the chemical profile of extracts of this mushroom has been actively studied and reported by kawagishi and co - researchers. extracts of h. erinaceus induced the expression of neurotrophic factors such as ngf in astrocytes. in their studies, hericenones were isolated from the fruiting bodies of lion 's mane while erinacines were isolated from the mycelium of the mushroom. endoplasmic reticulum stress - attenuating compounds and dilinoleoyl phosphatidylethanolamine, a phospholipid with linoleic acid, an unsaturated fatty acid, purified from extracts of dried fruiting bodies of h. erinaceus, reduce endoplasmic reticulum stress - induced cell death. investigated the neuroprotective effect of h. erinaceus on the ischemic brain damage in a middle cerebral artery occlusion model in mice. hericium erinaceus (300 mg / kg, continuously fed for 14 days after surgery) significantly decreased the size of the cerebral infarcts one day after the occlusion. in another study, a double - blind trial was conducted with 50- to 80-year - old japanese men and women diagnosed with mild cognitive impairment in order to examine the efficacy of oral administration of h. erinaceus, for improving cognitive impairment, using a cognitive function scale based on the revised hasegawa dementia scale (hds - r). the subjects in the h. erinaceus group took four 250 mg tablets containing 96% of yamabushitake dry powder three times a day for 16 weeks. extracts of h. erinaceus induced phosphorylation of c - jun n - terminal kinase (jnk) and its downstream substrate c - jun, and increased c - fos expression, suggesting that h. erinaceus promotes nerve growth factor gene expression via jnk signaling. furthermore, the efficacy of h. erinaceus in vivo has been examined. mice given feed regime containing 5% (w / w) h. erinaceus dry powder for seven days showed an increase in the level of ngf mrna expression in the hippocampus. these studies are very encouraging, and the consumption of h. erinaceus - fresh or processed is acceptable. however, there should be guidelines and monitoring to ensure safety, efficacy, and other parameters to ensure that these preparations are functional as claimed. though hericium erinaceus is a temperate mushroom, it can be cultivated in malaysia, a tropical country. the consumers at large do not know the potential of this rather strange - looking mushroom. realizing the potential of this mushroom, research on the locally grown mushroom will help spearhead the growing as well as the consumption of this mushroom in malaysia. a : h. erinaceus ; b : p. giganteus ; c : g. lucidium ; d : l. rhinocerotis ; d : g.neo-japonicum the stimulation of neurite extension using in vitro model using ng 108 - 15 cells by h. erinaceus grown in hot climate was investigated. though the mushroom is now grown at higher temperatures, the unique property of this mushroom is retained. in fact, there was no difference in the neurite outgrowth stimulatory activity between the low- and high temperature - grown mushrooms. this is very important to note, as the commercially available h. erinaceus is oven- dried to increase its shelf life. the extracts of the fruiting bodies dried in an oven was demonstrated to have lost their ability to stimulate neurite outgrowth [figure 1 ]. the effects of an aqueous extract of fruiting bodies of h. erinaceus grown in a tropical climate. a : negative control (without treatment) ; b : ngf (20 ng / ml) ; c : aqueous extract of h. erinaceus (extensive neurite outgrowth) in another study, it was shown that extracts of h. erinaceus can enhance the activity of ngf in ng108 - 15 cells. a combination of 10 ng / ml ngf with 10 g / ml h. erinaceus aqueous extract induced the highest percentage increase of 59.5 5.0% neurite outgrowth compared to untreated neural cells (lai., 2012, unpublished). the effect of neurite outgrowth stimulation was enhanced in this combined mixture when compared to the concentration of extract or ngf applied individually. the neurite outgrowth stimulation activity increased approximately 1.2-fold when compared to treatment with only 10 ng / ml ngf (lai., 2012, unpublished). at higher concentrations, however, the enhancement of neurite stimulation activity of the aqueous mushroom extract and ngf was not observed. further, early observations of an on - going study showed that the mushroom extract triggered ngf secretion in the presence of small amounts of ngf. further studies, especially on mechanism of action at biochemical and molecular levels, are important. this far, there are no reports on toxicity due to long - term consumption of the mushroom. further, a number of reported studies showed that this mushroom has potential to be developed into a functional food. the processing techniques, however, have to be optimized so that maximum activity will be retained to enhance nerve health and possibly nerve regeneration. peripheral nerve injury leads to changes at the axonal site of injury and remotely in the dorsal root ganglia (drg) containing cell bodies of sensory afferent neurons. the regenerative ability of injured axons depends on their capacity to locally synthesize new proteins and to degrade others at the injury site autonomously from the cell body. nerve crush injury is a well - established axonotmetic model in experimental regeneration studies to investigate the impact of various pharmacological treatments. the results indicated that the intensity of nuclear ribonucleoprotein at the injured segments of crushed nerves in rats of treated groups was significantly higher than in negative control group. in addition, the principal signaling pathways that have been demonstrated to be involved in axon regeneration are protein kinase b (akt) and mitogen - activated protein kinase (mapk). akt cascade plays a major role in mediating neurotrophin - promoted cell survival while mapk cascade is involved in mediating neurite outgrowth. a subfamily of mapk, extracellular signal - regulated kinases 1 and 2 (erk1/2), controls cellular differentiation and proliferation. double - labeling immunofluorescence studies showed that small neurons in l5 drg ipsilateral to the crush injury in rats of treated groups expressed higher immunoreactivities for akt and erk1/2 compared to negative control group. these data suggest that daily oral administration of aqueous extract of h. erinaceus fresh fruiting bodies could promote the regeneration of injured rat peripheral nerve in the early stages of recovery. hericium erinaceus, the mushroom associated with brain and nerve health, has been well - studied, and preparations are available. however, there is a paucity of clinical trials to endorse its potential in mitigating / delaying neurodegenerative disorders. further, this mushroom may not be the only one that has beneficial effects on nerve and brain health. to tap the indigenous knowledge in the utilization of the macrofungal diversity of malaysia as food and medicine, the diversity of potential mushrooms used by the indigenous communities are being explored for neurohealth. lignosus rhinocerotis (cooke) ryvarden (plate1d) ; ganoderma neo - japonicum imazeki (plate1e) and pleurotus giganteus (berk.) karunarathna and k.d. hyde (plate 1b) based on indigenous knowledge, selected mushrooms have been used to prepare infusions that are believed to improve vitality, increase alertness and overall wellness of the individual. the sclerotium of l. rhinocerotis (known as tiger milk mushroom) is taken daily or when needed in small amounts to a national treasure mushroom, l. rhinocerotis, has been used to treat variety ailments by local and indigenous communities in malaysia. the mushroom is harvested in the wild and is believed in folklore that it grows where the tiger has spilled its milk. the mushroom, which is rarely encountered in the wild, has now been successfully domesticated. in on - going in vitro investigations, an aqueous extract of the sclerotium of l. rhinocerotis induced neurite outgrowths of 24.4% and 42.1% at 20 g / ml (w / v) of aqueous extract alone and a combination of 20 g / ml (w / v) aqueous extract and 30 ng / ml (w / v) of ngf, respectively, in rat pheochromocytoma cells (pc-12adh). further, a combination of ngf and sclerotium extract had additive effects and enhanced neurite outgrowth. neuronal differentiation was confirmed by indirect immunofluorescence of neurofilament protein. to our knowledge, this is the first report on neurite - stimulating activities of l. rhinocerotis, and it may be a potential mushroom to develop nerve and brain health nutraceuticals or nutriceuticals.. there are on - going studies to determine the chemical components involved and the mechanisms of activity that trigger neurite outgrowth. the mushroom was boiled, and the decoction was consumed to treat fever, epilepsy and to improve body strength (tan., 2012, unpublished). the neurite outgrowth stimulation activity of aqueous extracts of the fruiting bodies of g. lucidum (plate 1c) and g. neo - japonicum were investigated. varying concentrations of the mushrooms and 50 ng / ml (w / v) of ngf as positive control was added to pc-12adh cells. aqueous extract of g. neo - japonicum showed the highest percentage [figure 2 ] of neurite - bearing cells of 14.05 0.66% at 50 g / ml (w / v), followed by g. lucidum (12.08 0.85%) at a higher concentration of 75 g / ml (w / v). the percentage of neurite - bearing cells of g. neo - japonicum obtained with 50 g / ml (w / v) was 73% higher compared to the positive control. it was proposed that g. neo - japonicum contained ngf - like bioactive compound / s for maintaining and rebuilding the neural communications network that may help to prevent neurodegenerative diseases (syntyche ling. arrows indicate neurite extensions. a : negative control - f-12k medium only ; b : positive control - 50 ng / ml (w / v) of ngf ; c : 50 g / ml of g. neo - japonicum neurofilament staining to confirm neurite outgrowth effects of ngf and p. giganteus extracts on neurite outgrowth using pc12 as an in vitro model. p < 0.05 compared to ngf (the positive control) pleurotus giganteus (berk.) karunarathna and k.d. hyde formerly known as panus / lentinus giganteus is a popular mushroom consumed by indigenous communities in malaysia. a variety domesticated in china is being grown in malaysia, and it is a very popular culinary mushroom and has a nutritional profile that warrants its inclusion in human diets. the fruiting bodies of p. giganteus contained per gm per 100 gm : 67.2 carbohydrate, 15.4 protein, and 33.3 dietary fiber. to our knowledge, the medicinal benefits, if any, are yet to be explored. an aqueous extract at 25 g / ml had a significant (p < 0.05) effect (31.7 1.1%) in stimulating neuronal differentiation compared to 50 ng / ml of ngf (28.3 0.4%). the high potassium level in the fruiting bodies and the presence of bioactive compounds (mainly triterpenoids) could be responsible for the neuronal stimulatory activity. further, in the on - going study, it was observed that neurite outgrowth stimulated by p. giganteus was mediated via the cross - talk between mek / erks and pi3k / akt pathways. the mushroom may be a candidate for the development of functional food to reduce or prevent severity of age - related neurodegenerative diseases. prevention is preferred for this traumatic disorder ; once the symptoms are clinically apparent, the reversal is almost impossible. almost all culinary mushrooms are noted for their polysaccharide components, which play a role in stimulation of immune system and possible applications in cancer management. many edible genera, however, are also noted for their specific activities. among the special activities, a number of mushrooms including sarcodon scabrosus, ganoderma lucidum, grifola frondosa, and hericium erinaceus are reported to have activities related to nerve and brain health. in this paper, the potentials of mushrooms as nutraceutical or nutriceuticals to help in the reduction or even prevention of age - related neurodegenerative diseases are discussed. when rat pheochromocytoma cells (pc12) were treated with scabronines a and g, there was neurite outgrowth. extracts of g. lucidium contained neuroactive compounds that induced neuronal differentiation and prevented ngf - dependent apoptosis of rat pheochromocytoma pc12 neuronal cells. these effects are probably mediated through the ras / erk and camp - response element - binding protein (creb) signaling pathways, because g. lucidum extracts induced phosphorylation of erk 1, erk 2, and creb. the culinary mushroom that has been extensively studied for its neurohealth properties is h. erinaceus (lion 's mane mushroom). the polysaccharides in an aqueous extract of the lion 's mane mushroom could induce neuronal differentiation and promote neuronal survival. the chemical profile of extracts of this mushroom has been actively studied and reported by kawagishi and co - researchers. extracts of h. erinaceus induced the expression of neurotrophic factors such as ngf in astrocytes. in their studies, hericenones were isolated from the fruiting bodies of lion 's mane while erinacines were isolated from the mycelium of the mushroom. endoplasmic reticulum stress - attenuating compounds and dilinoleoyl phosphatidylethanolamine, a phospholipid with linoleic acid, an unsaturated fatty acid, purified from extracts of dried fruiting bodies of h. erinaceus, reduce endoplasmic reticulum stress - induced cell death. investigated the neuroprotective effect of h. erinaceus on the ischemic brain damage in a middle cerebral artery occlusion model in mice. hericium erinaceus (300 mg / kg, continuously fed for 14 days after surgery) significantly decreased the size of the cerebral infarcts one day after the occlusion. in another study, a double - blind trial was conducted with 50- to 80-year - old japanese men and women diagnosed with mild cognitive impairment in order to examine the efficacy of oral administration of h. erinaceus, for improving cognitive impairment, using a cognitive function scale based on the revised hasegawa dementia scale (hds - r). the subjects in the h. erinaceus group took four 250 mg tablets containing 96% of yamabushitake dry powder three times a day for 16 weeks. extracts of h. erinaceus induced phosphorylation of c - jun n - terminal kinase (jnk) and its downstream substrate c - jun, and increased c - fos expression, suggesting that h. erinaceus promotes nerve growth factor gene expression via jnk signaling. furthermore, the efficacy of h. erinaceus in vivo has been examined. mice given feed regime containing 5% (w / w) h. erinaceus dry powder for seven days showed an increase in the level of ngf mrna expression in the hippocampus. these studies are very encouraging, and the consumption of h. erinaceus - fresh or processed is acceptable.. however, there should be guidelines and monitoring to ensure safety, efficacy, and other parameters to ensure that these preparations are functional as claimed. though hericium erinaceus is a temperate mushroom, it can be cultivated in malaysia, a tropical country. the consumers at large do not know the potential of this rather strange - looking mushroom. realizing the potential of this mushroom, research on the locally grown mushroom will help spearhead the growing as well as the consumption of this mushroom in malaysia. a : h. erinaceus ; b : p. giganteus ; c : g. lucidium ; d : l. rhinocerotis ; d : g.neo-japonicum the stimulation of neurite extension using in vitro model using ng 108 - 15 cells by h. erinaceus grown in hot climate was investigated. though the mushroom is now grown at higher temperatures, the unique property of this mushroom is retained. in fact, there was no difference in the neurite outgrowth stimulatory activity between the low- and high temperature - grown mushrooms. this is very important to note, as the commercially available h. erinaceus is oven- dried to increase its shelf life. the extracts of the fruiting bodies dried in an oven was demonstrated to have lost their ability to stimulate neurite outgrowth [figure 1 ]. the effects of an aqueous extract of fruiting bodies of h. erinaceus grown in a tropical climate. a : negative control (without treatment) ; b : ngf (20 ng / ml) ; c : aqueous extract of h. erinaceus (extensive neurite outgrowth) in another study, it was shown that extracts of h. erinaceus can enhance the activity of ngf in ng108 - 15 cells. a combination of 10 ng / ml ngf with 10 g / ml h. erinaceus aqueous extract induced the highest percentage increase of 59.5 5.0% neurite outgrowth compared to untreated neural cells (lai., 2012, unpublished). the effect of neurite outgrowth stimulation was enhanced in this combined mixture when compared to the concentration of extract or ngf applied individually. the neurite outgrowth stimulation activity increased approximately 1.2-fold when compared to treatment with only 10 ng / ml ngf (lai. however, the enhancement of neurite stimulation activity of the aqueous mushroom extract and ngf was not observed. further, early observations of an on - going study showed that the mushroom extract triggered ngf secretion in the presence of small amounts of ngf. further studies, especially on mechanism of action at biochemical and molecular levels, are important. this far, there are no reports on toxicity due to long - term consumption of the mushroom. further, a number of reported studies showed that this mushroom has potential to be developed into a functional food. the processing techniques, however, have to be optimized so that maximum activity will be retained to enhance nerve health and possibly nerve regeneration. peripheral nerve injury leads to changes at the axonal site of injury and remotely in the dorsal root ganglia (drg) containing cell bodies of sensory afferent neurons. the regenerative ability of injured axons depends on their capacity to locally synthesize new proteins and to degrade others at the injury site autonomously from the cell body. nerve crush injury is a well - established axonotmetic model in experimental regeneration studies to investigate the impact of various pharmacological treatments. the results indicated that the intensity of nuclear ribonucleoprotein at the injured segments of crushed nerves in rats of treated groups was significantly higher than in negative control group. in addition, the principal signaling pathways that have been demonstrated to be involved in axon regeneration are protein kinase b (akt) and mitogen - activated protein kinase (mapk). akt cascade plays a major role in mediating neurotrophin - promoted cell survival while mapk cascade is involved in mediating neurite outgrowth. a subfamily of mapk, extracellular signal - regulated kinases 1 and 2 (erk1/2), controls cellular differentiation and proliferation. double - labeling immunofluorescence studies showed that small neurons in l5 drg ipsilateral to the crush injury in rats of treated groups expressed higher immunoreactivities for akt and erk1/2 compared to negative control group. these data suggest that daily oral administration of aqueous extract of h. erinaceus fresh fruiting bodies could promote the regeneration of injured rat peripheral nerve in the early stages of recovery. hericium erinaceus, the mushroom associated with brain and nerve health, has been well - studied, and preparations are available. however, there is a paucity of clinical trials to endorse its potential in mitigating / delaying neurodegenerative disorders. further, this mushroom may not be the only one that has beneficial effects on nerve and brain health. to tap the indigenous knowledge in the utilization of the macrofungal diversity of malaysia as food and medicine, the diversity of potential mushrooms used by the indigenous communities are being explored for neurohealth. lignosus rhinocerotis (cooke) ryvarden (plate1d) ; ganoderma neo - japonicum imazeki (plate1e) and pleurotus giganteus (berk.) karunarathna and k.d. hyde (plate 1b) based on indigenous knowledge, selected mushrooms have been used to prepare infusions that are believed to improve vitality, increase alertness and overall wellness of the individual. the sclerotium of l. rhinocerotis (known as tiger milk mushroom) is taken daily or when needed in small amounts to a national treasure mushroom, l. rhinocerotis, has been used to treat variety ailments by local and indigenous communities in malaysia. the mushroom is harvested in the wild and is believed in folklore that it grows where the tiger has spilled its milk. the mushroom, which is rarely encountered in the wild, has now been successfully domesticated. in on - going in vitro investigations, an aqueous extract of the sclerotium of l. rhinocerotis induced neurite outgrowths of 24.4% and 42.1% at 20 g / ml (w / v) of aqueous extract alone and a combination of 20 g / ml (w / v) aqueous extract and 30 ng / ml (w / v) of ngf, respectively, in rat pheochromocytoma cells (pc-12adh). further, a combination of ngf and sclerotium extract had additive effects and enhanced neurite outgrowth. neuronal differentiation was confirmed by indirect immunofluorescence of neurofilament protein. to our knowledge, this is the first report on neurite - stimulating activities of l. rhinocerotis, and it may be a potential mushroom to develop nerve and brain health nutraceuticals or nutriceuticals.. there are on - going studies to determine the chemical components involved and the mechanisms of activity that trigger neurite outgrowth. the mushroom was boiled, and the decoction was consumed to treat fever, epilepsy and to improve body strength (tan., 2012, unpublished). the neurite outgrowth stimulation activity of aqueous extracts of the fruiting bodies of g. lucidum (plate 1c) and g. neo - japonicum were investigated. varying concentrations of the mushrooms and 50 ng / ml (w / v) of ngf as positive control was added to pc-12adh cells. aqueous extract of g. neo - japonicum showed the highest percentage [figure 2 ] of neurite - bearing cells of 14.05 0.66% at 50 g / ml (w / v), followed by g. lucidum (12.08 0.85%) at a higher concentration of 75 g / ml (w / v). the percentage of neurite - bearing cells of g. neo - japonicum obtained with 50 g / ml (w / v) was 73% higher compared to the positive control. it was proposed that g. neo - japonicum contained ngf - like bioactive compound / s for maintaining and rebuilding the neural communications network that may help to prevent neurodegenerative diseases (syntyche ling., arrows indicate neurite extensions. a : negative control - f-12k medium only ; b : positive control - 50 ng / ml (w / v) of ngf ; c : 50 g / ml of g. neo - japonicum neurofilament staining to confirm neurite outgrowth effects of ngf and p. giganteus extracts on neurite outgrowth using pc12 as an in vitro model. p < 0.05 compared to ngf (the positive control) pleurotus giganteus (berk.) hyde formerly known as panus / lentinus giganteus is a popular mushroom consumed by indigenous communities in malaysia. a variety domesticated in china is being grown in malaysia, and it is a very popular culinary mushroom and has a nutritional profile that warrants its inclusion in human diets. the fruiting bodies of p. giganteus contained per gm per 100 gm : 67.2 carbohydrate, 15.4 protein, and 33.3 dietary fiber. to our knowledge, the medicinal benefits, if any, are yet to be explored. an aqueous extract at 25 g / ml had a significant (p < 0.05) effect (31.7 1.1%) in stimulating neuronal differentiation compared to 50 ng / ml of ngf (28.3 0.4%). the high potassium level in the fruiting bodies and the presence of bioactive compounds (mainly triterpenoids) could be responsible for the neuronal stimulatory activity. further, in the on - going study, it was observed that neurite outgrowth stimulated by p. giganteus was mediated via the cross - talk between mek / erks and pi3k / akt pathways. the mushroom may be a candidate for the development of functional food to reduce or prevent severity of age - related neurodegenerative diseases. prevention is preferred for this traumatic disorder ; once the symptoms are clinically apparent, the reversal is almost impossible. the studies done by many researchers as well as on - going studies show that selected mushrooms do have neurotrophic properties that can be beneficial to humans. this is particularly useful during injury (as in an accidents) or as we age. further, there is a need to study the potential mushrooms to elucidate their mechanisms of activities as well as clinical trials. the identification of more varieties of mushroom with these unique properties may contribute to identifying novel chemical agents that can have preventative or therapeutic functions. | hericium erinaceus a culinary and medicinal mushroom is a well established candidate for brain and nerve health. ganoderma lucidum, grifola frondosa and sarcodon scabrosus have been reported to have neurite outgrowth and neuronal health benefits. the number of mushrooms, however, studied for neurohealth activity are few compared to the more than 2 000 species of edible and / or medicinal mushrooms identified. in the on - going search for other potent culinary and / or medicinal mushrooms, indigenous mushrooms used in traditional medicines such as lignosus rhinocerotis and ganoderma neo - japonicum are also being investigated. further, the edible mushroom, pleurotus giganteus can be a potential candidate, too. can these edible and medicinal mushrooms be tapped to tackle the health concerns of the aging population which is projected to be more than 80 - 90 million of people age 65 and above in 2050 who may be affected by age - related neurodegenerative disorders. scientific validation is needed if these mushrooms are to be considered and this can be achieved by understanding the molecular and biochemical mechanisms involved in the stimulation of neurite outgrowth. though it is difficult to extrapolate the in vitro studies to what may happen in the human brain, studies have shown that there can be improvement in cognitive abilities of the aged if the mushroom is incorporated in their daily diets. |
mycoplasmas are in a class of bacteria designated as mollicutes, which lack cell walls, and this characteristic along with their minute size separates them from other bacteria. mycoplasma hominis, ureaplasma urealyticum, u. parvum, and m. genitalium are potentially pathogenic species frequently isolated from the genitourinary tract and are known as urogenital mycoplasmas (1). these bacteria together with neisseria gonorrhoeae and chlamydia trachomatis are considered among the most prevalent sexually transmitted pathogens that have a global distribution (2 3). urogenital mycoplasmas are associated with some symptomatic and asymptomatic genitourinary tract infections in both males and females ; for example, they may cause pelvic inflammatory disease (pid) resulting in damage to the fallopian tubes, which may lead to ectopic pregnancy. these bacteria are also associated with non - gonococcal urethritis, endometritis, bacterial vaginosis, preterm delivery, postpartum, or post - abortal fever, as well as perinatal disorders such as low birth weight and neonatal bacteremia / meningitis (4 12). the effects of urogenital mycoplasmas on spermatozoa and seminological variables and their role in male or female infertility are controversial and remain unclear (13 18) ; however, there is some evidence that m. genitalium may cause female infertility, particularly tubal infertility (19 20). some of investigators believe that mycoplasmas are genitourinary tract commensals ; thus, one of the important problems concerning urogenital mycoplasmas is that there are many clinically asymptomatic carriers silently colonized by these bacteria while these microorganisms are potentially pathogenic and may play a role in urogenital tract infection or affect fertility potential as an opportunistic pathogen, under certain circumstances (21 25). nevertheless, the majority of asymptomatic infections may remain undetected and consequently untreated. in iran, to date, several studies have reported the frequency of urogenital mycoplasmas infections in males and/or females, in which the frequency of these pathogens varies significantly in different surveys. however, most of these studies are local and limited to an individual hospital or a special province or city, and a comprehensive analysis of the overall prevalence of these bacteria, which may be useful to set up control programs for the prevention of sexually transmitted infections (stis), has not yet been performed. thus, the present study was designed to determine the prevalence of urogenital mycoplasmas infections in iran using a systematic review and meta - analysis according to the preferred reporting items for systematic reviews and meta - analyses (prisma) statement (26). we searched electronic databases, including ovid databases, pubmed, web of science, scopus, and google scholar for the prevalence of urogenital mycoplasmas infections in iran from nov 1999 to feb 2015. the search was restricted to original articles published in english that present the prevalence, frequency, or incidence of urogenital mycoplasmas infections in iranian males or females using the following keywords with the help of boolean operators (and, or) : mycoplasma, ureaplasma, urogenital mycoplasmas, genital mycoplasmas, m. hominis, m. genitalium, u. urealyticum, u. parvum, prevalence, incidence, frequency, epidemiology, and m. fermentans, m. penetrans, m. primatum, and m. spermatophilum. in addition to articles published in english, we also looked for relevant articles in persian published and indexed in iranian databases, such as scientific information database (sid) (http://www.sid.ir/), magiran (http://www.magiran.com/), irandoc (http://www.irandoc.ac.ir/), regional information center for science and technology (ricst) (http://en.ricest.ac.ir/), and iranian national library (http://www.nlai.ir/), with similar strategies and related appropriate persian keywords. control, or cohort studies on the prevalence of symptomatic or asymptomatic urogenital mycoplasmas infections in iranian males / females in which the methods for diagnoses were molecular amplification techniques, such as pcr, pcr - restriction fragment length polymorphism (pcr - rflp), and multiplex pcr. excluded studies were : 1) those that used detection methods other than nucleic acid amplification techniques (naat), including culture or serological methods, such as enzyme - linked immunosorbent assay (elisa) or immunofluorescence (if), 2) studies reporting prevalence of myco - plasma species other than urogenital mycoplasmas such as m. pneumoniae, 3) studies that included mycoplasma infections in organs or body sites other than the genitourinary tract, and 4) studies reporting the prevalence of urogenital mycoplasmas at the genus level and not the species of mycoplasma. review articles, congress abstracts, studies reported in languages other than english or persian, meta - analyses or systematic reviews, duplicate publications of the same study, and articles available only in abstract form were also excluded. variables and information extracted from each study included first author s name, year of publication, study setting, geographical location, participants characteristic, gender, specimen type, number of patients investigated (sample size), bacterial species investigated, type of detection method, and number of positive samples. the data were analyzed using comprehensive meta - analysis software version 2.0 (biostat, englewood, nj, usa). cochrane q - statistic test and i test were performed to estimate heterogeneity between studies, and the random effect model was chosen to estimate the average prevalence because of its conservative summary estimate and because in all calculations (except one in which the fixed effect model was used), i was above 50%. to assess possible publication bias, a funnel plot along with begg s rank correlation and egger s weighted regression methods were used. we searched electronic databases, including ovid databases, pubmed, web of science, scopus, and google scholar for the prevalence of urogenital mycoplasmas infections in iran from nov 1999 to feb 2015. the search was restricted to original articles published in english that present the prevalence, frequency, or incidence of urogenital mycoplasmas infections in iranian males or females using the following keywords with the help of boolean operators (and, or) : mycoplasma, ureaplasma, urogenital mycoplasmas, genital mycoplasmas, m. hominis, m. genitalium, u. urealyticum, u. parvum, prevalence, incidence, frequency, epidemiology, and m. fermentans, m. penetrans, m. primatum, and m. spermatophilum. in addition to articles published in english, we also looked for relevant articles in persian published and indexed in iranian databases, such as scientific information database (sid) (http://www.sid.ir/), magiran (http://www.magiran.com/), irandoc (http://www.irandoc.ac.ir/), regional information center for science and technology (ricst) (http://en.ricest.ac.ir/), and iranian national library (http://www.nlai.ir/), with similar strategies and related appropriate persian keywords. included studies were all original articles presenting cross - sectional, case control, or cohort studies on the prevalence of symptomatic or asymptomatic urogenital mycoplasmas infections in iranian males / females in which the methods for diagnoses were molecular amplification techniques, such as pcr, pcr - restriction fragment length polymorphism (pcr - rflp), and multiplex pcr. excluded studies were : 1) those that used detection methods other than nucleic acid amplification techniques (naat), including culture or serological methods, such as enzyme - linked immunosorbent assay (elisa) or immunofluorescence (if), 2) studies reporting prevalence of myco - plasma species other than urogenital mycoplasmas such as m. pneumoniae, 3) studies that included mycoplasma infections in organs or body sites other than the genitourinary tract, and 4) studies reporting the prevalence of urogenital mycoplasmas at the genus level and not the species of mycoplasma. review articles, congress abstracts, studies reported in languages other than english or persian, meta - analyses or systematic reviews, duplicate publications of the same study, and articles available only in abstract form were also excluded. variables and information extracted from each study included first author s name, year of publication, study setting, geographical location, participants characteristic, gender, specimen type, number of patients investigated (sample size), bacterial species investigated, type of detection method, and number of positive samples. the data were analyzed using comprehensive meta - analysis software version 2.0 (biostat, englewood, nj, usa). the prevalence was reported by 95% confidence interval (ci). cochrane q - statistic test and i test were performed to estimate heterogeneity between studies, and the random effect model was chosen to estimate the average prevalence because of its conservative summary estimate and because in all calculations (except one in which the fixed effect model was used), i was above 50%. to assess possible publication bias, a funnel plot along with begg s rank correlation and egger s weighted regression methods were used. a total of 99 articles (59 in english and 40 in persian) were collected. through the first screening, after the second assessment, 12 papers were discarded because they had reported mycoplasma infections in organs or body sites other than the genitourinary tract, or investigated other mycoplasma species (such as m. pneumonia). finally, after full - text evaluation, 30 studies were ruled out on the basis of their detection methods, or because they had reported the prevalence of urogenital mycoplasmas at the genus level and not specified the species, and therefore 29 articles (19 in english and 10 in persian) published between 2005 and 2015 were selected and included in our analysis (fig. 1 and table 1). studies reporting prevalence of mycoplasma species other than urogenital mycoplasmas, such as m. pneumonia ; surveys reporting the prevalence of urogenital mycoplasmas at genus level studies included in meta - analysis after final evaluation m : males ; f : females ; fvu : first void urine ; mh : m. hominis ; uu : u. urealyticum ; mg : m. genitalium ; up : u. parvum ; of the 29 articles included, 16 had studied the prevalence of urogenital mycoplasmas infections in women, 11 in men, and two in both genders ; four articles had studied only m. hominis ; five only studied u. urealyticum, and eight only studied m. genitalium ; nine articles had studied both m. hominis and u. urealyticum, two had investigated all three bacteria simultaneously, and there was only one article, explored u. urealyticum and u. parvum concurrently. we did nt find any articles conducted in iran about other urogenital tract associated mycoplasma species, including : m. fermentans, m. penetrans, m. primatum, and m. spermatophilum. seven studies (five for men and two for women) were case control (investigating the frequency of urogenital mycoplasmas in symptomatic asymptomatic or fertile infertile individuals, or in females having spontaneous abortion normal pregnancy), and the rest were cross - sectional. the participants of the cross - sectional studies varied from asymptomatic and fertile individuals to symptomatic men and women having urogenital infections and complications including urethritis, prostatitis, cervicitis, vaginitis, vaginosis, and infertility. the most commonly collected sample for detection was cervical or endocervical swabs for women, and semen samples for men, but other samples included vaginal discharge or vaginal swabs for women, prostate tissue for men, and first void urine for both genders. five papers studied other agents in addition to urogenital mycoplasmas, such as gardnerella vaginalis, neisseria gonorrhoeae, and chlamydia trachomatis, simultaneously. most of the included studies had been performed in tehran (n=18), which is the capital of iran, in comparison with ones performed in western (n=3), southeastern (n=3), southwestern (n=2), northern (n=2), and northeastern (n=1) iran. the prevalence of urogenital mycoplasma species in different regions of iran is shown in table 2. prevalence of urogenital mycoplasma species in different regions of iran m : male ; f : female ; mh : m. hominis ; uu : u. urealyticum ; mg : m. genitalium ; up : u. parvum ; based on random effects, (95% ci) ; there was only one article among included studies, investigated the prevalence of u. parvum in males ; there was only one study among included articles in this gender, from this city ; ns : no study conducted in this city, about this gender. the age for women ranged from 1460 yr (median : 37 yr), and for men from 1765 yr (median : 41 yr). three studies had not reported the ages of participants, and nine did not have age - stratified data.. however, the highest prevalence of urogenital mycoplasmas between the age groups belonged to 2636 yr [38.2% (95% ci, range : 18.8%57.6%) ] and 2535 yr [42.6% (95%ci, range : 33.7%51.5%) ] for men and women, respectively. the numbers of participants (sample sizes) in the included studies varied from 45246 in men and 44465 in women. prevalence of urogenital mycoplasmas for men and women ranged from 2%40.5% and 2%44.3%, respectively (95% ci) ; the lowest rate was related to m. genitalium and the highest one was associated to u. urealyticum, in both genders. the pooled prevalence of these bacteria in men was 11.1% (95% ci, range : 7.4%16.4%) and in women was 12.8% (95% ci, range : 9.8%16.5%) ; moreover, a high level of heterogeneity was observed in men (i = 92.4% ; p<0.001) and women (i = 93.3% ; p<0.001). fig. 2 (a and b) shows the forest plots of the meta - analyses of urogenital mycoplasmas prevalence for men and women, respectively. the funnel plots for meta - analysis of urogenital mycoplasmas prevalence in both men and women suggest some evidence of publication bias [fig. 4 (a and b) ] ; two - tailed p for begg s (based on continuity - corrected normal approximation) and egger s tests in men was 0.0151 and 0.0007, respectively, and in women was 0.0086 and 0.0006, correspondingly. we also analyzed the prevalence of each mycoplasma species in men and women, separately ; the results are shown in table 3 and corresponding forest and funnel plots are shown in fig. 3 (a f) and 4 (c h), respectively. some evidence of publication bias was observed in analysis of u. urealyticum prevalence in men (but not in women) [egger s test (two - tailed p=0.0085), and begg s test (two - tailed p=0.0242) ]. conversely, no evidence of publication bias was observed in the analysis of other mycoplasma species in men or women [table 3 and fig. 4 (ch) ] ; however, the number of studies on the prevalence of m. genitalium in both men and women and prevalence of m. hominis and u. urealyticum in men was fewer than 10, and insufficient for an accurate conclusion. forest plots of the meta - analysis of urogenital mycoplasmas prevalence for men (a) and women (b). mh : m. hominis ; uu : u. urealyticum ; mg : m. genitalium ; up : u. parvum funnel plots of the meta - analysis of urogenital mycoplasmas prevalence. a and b : funnel plots for meta - analysis of urogenital mycoplasmas prevalence in men and women, respectively. c, e, and g : funnel plots for meta - analysis of prevalence of m. genitalium, m. hominis, and u. urealyticum, respectively in men. d, f, and h : funnel plots for meta - analysis of prevalence of m. genitalium, m. hominis, and u. urealyticum, respectively in women. a, c, and e : meta - analysis of prevalence of m. genitalium, m. hominis, and u. urealyticum, respectively in men. b, d, and f : meta - analysis of prevalence of m. genitalium, m. hominis, and u. urealyticum, respectively in women meta - analysis results for prevalence of each mycoplasma species in included studies m : male ; f : female ; min : minimum ; max : maximum ; a : kendall s tau without continuity correction ; b : kendall s tau with continuity correction. there was only one article studied the prevalence of u. parvum and reported its prevalence to be 2% and 3% in fertile and infertile men, respectively ; pooled prevalence (based on random effects) ; begg and mazumdar rank correlation ; egger s regression intercept. there were only five case control studies among the included articles investigated the prevalence of some urogenital mycoplasma species in both fertile and infertile men ; the forest plot of meta - analysis showed that the odds ratios for all these studies were above 1, and p values for three surveys were below 0.05 (fig. 5a), indicating that the prevalence of urogenital mycoplasmas was significantly higher in the case group (infertile men) compared with that in the control group (fertile men) ; as i was 27.2% (below 50%), the fixed effect model was used ; however, the number of studies was fewer than 10, and insufficient for an accurate conclusion ; the resulting funnel plot is shown in fig. 5b ; [no publication bias was observed : egger s test (two - tailed p=0.8232), and begg s test (two - tailed p=1.000) ]. moreover, we found just one article among the included studies that investigated the prevalence of u. parvum and reported its prevalence to be 2% and 3% in fertile and infertile men, respectively (27). meta - analysis of urogenital mycoplasmas prevalence for six case - control studies about men. a : forest plot, b : funnel plot ; uu : u. urealyticum ; mg : m. genitalium ; up : u. parvum urogenital mycoplasma species are primarily mucus - associated organisms inhabiting the urogenital tracts of their hosts in close relation with epithelial cells. they may invade host cells and reside intracellularly ; a trait that may assist them to cause chronic infections and give them the ability to evade from host immune responses (5, 27 28). these microorganisms may also cause clinically asymptomatic infections as an opportunistic pathogen, while considering as commensals (21 25) ; perhaps due to an imbalance occurred among vaginal microbiota in some circumstances in which some bacteria can multiply and cause diseases (29). however, a considerable amount of urogenital infections caused by these bacteria is asymptomatic and remain undetected and untreated, and consequently the infection may be transmitted to the sexual partner(s). thus, designing and implementing national control programs to prevent subsequent complications is thought to be necessary. comprehensive analyses of the overall prevalence of these bacteria, particularly in developing countries (including iran), may help to carry out such a strategy. through this systematic review and meta - analysis, which is the first such study in iran, we found that prevalence and frequency of urogenital mycoplasma species in men as well as women was highly variable in various studies. there was also a high grade of heterogeneity in participants characteristics, samples taken for tests, detection methods, sample sizes, and study settings. as men and women are distinct populations with different indicators of prevalence, corresponding data about each of them were analyzed separately in the present study. among articles finally included in this analysis, there were no studies conducted in central, southern, or northwestern iran, and most of them were performed in the capital of iran (tehran) ; this was one of the limitations of our study, which may suggest some participation bias in the generalization of the meta - analysis results. another restriction of the present study was that many papers conducted in different regions of the country had to be excluded from analysis because their methods of detection were serology (for example, elisa and if) or culture, which have lower sensitivity and specificity in comparison with molecular techniques. furthermore, the samples used for detections were different, and sample sizes in the included studies were different and ranged from 45246 and 44 465 for men and women, respectively ; this may have impacted on the results of analysis, thus we calculated and reported the relative weight for each study. an additional restriction encountered in the current study was that the numbers of included studies investigated the prevalence of m. genitalium in both men and women, and m. hominis and u. urealyticum in men were fewer than 10 and insufficient for a good meta - analysis and an accurate conclusion. in order to find out about possible effects of urogenital mycoplasmas on fertility potential, we analyzed five case control studies among the included articles investigated the prevalence of urogenital mycoplasmas in both fertile and infertile men ; the odds ratios for all of these studies were above 1, p values for three surveys (investigated u. urealyticum) were below 0.05, the overall odds ratio was 3.2 (95% ci, range : 1.76.1), and the overall p value was zero ; meaning that the prevalence of these bacteria is significantly higher in the case group (infertile men) compared with that in the control group (fertile men), and these bacteria may play a role in male infertility. nevertheless, because the number of these studies was fewer than 10, we could nt achieve an exact conclusion about the correlation between urogenital mycoplasmas and male infertility ; however, as mentioned in the introduction, this topic is controversial and further case control or cohort studies are needed to conclude accurately and generalize the results. moreover, we could not find any interventional or randomized - controlled clinical trial studies assessing the effects of antibiotic therapy for urogenital mycoplasmas infections (particularly asymptomatic infections) on the treatment of probable infertility due to these bacteria. according to the results of our meta - analysis, the highest pooled prevalence among urogenital mycoplasma species belonged to m. hominis in men and u. urealyticum in women, and the pooled prevalence of m. genitalium was lower than m. hominis and u. urealyticum in both men and women (table 3). as shown in table 2, the study of urogenital mycoplasmas prevalence in the male population was conducted only in two provinces of iran, in which the pooled prevalence rate was higher in kerman [pooled prevalence : 16.2%, (95% ci, range : 11.8%22%) ], rather than tehran [pooled prevalence : 10.1%, (95% ci, range : 6.2%16.1%) ] ; although the number of studies was more in tehran (n=14) rather than the kerman province (n=3), and there was no study about u. urealyticum from kerman. in the case of women, the highest prevalence rate belonged to tonekabon city [22.7%, (95% ci, range : 12.7%37.3%) ], although only one study had been reported from this city on women, and which only investigated the prevalence rate of m. genitalium ; after that, tehran province had 12 studies about three mycoplasma species [pooled prevalence : 19.6%, (95% ci, range : 15.1%25.1%) ], and the lowest one belonged to sabzevar city [prevalence rate : 2%, (95% ci, range : 0.8%5.3%) ], though only one survey had been performed in this city on women about m. genitalium prevalence (table 2). the world health organization has estimated that more than 340 million new cases of stis occur annually throughout the world, with the highest incidence in developing countries (30). we could not find similar meta - analysis studies on the prevalence of urogenital mycoplasmas conducted either in neighboring or other countries, to be compared with our analysis in iran. as mentioned in the results, the highest prevalence of urogenital mycoplasmas in our analysis was in young individuals of 2536 yr ; this finding is consistent with other studies performed in other countries (31 36). however, the overall prevalence of urogenital mycoplasmas varies in different countries and international reports suggest an increase in infections due to these bacteria over the last decade (37). this variability in prevalence rates reported in different countries is perhaps due to a variety in ethnic and social populations, differences in detection methods, types of samples studied, sample sizes, hygiene issues, socioeconomic status, age of participants, and absence of regular screening, treatment, and control programs, particularly in some of the developing countries for dealing with the infections caused by these bacteria. the results show a relatively high prevalence of urogenital mycoplasma species in male as well as female populations in iran, particularly in youth (2536 yr). urogenital mycoplasmas may play a role in male infertility ; this highlights the necessity of planning national programs for adequate diagnosis and screening for genitourinary infections due to these bacteria, particularly asymptomatic infections, and treating infected individuals (including sexual partners) to control stis and the consequent complications, reduce the carrier rate, and maintain reproductive health and fertility potential. moreover, further case control studies or randomized - controlled trials are needed, particularly on the likely influences of these bacteria on reproductive health and their correlation with male / female infertility. ethical issues (including plagiarism, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors. ethical issues (including plagiarism, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors. | background : urogenital mycoplasmas are potentially pathogenic species causing genitourinary tract infections that may be initially asymptomatic but can progress and lead to severe complications and threaten reproductive health. however, the overall prevalence rate of this bacterium and its probable impacts on fertility potential have yet to be determined.methods:we searched both english and persian electronic databases using key words such as mycoplasma, ureaplasma, m. hominis, m. genitalium, u. urealyticum, u. parvum, prevalence, and iran. finally, after some exclusion, 29 studies from different regions of iran were included in our study, and a meta - analysis was performed on collected data.results:urogenital mycoplasmas prevalence for women and men was high and ranged from 2%40.5% and 2%44.3%, respectively. the pooled prevalence in the male population was 11.1% (95% ci, 7.4%16.4%) and in female was 12.8% (95% ci, 9.8%16.5%). the prevalence of these bacteria was significantly higher in infertile men compared with that in fertile men. a high level of heterogeneity was observed for both men (i2 = 92.4% ; p<0.001) and women (i2 = 93.3% ; p<0.001). some evidence for publication bias was observed in both men [egger s test (two - tailed p=0.0007), and begg s test (two - tailed p=0.0151) ] and women [egger s test (two - tailed p=0.0006), and begg s test (two - tailed p=0.0086) ] analysis.conclusion:since urogenital mycoplasmas may play a role in male infertility, screening strategies, particularly for asymptomatic individuals, and treatment of infected ones, which can reduce consequent complications, looks to be necessary. |
pulmonary vein isolation (pvi) is an established strategy of paroxysmal atrial fibrillation (paf) ablation,,. radiofrequency catheters have been the standard for af ablation ; however, pvi using balloon - based ablation has been increasingly adopted by electrophysiology laboratories around the world,,. the second - generation cryoballoon (cb2) (arctic front advance, medtronic, mn, usa), which is designed to achieve more uniform cooling across the entire distal hemisphere of the balloon, has been recently launched on the market for the treatment of paf. cb2 ablation is effective for the treatment of paf ; however, there is some debate concerning the optimal number of freezing cycles, the ideal freezing duration of each cycle, and the appropriate patient characteristics for cb2 ablation. no prospective, randomized clinical cb2 trials have been conducted to investigate the need for an additional (bonus) freezing cycle after pvi is achieved. we therefore conducted an investigation of the optimal freezing cycles in af patients treated with cb2 ablation. the ad - balloon study was designed as a prospective, multicenter, randomized clinical trial of the optimal strategy (freezing cycles) of cb2 ablation (umin clinical trials registry umin000020130). the objectives of this study are, firstly, to investigate the need for an additional freezing cycle after pvi in patients treated with cb2 ablation ; and secondly, to investigate the usefulness of delayed - enhancement magnetic resonance imaging (mri) to assess the ablation lesions created by cb2 ablation. the patients will be randomly assigned in a 1:1 ratio to treatment with an additional freezing (ad group) cycle or without additional freezing (non - ad group). in the ad group, 3 min of additional freezing time will be applied in all pulmonary veins (pvs) after pvi is confirmed during the previous freezing cycle. in the non - ad group, no additional freezing will be applied in any of the pvs after pvi is confirmed. we will use efrons biased coin design to allocate patients to either group with computer generated random numbers in order to reduce the predictability and selection bias. the primary endpoint of this study is the occurrence of atrial tachyarrhythmias (ats) at 1 year with a blanking period of 90 days after cb2 ablation. based on previous studies in paf patients, the incidence of recurrent ats after a single cb2 ablation procedure with a blanking period of 90 days will be assumed to be 20%,,. considering the primary endpoint as a binary outcome, 55 patients in each group, and 110 patients in total will be required to achieve a 90% confidence interval in this study. the secondary efficacy endpoint is repeat ablation for ats at 1 year without the blanking period. the secondary safety endpoints include procedural complications, total procedure time, and total fluoroscopy time. all participants are required to provide written informed consent (m26 - 159, nov 27, 2015). the patient inclusion criteria for enrollment are as follows : (1) a diagnosis of paf in accordance with the 2014 aha / acc / hrs guideline on the management of patients with atrial fibrillation ; (2) treatment with cb2 ablation for paf ; and (3) aged 2085 years, regardless of the sex. the exclusion criteria of this study are as follows : (1) aged 30 s after the index procedure recorded by a standard ecg, 24-h ecg holter monitoring, or cardiac event recorder during both a planned and symptom driven consultation will be considered as a recurrence. when an at recurrence occurs and the patient agrees, a second catheter ablation will performed. the patient characteristics and status of the medical treatment at baseline in each group will be collected as presented in table 1. the cb2 ablation procedures, results, complications, status of medical treatment after cb2 ablation, and follow - up data in each group will be collected as presented in table 2, table 3. registration for this study will be conducted by the medical staff of each center, who will be required to fill out a hard copy data sheet at enrollment and send it to the secretary of this study. the secretary of this study will assign the patients a specific i d for this study, which will be shared electronically with the medical staff at each center. the medical staff may input the patient data into the website via their personal computers or hard copy data sheet. the data will be provided via traditional or electronic mail using only the specific i d without the patients name, and personal confidentiality will be protected. the data collection will begin after the ethical aspects of this study have been approved by the ethics committees at each center. all patients will be prospectively followed - up by the participating centers for at least 1 year. a sample size of 55 patients per group (equally sized - groups) will provide a two - sided 95% confidence interval (ci) for the difference in population proportions with a width that is equal to 0.35 when the two estimated group sample proportions are equally 0.20. analyses will be done according to an intention - to - treat (itt) principle. analysis of the per - protocol set will be also done to assess the robustness of the itt analysis. patient demographic data will be analyzed descriptively ; categorical variables will be assessed with the chi - square test or fishers exact test, whereas continuous variables will be assessed with the students t - test or the wilcoxon rank - sum test, as appropriate. the difference in the occurrence rate of at between the two groups will be calculated with the 95% ci, and the two occurrence rates will be compared using the chi - square test. also, the at - free survival will be summarized using the kaplan meier method in each group, and the survival curves will be compared by the log rank test. an additional exploratory logistic regression or cox regression model will be fitted for planned subgroup analysis in which the odds ratio or hazard ratio will be stratified as described and, when applicable, with treatment as a covariate. the ad - balloon study was designed as a prospective, multicenter, randomized clinical trial of the optimal strategy (freezing cycles) of cb2 ablation (umin clinical trials registry umin000020130). the objectives of this study are, firstly, to investigate the need for an additional freezing cycle after pvi in patients treated with cb2 ablation ; and secondly, to investigate the usefulness of delayed - enhancement magnetic resonance imaging (mri) to assess the ablation lesions created by cb2 ablation. the patients will be randomly assigned in a 1:1 ratio to treatment with an additional freezing (ad group) cycle or without additional freezing (non - ad group). in the ad group, 3 min of additional freezing time will be applied in all pulmonary veins (pvs) after pvi is confirmed during the previous freezing cycle. in the non - ad group, no additional freezing will be applied in any of the pvs after pvi is confirmed. we will use efrons biased coin design to allocate patients to either group with computer generated random numbers in order to reduce the predictability and selection bias. the primary endpoint of this study is the occurrence of atrial tachyarrhythmias (ats) at 1 year with a blanking period of 90 days after cb2 ablation. based on previous studies in paf patients, the incidence of recurrent ats after a single cb2 ablation procedure with a blanking period of 90 days will be assumed to be 20%,,. considering the primary endpoint as a binary outcome, 55 patients in each group, and 110 patients in total will be required to achieve a 90% confidence interval in this study. the secondary efficacy endpoint is repeat ablation for ats at 1 year without the blanking period. the secondary safety endpoints include procedural complications, total procedure time, and total fluoroscopy time all participants are required to provide written informed consent (m26 - 159, nov 27, 2015). the patient inclusion criteria for enrollment are as follows : (1) a diagnosis of paf in accordance with the 2014 aha / acc / hrs guideline on the management of patients with atrial fibrillation ; (2) treatment with cb2 ablation for paf ; and (3) aged 2085 years, regardless of the sex. the exclusion criteria of this study are as follows : (1) aged 30 s after the index procedure recorded by a standard ecg, 24-h ecg holter monitoring, or cardiac event recorder during both a planned and symptom driven consultation will be considered as a recurrence. when an at recurrence occurs and the patient agrees, a second catheter ablation will performed. the patient characteristics and status of the medical treatment at baseline in each group will be collected as presented in table 1. the cb2 ablation procedures, results, complications, status of medical treatment after cb2 ablation, and follow - up data in each group will be collected as presented in table 2, table 3. registration for this study will be conducted by the medical staff of each center, who will be required to fill out a hard copy data sheet at enrollment and send it to the secretary of this study. the secretary of this study will assign the patients a specific i d for this study, which will be shared electronically with the medical staff at each center. the medical staff may input the patient data into the website via their personal computers or hard copy data sheet. the data will be provided via traditional or electronic mail using only the specific i d without the patients name, and personal confidentiality will be protected. the data collection will begin after the ethical aspects of this study have been approved by the ethics committees at each center. all patients will be prospectively followed - up by the participating centers for at least 1 year. a sample size of 55 patients per group (equally sized - groups) will provide a two - sided 95% confidence interval (ci) for the difference in population proportions with a width that is equal to 0.35 when the two estimated group sample proportions are equally 0.20. analyses will be done according to an intention - to - treat (itt) principle. analysis of the per - protocol set will be also done to assess the robustness of the itt analysis. patient demographic data will be analyzed descriptively ; categorical variables will be assessed with the chi - square test or fishers exact test, whereas continuous variables will be assessed with the students t - test or the wilcoxon rank - sum test, as appropriate. the difference in the occurrence rate of at between the two groups will be calculated with the 95% ci, and the two occurrence rates will be compared using the chi - square test. also, the at - free survival will be summarized using the kaplan meier method in each group, and the survival curves will be compared by the log rank test. an additional exploratory logistic regression or cox regression model will be fitted for planned subgroup analysis in which the odds ratio or hazard ratio will be stratified as described and, when applicable, with treatment as a covariate. cb2 ablation provides more reproducible results and reduced procedural times than conventional radiofrequency catheter ablation,. in addition, some studies have reported a high rate of pvi durability and a high long - term af free rate of about 80% using the cb2 ablation procedure. some clinical reports describing cryobiology stress the need for repetitive freezing cycles to extend the lethal effect at the periphery of the target tissue,. on the other hand, recent studies on the cb2 have shown that a single 3-min freezing cycle may be sufficient for a durable pvi,. fifty - two consecutive patients were included in the study. in the first 24 (46%) consecutive patients, an additional freezing cycle after pvi was performed, and it was not given in the subsequent 28 patients (54%). as a result, there were no differences in the recurrence rate between patients with and without an additional freezing cycle after pvi. however, patients were not randomly assigned, and the learning curve might have affected the results in the study about the initial experience with cb2 ablation. we therefore assess the optimal freezing cycles in af patients treated with cb2 ablation in this prospective, multicenter, and randomized clinical trial. the ad - balloon study will be the first prospective investigation to assess the need for an additional (bonus) freezing cycle after pvi is achieved. the ad - balloon study is partly supported by the intramural research fund (25 - 4 - 7, kusano) for cardiovascular diseases of national cerebral and cardiovascular center. there is no specific sponsor that has a role in the study design or conduct of the study, collection of the data, its analysis or its interpretation, or in the preparation of the present manuscript. the authors have full access to all data and take full responsibility for the integrity of the data in this study, and for the decision to submit this manuscript for publication. | backgroundpulmonary vein isolation (pvi) is a cornerstone of catheter ablation in patients with paroxysmal atrial fibrillation (paf), and balloon - based ablation has been recently performed worldwide. ablation using the second - generation cryoballoon (cb2) (arctic front advancetm, medtronic, mn, usa) is useful for pvi ; however, there is some debate concerning the optimal freezing time and number of cycles after pvi is achieved.methodsthe ad - balloon study was designed as a prospective, multicenter, randomized clinical trial to evaluate the optimal strategy (freezing cycles) of cb2 ablation (umin clinical trials registry umin000020130). the main objective of this study is to investigate the need for an additional freezing cycle after pvi in patients treated with cb2 ablation. patients will be randomly assigned in a 1:1 ratio to treatment with additional freezing (ad group) or without additional freezing (non - ad group). in the ad group, 3 min of additional freezing time will be applied in all pulmonary veins after pvi is confirmed at the previous freezing cycle. in the non - ad group, no additional freezing will be applied in all pulmonary veins after pvi is confirmed. the primary endpoint of this study is the occurrence of atrial tachyarrhythmias within a 1-year follow - up period. we will enroll 110 consecutive patients with paf. we will also investigate the usefulness of delayed - enhancement magnetic resonance imaging to assess the ablation lesions caused by cb2 ablation.resultsthe results of this study are currently under investigation.conclusionthe ad - balloon study would assess the need for an additional freezing cycle after pvi is achieved. our findings may contribute to further improvement of the cb2 ablation procedure. |
despite a careful selection of patients, the failure rates associated with lumbar fusion surgery have been reported to range from 5 to 30%5,27). among the different sources of low back pain following lumbar fusion surgery, the sacroiliac (sij) joint has been suggested as a possible source by several authors10,11,12,27). maigne and planchon27) suggested three possible causes of sij pain following lumbar fusion surgery : 1) mechanical load transfer on sij after fusion ; 2) a consequence of bone graft harvesting in the iliac crest close the joint ; and 3) the misdiagnosing of a sacroiliac syndrome before fusion (i.e., the lumbar spine being fused erroneously). the adoption by humans of upright position resulted in the pelvis becoming a paramount structure within human motor apparatus. the pelvis forms the bond between the trunk and the posterior limbs by femoral head and can rotate around it (bicoxo - femoral axis). this rotatory ability of the pelvis around the femoral heads is one of mechanisms of regulation of sagittal balance. because the position of lumbar spine, which is attached to the sacral plateau, is affected by the pelvic position19). recently, owing to their close relationship with the spine, many authors have raised the role of sacropelvic morphology and balance in the pathogenesis of various spinal disorders including degenerative spondylolisthesis3,28), adult spinal deformity14,17) and adolescent idiopathic scoliosis25). however, as far as we know, there has been no study specifically describing the sacropelvic morphology and balance of the patients with sij pain following lumbar fusion surgery. this study aims to investigate the sagittal sacropelvic morphology and balance of the patients with sij pain following lumbar fusion surgery. the approval of the institutional review board at our hospital was obtained for the current study. between june 2009 and january 2013, a total of 452 consecutive patients who underwent posterior lumbar interbody fusion (plif) at our institution were investigated. all cases of plif surgery were performed using a peek cage packed with autologous bones obtained from lamina or spinous process and pedicle screws were inserted. there were no injury to sij and all procedures of plif were done by the same surgeon. patients were included into sij pain group if their reported postoperative pain met following criteria16,26) : pain begins with standing or sitting position after surgery and it differs from preoperative one ; unilateral or bilateral prevalence persistent pain which had compatible distribution with a sacroiliac joint (below the l5 spinous process, over the posterior aspect of one or both sijs) ; with or without radiation to the posterior thigh or groin but not below the knee ; with or without tenderness of the sacroiliac sulcus at palpation ; and no evidence of lumbar cause (no breach of screw and no residual nerve root impingement on postoperative ct scan images. then, for the patients whose symptoms were consistent with above mentioned criteria, a fluoroscopically guided sij block was performed and after block, the patient with positive response was classified into sij pain group. a positive response was defined as characteristic pain reduction of 75% or greater for 1 - 4 hours following the sij block22). all sij blocks were performed by two spine fellows and the procedures of sij block was performed as described in previously report31). we excluded patients with clear nerve root compression sign (motor or sensory deficits), with proven osteoarthritis of hip with clinical symptoms, with pain related to disc or facet origin, and with signs of sacroilitis (ankylosing spondylitis, reactive arthritis, or enteropathic arthritis). to compare the characteristics of sagittal sacropelvic morphology and balance between patients with sij pain and those without sij pain after plif, one of spine fellows who was not informed of current study participated in extracting a control group (non - sij pain group consisted of patients without postoperative sij pain). at first, he randomly selected the patients who were matched for sex, age group, the number of fused level and fusion to sacrum. the ratio of case to control was 1 to 2 and we expected this frequency matching extraction would enhance statistical power6). patient clinical data (including age, sex, diagnosis, bmd and bmi) was collected by chart review. before and after operation, full - length lateral radiographs of the spine were taken. in the standing position, the patients were asked to stand straight but relaxed with elbows fully flexed, fists resting on the clavicles, and the knees fully extended as possible. not knowing whether the patient belonged to sij pain or non - sij pain group, the other spine fellow who was not involved in patient selection performed radiological assessment on a digital radiographic image displayed on a picture archiving and communication system (pacs) terminal (marosis 2003, seoul, korea). measuring angles of radiological parameters was done by using an automated program of the pacs computer software system and additionally, data regarding levels of fusion and involvement of sacrum were also collected. the lumbopelvic parameters including lumbar lordosis (ll), pelvic incidence (pi), pelvic tilt (pt) and sacral slope (ss), were assessed as reported by duval - beaupre.9) on the standing radiographs. the angle of ll was measured from the inferior endplate of t12 to the superior endplate of s1 using the cobb method. pi was defined as the angle formed by a line drawn between the center of the femoral head and the sacral endplate. pt was defined as the angle formed by a line drawn from the midpoint of the sacral endplate to the center of the bicoxofemoral axis and a vertical and a vertical plumb line. ss was defined as the angle formed by a line drawn along the endplate of the sacrum and a horizontal reference line (fig. 1). in order to evaluated the sagittal sacropelvic morphology and balance, as advocated by mac - thiong.23), the ratio of pt to pi (pt / pi), ss to pi (ss / pi) and pt to ss (pt / ss) were calculated and compared between sij pain and non - sij pain group. for comparisons of categorical variables, pearson 's chi - square test was used and continuous variables were compared either using student 's t - test after verification of normal distribution by the kolmogorov - smirnov test or using the non - parametric mann - whitney u test. to compare the changes between before and after plif surgery, paired t - test or wilcoxon signed rank test was used depending on result of normality test. the approval of the institutional review board at our hospital was obtained for the current study. between june 2009 and january 2013, a total of 452 consecutive patients who underwent posterior lumbar interbody fusion (plif) at our institution were investigated. all cases of plif surgery were performed using a peek cage packed with autologous bones obtained from lamina or spinous process and pedicle screws were inserted. there were no injury to sij and all procedures of plif were done by the same surgeon. patients were included into sij pain group if their reported postoperative pain met following criteria16,26) : pain begins with standing or sitting position after surgery and it differs from preoperative one ; unilateral or bilateral prevalence persistent pain which had compatible distribution with a sacroiliac joint (below the l5 spinous process, over the posterior aspect of one or both sijs) ; with or without radiation to the posterior thigh or groin but not below the knee ; with or without tenderness of the sacroiliac sulcus at palpation ; and no evidence of lumbar cause (no breach of screw and no residual nerve root impingement on postoperative ct scan images. then, for the patients whose symptoms were consistent with above mentioned criteria, a fluoroscopically guided sij block was performed and after block, the patient with positive response was classified into sij pain group. a positive response was defined as characteristic pain reduction of 75% or greater for 1 - 4 hours following the sij block22). all sij blocks were performed by two spine fellows and the procedures of sij block was performed as described in previously report31). we excluded patients with clear nerve root compression sign (motor or sensory deficits), with proven osteoarthritis of hip with clinical symptoms, with pain related to disc or facet origin, and with signs of sacroilitis (ankylosing spondylitis, reactive arthritis, or enteropathic arthritis). to compare the characteristics of sagittal sacropelvic morphology and balance between patients with sij pain and those without sij pain after plif, one of spine fellows who was not informed of current study participated in extracting a control group (non - sij pain group consisted of patients without postoperative sij pain). at first, he randomly selected the patients who were matched for sex, age group, the number of fused level and fusion to sacrum. the ratio of case to control was 1 to 2 and we expected this frequency matching extraction would enhance statistical power6). patient clinical data (including age, sex, diagnosis, bmd and bmi) was collected by chart review. before and after operation, full - length lateral radiographs of the spine were taken. in the standing position, the patients were asked to stand straight but relaxed with elbows fully flexed, fists resting on the clavicles, and the knees fully extended as possible. not knowing whether the patient belonged to sij pain or non - sij pain group, the other spine fellow who was not involved in patient selection performed radiological assessment on a digital radiographic image displayed on a picture archiving and communication system (pacs) terminal (marosis 2003, seoul, korea). measuring angles of radiological parameters was done by using an automated program of the pacs computer software system and additionally, data regarding levels of fusion and involvement of sacrum were also collected. the lumbopelvic parameters including lumbar lordosis (ll), pelvic incidence (pi), pelvic tilt (pt) and sacral slope (ss), were assessed as reported by duval - beaupre.9) on the standing radiographs. the angle of ll was measured from the inferior endplate of t12 to the superior endplate of s1 using the cobb method. pi was defined as the angle formed by a line drawn between the center of the femoral head and the sacral endplate. pt was defined as the angle formed by a line drawn from the midpoint of the sacral endplate to the center of the bicoxofemoral axis and a vertical and a vertical plumb line. ss was defined as the angle formed by a line drawn along the endplate of the sacrum and a horizontal reference line (fig. in order to evaluated the sagittal sacropelvic morphology and balance, as advocated by mac - thiong.23), the ratio of pt to pi (pt / pi), ss to pi (ss / pi) and pt to ss (pt / ss) were calculated and compared between sij pain and non - sij pain group. for comparisons of categorical variables, pearson 's chi - square test was used and continuous variables were compared either using student 's t - test after verification of normal distribution by the kolmogorov - smirnov test or using the non - parametric mann - whitney u test. to compare the changes between before and after plif surgery, paired t - test or wilcoxon signed rank test was used depending on result of normality test. twenty - eight patients (10 males and 18 females) with postoperative sij pain, being response to sij block, were enrolled in sij pain group. by frequency matching by the ratio of 1 to 2, fifty - six patients (20 males and 36 females) without postoperative sij pain were selected for non - sij pain group. patients in sij pain group were older and more osteoporotic than those in non - sij pain group however, they were statistically not significant (p=0.38 and 0.81, respectively) and the remaining bmi, number of fused level, involvement of sacrum, and diagnosis were similar as well (p=0.81, 0.92, 0.64, and 0.95, respectively). table 2 shows the mean value of pre- and postoperative lumbopelvic parameters in two groups. preoperatively, there were no statistically significant differences between sij pain and non - sij pain group in terms of lumbopelvic parameters including ll, pi, pt and ss (p=0.58, 0.47, 0.44 and 0.83, respectively). however, postopertively, pt of sij pain group were significantly greater (p=0.02) than that of non - sij pain group and the remaining values of lumbopelvic parameters were similar. table 3 shows the comparison of ratios among pelvic parameters in sij pain and non - sij pain group. of the calculated ratios, preoperative pt / pi, ss / pi and pt / ss showed no significant differences between sij pain and non - sij pain group (p=0.52, 0.52 and 0.28, respectively). however postoperatively, pt / pi and ss / pi in sij pain group was significantly greater and smaller than those in non - sij pain group respectively (p=0.03, 0.02, respectively) except for pt / ss (p=0.05). sij pain group did not show significant postoperative changes of pt / pi and ss / pi (p=0.09 and 0.08, respectively) while non - sij pain group showed significantly decrease of pt / pi (p=0.00) and increase of ss / pi (p=0.00) postoperatively. in terms of pt / ss, both sij pain and non - sij pain group showed significant decrease of the ratio (p=0.02 and 0.00, respectively). twenty - eight patients (10 males and 18 females) with postoperative sij pain, being response to sij block, were enrolled in sij pain group. by frequency matching by the ratio of 1 to 2, fifty - six patients (20 males and 36 females) without postoperative sij pain were selected for non - sij pain group. patients in sij pain group were older and more osteoporotic than those in non - sij pain group however, they were statistically not significant (p=0.38 and 0.81, respectively) and the remaining bmi, number of fused level, involvement of sacrum, and diagnosis were similar as well (p=0.81, 0.92, 0.64, and 0.95, respectively). table 2 shows the mean value of pre- and postoperative lumbopelvic parameters in two groups. preoperatively, there were no statistically significant differences between sij pain and non - sij pain group in terms of lumbopelvic parameters including ll, pi, pt and ss (p=0.58, 0.47, 0.44 and 0.83, respectively). however, postopertively, pt of sij pain group were significantly greater (p=0.02) than that of non - sij pain group and the remaining values of lumbopelvic parameters were similar. table 3 shows the comparison of ratios among pelvic parameters in sij pain and non - sij pain group. of the calculated ratios, preoperative pt / pi, ss / pi and pt / ss showed no significant differences between sij pain and non - sij pain group (p=0.52, 0.52 and 0.28, respectively). however postoperatively, pt / pi and ss / pi in sij pain group was significantly greater and smaller than those in non - sij pain group respectively (p=0.03, 0.02, respectively) except for pt / ss (p=0.05). sij pain group did not show significant postoperative changes of pt / pi and ss / pi (p=0.09 and 0.08, respectively) while non - sij pain group showed significantly decrease of pt / pi (p=0.00) and increase of ss / pi (p=0.00) postoperatively. in terms of pt / ss, both sij pain and non - sij pain group showed significant decrease of the ratio (p=0.02 and 0.00, respectively). this study demonstrated the differences of sagittal sacropelvic morphology and balance between the patients with / without sij pain following lumbar fusion surgery. several reports1,12,16,26) have discussed the etiology of sij pain following lumbar fusion surgery however, it is true that there lacks of postural analysis in terms of sagittal sacropelvic morphology and balance. in prior to this study, authors reported the differences of lumbopelvic parameters between the patients with / without sij pain following lumbar fusion surgery and pointed out the importance of restoring appropriate ll based on pi to prevent postoperative sij pain32). in current study, we tried to reveal the sagittal sacropelvic morphology and balance of patients with sij pain after lumbar fusion surgery by using the ratios among pelvic parameters proposed by mac - thiong.23) and discussed about the comparison with normative values in the literature. the sagittal sacropelvic morphology can be defined by pi and its two component, namely pt and ss8). pi is a morphological, individually constant parameter that did not depends on the orientation of the pelvis and the patient 's position, whereas the ss and pt are directly related to pelvic position4). pi is strongly correlated to the ss (r=0.80) and lumbar lordosis (ll, r=0.60), and the ll is strongly correlated to the ss (r=0.86)21). through these strong correlations, pi plays a central role in the determination of pelvic orientation and sagittal curvature as a high pi necessitates a high ll. it is also known that, mathematically pi is the sum of pt and ss (pi = pt+ss)21). as pt increases, the ss decreases because the sacrum assumes a more vertical position about the femoral head axis namely, pelvic retroversion. pelvic retroversion is a compensatory process to rebalance the spine and maintain upright posture in case of sagittal imbalance2). in current study, both pre- and postoperative lumbopelvic parameters including ll, pi and ss showed no statistically significant differences in sij pain and non - sij pain group. however, pt of sij pain group was significantly greater than that of sij pain group postoperatively, whereas this value was similar preoperatively. lafage.18), after prospective investigation on 125 patients with spinal deformity, revealed increased pt was correlated with worsening health related quality of life (hrqol). they explained this result as a compensatory mechanism of pelvis (increase pt, namely pelvic retroversion) to keep a balanced standing posture and insisted resetting the pt to normal values was important to restore ambulatory function. schwab.30) reported correlation analysis between radiographic spinopelvic parameters and hrqol revealed no significance in terms of coronal plane parameters and confirmed that pelvic position measured via pt correlated with hrqol in adult deformity. they also regarded high values of pt expressed compensatory pelvic retroversion for sagittal spinal malalignment. compensatory increase in pt is considered as a part of the disease process. with retroversed pelvis, this allows the c7 plumb line to be behind the vertical line passing through the center between the femoral heads and the gravity line. although body is balanced, it is less economical because posterior back muscle should be strengthened to restore ll. this is an energy consuming process that becomes easily painful and could be one of reason for back pain in the line with facet constraints and over loading15). the findings in current study indicate that increased stress across sij articular surface by hip hyper - extension and a decrease in the anterior coverage of the hip caused by pelvic retroversion18) are attributable to the development of sij pain following lumbar fusion surgery. mac - thiong.23) investigated large cohort of 709 asymptomatic adult (354 males and 355 females, mean age of 36.8 years) who did not have spinal pathology and reported the ratios among pelvic parameters. in their study, they used the ratios of pt to pi (pt / pi), ss to pi (ss / pi) and pt to ss (pt / ss) in order to evaluate the relationships between parameters of sagittal sacropelvic morphology and balance. they found pt / pi was less than 0.5 and ss / pi was more than 0.5, indicating that the pt value is normally at most half of the pi and the ss value is normally at least half of the pi in asymptomatic populations. based on this result, they insisted the values outside those ranges could potentially predispose to the development of spinal pathology. in current study, pre- and post - operative pt / pi were less than 0.5 and ss / pi were more than 0.5 in both groups and we presumed this result is related with the fact that our data were not obtained from spinal deformed patients but from patients with degenerative spinal disease. however, comparison between sij pain and non - sij pain group showed significant differences of pt / pi and ss / pi. in sij pain group, postoperative pt / pi was greater and ss / pi was lesser than non - sij pain group. moreover, in contrast to non - sij pain group, those values in sij pain group did not showed significant changes postoperatively. these data implicate that patients with sij pain have more vertical sacrum (decreased ss) and more retroversed pelvis (increased pt) than patients with non - sij pain. this finding is accordant with that of lazennec 's study. in their comparative study between 81 patients who underwent lumbosacral fusion pain and control group, they found pt was almost twice the normal value and sacrum was more vertical in post - fusion pain group. they insisted that achieving a strong fusion should not be the only goal and appropriate position of the fused vertebrae is also of paramount importance to minimize muscle work during posture maintenance. the changes of ratios of pt / pi and ss / pi indicate the changes of the sagittal sacropelvic morphology and balance. the patients without sij pain showed postoperative significant decrease and increase of pt / pi and ss / pi respectively while the patients with sij pain did not show significant differences. the possible explanation for this could be that lumbar fusion surgery failed to achieve the balanced sacropelvic morphology in sij pain group. although specific factors related to surgical procedure which bring out these differences could not be revealed in current study and we hope well designed investigation should be done in the future. notably, the ratios of pt / ss were significantly decrease in both groups. the value of pi is fixed for a given patient and as pt decreases, ss necessarily decreases thus, it is natural that the postoperative ratio of pt / ss decreases in both groups. however, we assume that the decrease of pt / ss could be the actual sign of occurrence of the sagittal sacropelvic realignment by lumbar fusion surgery, regardless of its clinical significance. so far, normative values of pelvic parameters have been reported by several authors13,23,29) and previously published studies have provided a basic understanding of the normative values that these parameters should fall within. however, most of these data has been obtained not from asian but from whites. considering the fact that there exist somewhat differences in spine between asian and whites20), it is more reasonable to use the calculated ratio rather than using absolute values for precise comparison. table 4 shows ratios among pelvic parameters of asymptomatic populations, which was adopted from previously published data7,20,24,29,33). the ratios of sij pain group was notably different from those of normal populations while the ratios of non - sij pain group was similar with those of normal populations. this is because through the lumbar fusion surgery, sagittal sacropelvic morphology of non - sij pain group could become normal. on the contrary, the pelvis and sacrum of sij pain group were still abnormal postoperatively i.e., the pelvis was retroversed and the sacrum was vertical. we believe achieving a normal sagittal sacropelvic morphology and balance are important to avoid the development of sij pain following lumbar fusion surgery. the present study has several limitations including small sample size and a retrospective case - control study rather than randomized controlled study. however, frequent matching extraction by uninformed spine fellow and radiologic measurement by the other spine fellow without information of patient 's group could minimize the chances of bias. this study demonstrated the different sagittal sacropelvic morphology and balance between the patients with / without sij pain following lumbar fusion surgery. the patients with sij pain showed retroversed pelvis and vertical sacrum compared with non - sij pain group. in addition, the patients without sij pain have similar sagittal sacropelvic morphology and balance with asymptomatic populations in the literature, while the patients with sij pain did not. the postoperative compensatory mechanism by sacropelvis and imbalance lead to great energy expenditure and give birth to the sij pain following lumbar fusion surgery. | objectiveto investigate the sagittal sacropelvic morphology and balance of the patients with sij pain following lumbar fusion.methodsamong 452 patients who underwent posterior lumbar interbody fusion between june 2009 and january 2013, patients with postoperative sij pain, being responded to sij block were enrolled. for a control group, patients matched for sex, age group, the number of fused level and fusion to sacrum were randomly selected. patients were assessed radiologic parameters including lumbar lordosis, pelvic incidence (pi), pelvic tilt (pt) and sacral slope (ss). to evaluate the sagittal sacropelvic morphology and balance, the ratio of pt / pi, ss / pi and pt / ss were analyzed.resultsa total of 28 patients with sij pain and 56 patients without sij pain were assessed. postoperatively, sij pain group showed significantly greater pt (p=0.02) than non - sij pain group. postoperatively, pt / pi and ss / pi in sij pain group was significantly greater and smaller than those in non - sij pain group respectively (p=0.03, 0.02, respectively) except for pt / ss (p=0.05). sij pain group did not show significant postoperative changes of pt / pi and ss / pi (p=0.09 and 0.08, respectively) while non - sij pain group showed significantly decrease of pt / pi (p=0.00) and increase of ss / pi (p=0.00).conclusionthis study presents different sagittal sacropelvic morphology and balance between the patients with / without sij pain following lumbar fusion surgery. the patients with sij pain showed retroversed pelvis and vertical sacrum while the patients without sij pain have similar morphologic features with asymptomatic populations in the literature. |
a 61-year - old chinese man with a history of psoriasis and alcoholic liver cirrhosis sought treatment for left ankle swelling, erythema, and tenderness. he worked as a dishwasher in a restaurant and could not recall any antecedent trauma to the affected limb. he was febrile on admission and was empirically treated with intravenous penicillin and cloxacillin for cellulitis. despite antimicrobial drug therapy, dbridement was made. during surgery, grayish necrotic fascia, extensive subcutaneous tissue necrosis, loss of resistance of the normally adherent superficial fascia to blunt dissection, and foul - smelling dishwater the tissue specimen was spread onto blood agar plate (trypticase soy agar supplemented with 5% sheep blood, bbl [becton, dickinson and company, franklin lakes, nj ]) and ashdown s media, a selective media for b. pseudomallei. characteristic colonial morphologic findings of b. pseudomallei were noted on blood agar and ashdown s media. the organism s identity was confirmed with biochemical tests (positive oxidase reaction, simmons citrate) and microscopic morphologic findings. (the automated blood culture system used was the bactec fluorescent series blood culture system [becton, dickinson and company ].) tissue (taken during surgery) and blood cultures grew b. pseudomallei susceptible to ceftazidime, amoxicillin / clavulanic acid, imipenem, chloramphenicol, and cotrimoxazole but resistant to ampicillin, amikacin, and gentamicin. results of melioidosis serologic testing were also positive. a computed tomography scan of his abdomen and pelvis showed splenic abscesses, but no prostatic abscess was noted. he was treated with ceftazidime and doxycycline ; when his fever persisted, his treatment was converted to intravenous imipenem. he responded well and was discharged after he completed a 4-week course of imipenem. on discharge, one month later, the man was readmitted with fever associated with scrotal swelling and pain. on examination, a firm, tender right prostatic nodule was noted. a transrectal ultrasound showed abscesses in both prostatic lobes. the aspirate from the prostatic abscesses and blood cultures grew b. pseudomallei with antimicrobial drug susceptibilities identical to the organism cultured from his initial admission. he also underwent transurethral resection of the prostate ; the histologic results indicated benign prostatic hyperplasia with acute or chronic inflammation. on discharge, he was prescribed oral amoxicillin - clavulanate (for 4 weeks) and doxycycline (for 1 year). seventeen months after cessation of oral antibiotics, he reappeared with a left temporal scalp abscess and fever. no organism was isolated from the scalp pus culture, but the blood culture again grew b. pseudomallei with identical antimicrobial drug susceptibilities. he was given 4 weeks of ceftazidime followed by amoxicillin - clavulanate (for 4 weeks) and doxycycline (long - term). the disease has been called the great mimicker, with a wide range of clinical syndromes including pneumonia, visceral abscesses, soft tissue infections, septic arthritis, and septicemia (1,3,5). previously reported a case of melioidosis with a parotid abscess that was drained and subsequently progressed to necrotizing fasciitis (4). although not previously reported, necrotizing fasciitis can be the initial sign of melioidosis. while cutaneous infections by b. pseudomallei are usually indolent soft tissue infections manifesting as cellulitis or abscesses (13,6), these soft tissue infections can progress to necrotizing fasciitis in melioidosis. melioidosis can remain latent for prolonged periods and reactivate with severe sepsis years after the initial infection (1,7). melioidosis is characterized by relapses or recrudescence, especially in immunocompromised patients (1,7,8). for this patient, another relapse occurred 17 months after cessation of oral maintenance antimicrobial drug therapy. for patients with a known history of melioidosis b. pseudomallei are frequently intrinsically resistant to many antimicrobial agents, including aminoglycosides and first- or second - generation cephalosporins. current recommended therapy of severe melioidosis includes intravenous ceftazidime or imipenem for 10 days to 4 weeks, followed by maintenance oral antimicrobial agents (amoxicillin - clavulanate or a combination of trimethoprim - sulfamethoxazole and doxycycline) for 10 to 18 weeks (9). in the context of soft tissue infections, patients with a history of melioidosis developing cutaneous septic foci should be treated promptly and aggressively with antibiotics against b. pseudomallei in addition to the common organisms responsible for these infections such as staphylococci aureus and streptococcal species. in this setting, an intravenous regime consisting of crystalline penicillin and cloxacillin may not be the appropriate initial antimicrobial drug regime (3). awareness that soft tissue infection can progress to necrotizing fasciitis, a life- and limb - threatening condition, is important, and a high index of suspicion should be maintained. when necrotizing fasciitis is suspected, magnetic resonance imaging of the affected area should be performed to ensure early diagnosis, and aggressive dbridement of all nonviable tissue should follow for an improved outcome (10). melioidosis is a major problem and a rampant disease in rural parts of central and south america, north australia, and southeast asia. in northeast thailand, it is responsible for 20% to 40% of deaths from community - acquired septicemia (11). its true incidence is probably underestimated due to under detection and limited availability of culture facilities (12). awareness of the spectrum of soft tissue infections caused by b. pseudomallei, including the distinct possibility of necrotizing fasciitis at the extreme of the spectrum, is important. the successful management of necrotizing fasciitis requires appropriate antimicrobial drug selection and aggressive operative dbridement. | in areas where melioidosis is endemic, stress on the healthcare system is substantial. because clinical manifestations are protean, the illness is difficult to diagnose, and cutaneous burkholderia pseudomallei infections can progress to necrotizing fasciitis. while it is an uncommon complication of cutaneous melioidosis, necrotizing fasciitis is potentially fatal and requires successful management, including early diagnosis, appropriate antibiotics selection, and operative dbridement. |
in rats, of the wistar - porton strain, a single intravenous injection of 1.25 mg cd2 + between days 9 and 15 of gestation results in a high incidence (80% of hydrocephalus, together with other malformations in the fetuses, examined on day 20. this dose is critical, since 1.1 mg cd2+/kg is not teratogenic, while 1.35 mg cd2+/kg kills all the embryos. intravenous injection of cd2 + to the pregnant rat on day 12 causes a dose - dependent inhibition of placental zn2 + transport. at the teratogenic dose, zn2 + transport is inhibited by about 75% at 4 hr. thereafter, inhibition decreases with time but is still significant at 48 hr. at 20 hr after administration of cd2 + the embryonic concentration of zn2 + is depressed by 33%. in the whole embryo the activity of the zn2 + -dependent thymidine kinase is inhibited by about 60% at 4 hr and at 20 hr the dna concentration is reduced significantly. placental transport of 14c - leucine and 14c - uridine, as well as the embryonic incorporation of these precursors into protein and rna is unaffected at least at short times after the administration of cd2 +. it is possible therefore, that the teratogenic effects of cd2 + may be related to the inhibition of dna synthesis in the embryo.imagesfigure 1.figure 2. |
|
aneurysmal bone cyst (abc) is the rare, benign, vascular, multicystic lesion of the bone with unknown origin that usually involves the long bone and spine4,11). venous channel in the diploic space is enlarged, which caused destruction of the cortical bone. about 20% of abc occurs at the spine, but rarely in the cranium with the incidence of 1 to 6%5,11,19). clinical symptoms and signs of orbital abc is similar to those of other orbital mass, and radiological diagnosis can be made with computed tomographic (ct) images and magnetic resonance (mr) images, and plain films. after initial symptoms and signs noted, it progress slowly for a few months, followed by rapid - deterioration. the proper management is complete surgical excision, and any remnant lesion is the causes of recurrence. abc is highly vascular tumor and attention should be paid to the excessive blood loss during surgery11 - 13). authors experienced a rare case of orbital abc with intracranial extension, which was treated by surgical intervention and present its clinical, pathological, and radiological characteristics with literature review. a 12-year - old female patient presented to the ophthalmic clinic with mild proptosis, limitation of extraocular muscle movement (eom), and inferior eyeball deviation of the right eye. mr images showed a multiloculated mass, about 2.52.4 centimeter in the upper part of right eye which compressed right eyeball. the cyst was showed low - signal intensity on t1-weighted image (t1wi), and iso - signal intensity on t2-weighted image and flair image. cystic wall was enhanced strongly after injection of gd - dtpa (gadolinium enhancement) (fig. five months later, exophthalmos progressed rapidly with conjuctival ecchymosis, limitation of eom, and downward eyeball deviation, and she was refered to neurosurgery department. right eyeball was fixed in downward gaze state and visual acuity was 20/200 (fig. plain films of the skull showed radiolucent lesion in the orbital roof of right eye that was surrounded by thin marginal shell of sclerotic cortical bone (fig. both inner and outer tables were thinned, but their integrity was preserved (fig. brain mr images showed a enlarged mass, about 4.44.0 centimeter which extended superior to frontal lobe and inferior to right eyeball with compression. a well - circumscribed multicystic mass with peripheral hypointense capsule and internal hypointense septation on t1wi. the cystic cavities were filled with heterogenous, but hyperintense hemorrhagic fluid - fluid levels on all sequence. the dura of the frontal base was detached off the orbital roof which was tumor itself, was destructed severely, and was extended intracranially. orbital mass was removed completely, and resection was continued more deeply into the optic foramen. it was removed totally under the surgical microscope with special attention to preserve the optic nerve. bony margins, which was suspected portion to have the pathological change, were also totally resected. after removing the tumor, orbital roof was reconstructed using codman cranioplastic kits, leibinger plate, and polybone. the patient refused transfusion because of the religious reason, the surgery was planned according to the " bloodless surgery " manual. during surgery, cell saver (autologous blood recovery system) was used to collect the blood in operating field. the autologous blood transfusion was delivered during surgery, and any surgical bleeding was controlled immediately, using electrical coagulator and bone wax, and surgical procedures was performed as fast as possible. as for pathologic findings, blood - containing cystic spaces were separated by fibroblasts and reactive bone formation was also discovered. exophthalmos was subsided, limitation of eom was restored, and visual acuity improved to 20/33 on 14 days after the operation. follow - up mr image taken 7 months after the operation revealed no evidence of mass in the orbit (fig. mild downward eyeball deviation was remained, and diplopia was presented on left lateral gaze with minimal disturbance of eye motions on medical and upward gaze (fig. aneurysmal bone cyst causes lytic changes in the diploic space of bone, filled with products of blood components, destructing cortical bone11 - 14). in 1942, jaffe and lichitenstein first described the term abc as " peculiar blood - containing cysts of large size"13). most of abc involve the metaphysis of the long bone, such as a femur, and one - fifth of abc involve the spine3,5 - 7). only 1 to 6% of the cases was reported to occur in the cranial bone4,11,19). in up to 85% of cases, it occurs at the age of 20 or less, a mean age of 13. the cortical bone is expanded and destructed by the venous channel with increased pressure in the diploic space, even though the precise nature and origin of abc is unclear13). abc is defined as a distinct entity, classified into primary or secondary according to the presence or absence of an associated lesion, such as giant cell tumor, chondroblastoma, osteoblastoma, osteosarcoma, chondromyxoid fibroma, and fibrous dysplasia2,13,14,18). it causes local hemodynamic disturbance, like arterio - venous shunts or malformations, which would increase venous pressure. these lead the subsequent bony resorption and destruction of the vascular bed, which would form abc eventually11). the first cranial abc was reported by odeku and mainwaring in 1965, which occurred in occipital bone, presenting painful swelling on occipital area. surgical resection was attempted, but the patient died of cardiac arrest unfortunately. in the cranium, the vault is the frequent site for abc, but the sphenoid, ethmoid, and orbit are rarely involved. cranial abc causes destruction of both inner and outer table of the skull, resulting in painful or painless mass by external expansion and neurological symptoms by compressing the brain or cranial nerve4,11,16,18). the first abc involving the orbit was reported by fite dj. in 19684). eight - year - old female patient was presented with exophthalmos of the right eye, which was treated by total tumor resection. thereafter, orbital involvement was reported in 25 cases1,4,11,12,14,15,19,24). among these 25 cases, orbital roof was involved in 19 cases, and medial wall was involved in 4 cases, lateral wall 2 cases. exophthalmos is noted in all cases, and diplopia in 7 cases, decreased vision in 6 cases. tumor was removed totally in 19 cases and recurrence was noted in 2 of 19 (10.52%). subtotal resection was performed in 5 of 25 orbital abcs, and tumor recurred in all cases. in author 's case, tumor was resected totally and recurrence was not noted at 13 months after surgery4,11,14,18). after initial diagnosis, the progression of abc is gradual in the first several months, followed by rapid clinical deterioration12). of the 25 cases of orbital abc, period of initial slow - progression was average 14 weeks (8 days to 6 months), and then clinical symptoms and signs deteriorated rapidly, causing painful exophthalmos, ptosis, diplopia, limitation of eom and decreased vision by compressing on the orbital contents such as extraocular muscles, optic and oculomotor nerve. many patients consulted the doctor during the phase of rapid growth. in most cases, this suggests the importance of early treatment before neurological deficit develops4,11 - 13). in the diagnosis of abc, aneurysmal bone cyst was named according to the features of plain skull films, that is single or multiple blown - out lytic lesion. the lytic lesion is surrounded by a thin marginal shell of sclerotic cortical bone4,8). on the computed tomography of brain, it appears as an expansive multioculated osseous lesion with the enhancement on peripheral capsule and internal septation of lesion after intravenous contrast11,12,18). the lesions may be surrounded by a fibrous capsule with hypointense signal intensity on all sequences. and, they may have internal septations with hypointense signal intensity, separating multiple cystic components2,11,15,22,23). if associated fibro - osseous lesions are shown on ct and/or mri, the diagnosis of abc should be strongly suspected11). angiographic features can sometimes reveal the blood supply to these vascular lesions and may also occasionally reveal arteriovenous shunts. gross pathological evaluation typically shows an abc to be composed of individual cysts filled with unclotted liquid blood and blood - tinged serous fluid. microscopically, abc appears as blood - filled cavernous spaces with a paucity of endothelial cells. the cysts are seperated by septa composed of fibrous tissue which contains multinucleated giants cells and possibly osteoid tissue2,8,17,19). total surgical excision is the treatment of choice in the management of abc, and any remnant lesion is the reason of recurrence. it is related to the age of the patient, size of the lesion, the presence of mitosis, and more importantly, the incompleteness of the resection2,5,9,13,19). in the cranium and the spine, there is also an added difficulty in that it is often impossible to reach and excise the lesion completely. this is especially true if the lesion is located in the skull base, for example in the roof, the medial and lateral walls of the orbit, the paranasal sinuses, and the petrous temporal bone. in these cases, partial excision or intralesional curettage with adjunctive therapy such as preoperative embolization or postoperative radiotherapy or stereotactic radiosurgery also, abc is highly vascular tumor and special attention should be paid to the possible excessive blood loss during surgery, and preoperative embolization may be helpful to prevent blood loss. in conclusion, abc is rare benign vascular lesion of bone that can occur very rarely in the orbit. it may cause local swelling of bone and surrounding tissue as well as symptoms including diplopia, limitation of eom, and headache. authors report a rare case of abc involving the orbit, which was treated by surgical excision with good result. | aneurysmal bone cyst (abc) is benign vascular lesion destructing the cortical bone by the expansion of the vascular channel in the diploic space that usually involve long bone and spine. orbital abc is rare and the clinical symptoms deteriorate rapidly after initial slow - progression period for a few months. a 12-year - old female patient visited ophthalmologist due to proptosis and upward gaze limitation of the right eye, and orbital mass was noted in the upper part of right eye on orbital mri. five months later, exophthalmos was worsened rapidly with other features of ophthalmoplegia. orbital mass was enlarged on mri with intracranial extension. surgery was done through frontal craniotomy and intracranial portion of the tumor was removed. destructed orbital roof and mass in the orbit was also removed, and surrounding bone which was suspected to have lesion was resected as much as possible. histopathological diagnosis was aneurysmal bone cyst. postoperative course was satisfactory and the patient 's eye symptoms improved. authors report a rare case of orbital abc with review of the literature. exact diagnosis by imaging studies is important and it is recommended to perform surgical resection before rapid - progressing period and to resect the mass completely to prevent recurrence. |
atrial fibrillation (af) is one of the most common cardiac arrhythmias and causes a 5- to 7-fold increased risk of ischemic stroke and mortality. although warfarin is the most commonly prescribed oral anticoagulant worldwide since 1950, and despite the strong evidence of stroke prevention in patients with nonvalvular atrial fibrillation (nvaf), it remains underused in the real world, particularly china. the common known reasons for warfarin underuse include a narrow therapeutic window, multiple interactions with a variety of common medicines and foods and inconsistent pharmacokinetics and pharmacodynamics that can cause hemorrhage. in addition, specific warfarin - related renal damage has recently garnered attention and has been reported in patients with or without chronic kidney disease, further preventing its use in af patients. on the other hand, a retrospective study revealed that long - term warfarin therapy for as long as 18 months delays the deterioration of kidney function and achieves a longer survival time in older patients with ckd and af. the effects of warfarin on the kidneys reportedly involve not only renal tubular obstruction by red blood cell, but also other potential mechanisms, such as oxidative stress damage to the renal tubules, inhibition of the activation of growth arrest - specific gene 6 products to protect the kidney. warfarin, a vitamin k antagonist, has been proven to inhibit glomerular mesangial cells by interfering with the activation of growth arrest - specific gene 6 products, which stimulates glomerular mesangial cell proliferation and hypertrophy. however, because of the lack of prospective studies, the effects of warfarin on renal function in nvaf patients remain unclear. therefore, we conducted this prospective study to evaluate the effects of warfarin on renal function and explore the factors associated with kidney dysfunction in adult patients with electrocardiography - detected nvaf and no dialysis therapy. from january 2011 to december 2013, 951 nvaf patients from 18 hospitals led by beijing anzhen hospital were enrolled. all patients in the study were screened according to the following criteria : diagnosis of nvaf, no history of anticoagulant therapy before enrolment and available baseline, and multiple follow - up serum creatinine (scr) levels. patients with consumption of warfarin or no anticoagulant for <3 months, metastatic cancer, dementia, cirrhosis, renal failure caused by end - stage renal disease or requiring dialysis, previous hemorrhagic disease, and/or peptic ulcers were excluded. this prospective observational cohort study was approved by the ethics committee of beijing anzhen hospital, constituted in accordance with the national health and medical research council guidelines. the risk of stroke was estimated using the chads2(c - cardiac failure, h - hypertension, a - age 75 years, d - diabetes mellitus, and s - stroke) score derived as follows : congestive heart failure (chf) (1 point), hypertension (1 point), age 75 years (1 point), diabetes mellitus (1 point), and previous stroke or tia (2 points). to assess the estimated glomerular filtration rate (egfr), scr levels of all eligible patients were measured at baseline and at 3, 6, 12, 18, and 24 months, up to the end of the observation period. all egfr values available from enrollment to the end of the observation period were included in our calculations. egfr was calculated using the modified glomerular filtration rate estimating equation for chinese patients : egfr (mlmin1.73 m) = 186 (scr [mol / l ] 0.0113) age 0.742 (if female) 1.233 (if chinese). the decrease ratio was calculated as follows : (first egfr last egfr)/(first egfr) 100%. the study endpoint was a 25% decline in egfr from baseline during the follow - up period, which suggested the deterioration of renal function according to the national kidney foundation 's kdigo guidelines of 2012. the patients having taken warfarin were monitored for their international normalized ratio (inr) values at least every 2 weeks for the first 3 months and at least monthly thereafter, with an inr target of 23. all patients were followed every 36 months at the cardiology clinic or by telephone, and their data were recorded under strict surveillance. all analyses were conducted using sas 9.2 version (sas institute, cary, nc, usa). data are expressed as mean standard deviations (sds) for normally distributed continuous variables and as proportions for categorical variables. baseline values and time - independent outcomes were compared between the two groups using chi - square tests (for categorical data) or two - sample independent t - tests (for continuous data). log - rank tests were used to determine statistical significance (set at p < 0.05). univariate and multivariate cox regression analyses of the various clinical variables were performed to identify the predictors of a 25% decline in egfr. stepwise models of the candidate variables were used to determine the final variables for inclusion in the multivariate models ; these included variables with a p < 0.2 in univariate analysis and clinically relevant variables such as age, gender, hazard ratio (hr), systolic blood pressure (sbp) 140 mmhg (1 mmhg = 0.133 kpa), chads2 score and history of af ablation, stroke / transient ischemic attack (tia), hypertension, diabetes, chf, coronary heart disease (chd), hypertrophic cardiomyopathy, dilated cardiomyopathy, smoking, -blocker use, angiotensin - converting enzyme inhibitor / angiotensin receptor blocker use, and statin use. a value of p < 0.05 was considered statistically significant. from january 2011 to december 2013, 951 nvaf patients from 18 hospitals led by beijing anzhen hospital were enrolled. all patients in the study were screened according to the following criteria : diagnosis of nvaf, no history of anticoagulant therapy before enrolment and available baseline, and multiple follow - up serum creatinine (scr) levels. patients with consumption of warfarin or no anticoagulant for <3 months, metastatic cancer, dementia, cirrhosis, renal failure caused by end - stage renal disease or requiring dialysis, previous hemorrhagic disease, and/or peptic ulcers were excluded. this prospective observational cohort study was approved by the ethics committee of beijing anzhen hospital, constituted in accordance with the national health and medical research council guidelines. the risk of stroke was estimated using the chads2(c - cardiac failure, h - hypertension, a - age 75 years, d - diabetes mellitus, and s - stroke) score derived as follows : congestive heart failure (chf) (1 point), hypertension (1 point), age 75 years (1 point), diabetes mellitus (1 point), and previous stroke or tia (2 points). to assess the estimated glomerular filtration rate (egfr), scr levels of all eligible patients were measured at baseline and at 3, 6, 12, 18, and 24 months, up to the end of the observation period. all egfr values available from enrollment to the end of the observation period were included in our calculations. egfr was calculated using the modified glomerular filtration rate estimating equation for chinese patients : egfr (mlmin1.73 m) = 186 (scr [mol / l ] 0.0113) age 0.742 the decrease ratio was calculated as follows : (first egfr last egfr)/(first egfr) 100%. the study endpoint was a 25% decline in egfr from baseline during the follow - up period, which suggested the deterioration of renal function according to the national kidney foundation 's kdigo guidelines of 2012. the patients having taken warfarin were monitored for their international normalized ratio (inr) values at least every 2 weeks for the first 3 months and at least monthly thereafter, with an inr target of 23. all patients were followed every 36 months at the cardiology clinic or by telephone, and their data were recorded under strict surveillance. all analyses were conducted using sas 9.2 version (sas institute, cary, nc, usa). data are expressed as mean standard deviations (sds) for normally distributed continuous variables and as proportions for categorical variables. baseline values and time - independent outcomes were compared between the two groups using chi - square tests (for categorical data) or two - sample independent t - tests (for continuous data). log - rank tests were used to determine statistical significance (set at p < 0.05). univariate and multivariate cox regression analyses of the various clinical variables were performed to identify the predictors of a 25% decline in egfr. stepwise models of the candidate variables were used to determine the final variables for inclusion in the multivariate models ; these included variables with a p < 0.2 in univariate analysis and clinically relevant variables such as age, gender, hazard ratio (hr), systolic blood pressure (sbp) 140 mmhg (1 mmhg = 0.133 kpa), chads2 score and history of af ablation, stroke / transient ischemic attack (tia), hypertension, diabetes, chf, coronary heart disease (chd), hypertrophic cardiomyopathy, dilated cardiomyopathy, smoking, -blocker use, angiotensin - converting enzyme inhibitor / angiotensin receptor blocker use, and statin use. by the end of december 2013, a total of 951 af subjects were enrolled in the study. the eligible patients were then divided by observation into a warfarin group with 655 (68.9%) patients and a no anticoagulation group with 296 (31.1%) patients. the patients in the no anticoagulation group were older than those in the warfarin group. the level of sbp, number of sbp 140 mmhg, and chads2 scores were lower in the warfarin group than those in no anticoagulation group. moreover, the number in a history of chf, hypertension, diabetes, stoke / tia, and chd were less in the warfarin group than those in no anticoagulation group. there were no significant differences in gender, egfr and diastolic blood pressure values, a history of hypercholesterolemia, and -blocker use between the two groups. the use of statins and renin angiotensin system inhibitors was more frequent in the no anticoagulation group, while the use of antiarrhythmics was more frequent in the warfarin group. baseline characteristics of nvaf patients receiving warfarin therapy and those without any anticoagulation therapy data are presented as mean sds or n (%). : t values ; : values ; nvaf : nonvalvular atrial fibrillation ; bmi : body mass index ; egfr : estimated modified glomerular filtration rate ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; af : atrial fibrillation ; scr : serum creatinine ; chf : congestive heart failure ; tia : transient ischemic attack ; chd : coronary heart disease ; hcm : hypertrophic cardiomyopathy ; dcm : dilated cardiomyopathy ; chads2 : c - cardiac failure, h - hypertension, a - age 75 years, d - diabetes mellitus, s - stroke ; acei : angiotensin - converting enzyme inhibitor ; arbs : angiotensin receptor blockers ; sds : standard deviations. after an average of 19.8 10.8 months ' follow - up, 120 (12.6%) patients experienced renal endpoint. there was no significant difference of 25% decline in egfr between the warfarin group and the anticoagulation group [11.9% vs. 14.2%, log - rank p = 0.673, figure 1 ]. but a kaplan meier curve showed a significant difference in renal endpoint between patients with sbp < 140 mmhg and sbp 140 mmhg [= 4.903, log - rank p = 0.027, figure 2 ]. kaplan meier survival curve for time to a 25% decline in estimated glomerular filtration rate in nonvalvular atrial fibrillation patients receiving warfarin therapy and those without any anticoagulant therapy. meier survival curve for time to a 25% decline in estimated glomerular filtration rate in nonvalvular atrial fibrillation patients systolic blood pressure < 140 mmhg and those systolic blood pressure 140 mmhg (p < 0.05). in univariate cox regression analysis, variates of female, egfr, sbp, sbp 140 mmhg, and hypertension, respectively, predicted the incidence of 25% decrease in egfr in nvaf patients. multivariate cox regression analyses [table 2 ] revealed egfr and sbp as independent predictors of a 25% decline in egfr, with warfarin therapy found not to be a risk factor for this renal endpoint in nvaf patients. univariate and multivariate cox proportional hazard regression analyses for a 25% decline in egfr ci : confidence interval ; bmi : body mass index ; egfr : estimated modified glomerular filtration rate ; sbp : systolic blood pressure ; scr : serum creatinine ; chf : congestive heart failure ; tia : transient ischemic attack ; chd : coronary heart disease ; hcm : hypertrophic cardiomyopathy ; dcm : dilated cardiomyopathy ; chads2 : c - cardiac failure, h - hypertension, a - age 75 years, d - diabetes mellitus, s - stroke ; acei : angiotensin - converting enzyme inhibitor ; arbs : angiotensin receptor blockers. by the end of december 2013, a total of 951 af subjects were enrolled in the study. the eligible patients were then divided by observation into a warfarin group with 655 (68.9%) patients and a no anticoagulation group with 296 (31.1%) patients. the patients in the no anticoagulation group were older than those in the warfarin group. the level of sbp, number of sbp 140 mmhg, and chads2 scores were lower in the warfarin group than those in no anticoagulation group. moreover, the number in a history of chf, hypertension, diabetes, stoke / tia, and chd were less in the warfarin group than those in no anticoagulation group. there were no significant differences in gender, egfr and diastolic blood pressure values, a history of hypercholesterolemia, and -blocker use between the two groups. the use of statins and renin angiotensin system inhibitors was more frequent in the no anticoagulation group, while the use of antiarrhythmics was more frequent in the warfarin group. baseline characteristics of nvaf patients receiving warfarin therapy and those without any anticoagulation therapy data are presented as mean sds or n (%). : t values ; : values ; nvaf : nonvalvular atrial fibrillation ; bmi : body mass index ; egfr : estimated modified glomerular filtration rate ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; af : atrial fibrillation ; scr : serum creatinine ; chf : congestive heart failure ; tia : transient ischemic attack ; chd : coronary heart disease ; hcm : hypertrophic cardiomyopathy ; dcm : dilated cardiomyopathy ; chads2 : c - cardiac failure, h - hypertension, a - age 75 years, d - diabetes mellitus, s - stroke ; acei : angiotensin - converting enzyme inhibitor ; arbs : angiotensin receptor blockers ; sds : standard deviations. after an average of 19.8 10.8 months ' follow - up, 120 (12.6%) patients experienced renal endpoint. there was no significant difference of 25% decline in egfr between the warfarin group and the anticoagulation group [11.9% vs. 14.2%, log - rank p = 0.673, figure 1 ]. but a kaplan meier curve showed a significant difference in renal endpoint between patients with sbp < 140 mmhg and sbp 140 mmhg [= 4.903, log - rank p = 0.027, figure 2 ]. kaplan meier survival curve for time to a 25% decline in estimated glomerular filtration rate in nonvalvular atrial fibrillation patients receiving warfarin therapy and those without any anticoagulant therapy. kaplan meier survival curve for time to a 25% decline in estimated glomerular filtration rate in nonvalvular atrial fibrillation patients systolic blood pressure < 140 mmhg and those systolic blood pressure 140 mmhg (p < 0.05). in univariate cox regression analysis, variates of female, egfr, sbp, sbp 140 mmhg, and hypertension, respectively, predicted the incidence of 25% decrease in egfr in nvaf patients. multivariate cox regression analyses [table 2 ] revealed egfr and sbp as independent predictors of a 25% decline in egfr, with warfarin therapy found not to be a risk factor for this renal endpoint in nvaf patients. univariate and multivariate cox proportional hazard regression analyses for a 25% decline in egfr ci : confidence interval ; bmi : body mass index ; egfr : estimated modified glomerular filtration rate ; sbp : systolic blood pressure ; scr : serum creatinine ; chf : congestive heart failure ; tia : transient ischemic attack ; chd : coronary heart disease ; hcm : hypertrophic cardiomyopathy ; dcm : dilated cardiomyopathy ; chads2 : c - cardiac failure, h - hypertension, a - age 75 years, d - diabetes mellitus, s - stroke ; acei : angiotensin - converting enzyme inhibitor ; arbs : angiotensin receptor blockers. the long - term follow - up of nvaf patients without dialysis therapy revealed that warfarin therapy had no relation to increase the risk of a 25% decline in egfr compared with no anticoagulation. furthermore, after adjustment for potential clinical risk factors for renal dysfunction, baseline egfr and sbp were found to be the risk factors associated with the deterioration of kidney function in chinese nvaf patients. despite its relatively unpredictable response, narrow therapeutic range and drug interactions, warfarin is the most widely prescribed oral anticoagulant for patients with af, deep vein thrombosis or thrombi in other vascular beds, and antiphospholipid syndrome or a cardiac valve replacement. bleeding is the major adverse effect of warfarin therapy, but other nonhemorrhagic adverse reactions such as warfarin - induced allergic interstitial nephritis and warfarin - related nephropathy are also being reported. though there are many reports of renal damage caused by anticoagulation with warfarin, chang. conducted a retrospective study and found that after controlling for inr (1.95 1.01 ; goal, 23) and adjusting for potential confounders, warfarin therapy over 18 months could decrease the rate of deterioration of kidney function in older patients with ckd and af. to our knowledge, there is no prospective study demonstrating whether or not warfarin therapy is associated with damage of renal function in chinese nvaf patients. after following a large number of patients and adjusting all potential risk factors for renal dysfunction in cox regression model, we discovered that warfarin was not associated with the deterioration of renal survival duration of nvaf patients. in this respect, the results of our prospective study were different from known previous studies. even though our study was a prospective observational study, it was difficult to control factors at baseline and after treatment initiation in the two groups. the patients in the no anticoagulation group exhibited more severe clinical features compared with those in the warfarin group, such as an older age, higher sbp values and more comorbidities, all of which may confuse the possible effects of warfarin on kidney function. therefore, appropriate statistical analyses methods for long - term follow - up data were indispensable to evaluate the renal outcomes and treatment effectiveness, and an effective multivariate cox model was considered essential for our study. after adjusting for all confounding factors in multivariate cox regression analyses, we observed that baseline egfr and sbp were risk factors associated with kidney dysfunction and/or aggravated its deterioration in nvaf patients. in a prospective cohort study of type 2 diabetic mellitus, sbp is one of the most powerful independent risk factors for a rapid renal function decline. in our prospective study, first, the number of patients in the current study was not large enough with inevitable confounding factors ; therefore, the association between warfarin therapy and exacerbation in renal function may not be causal. second, the follow - up period and number of patients with renal dysfunction were limited. the long follow - up period and large number of patients with renal dysfunction may influence the significance of our results in future. third, as an important risk factor for cardiovascular disease and impaired renal function, proteinuria values were missing and not included for the study. further studies are necessary to discover the effects of warfarin therapy and no anticoagulant therapy with regard to different degrees of renal function deterioration in nvaf patients. in conclusion, the results of our study of a chinese cohort suggest that baseline egfr and sbp are associated with the deterioration of kidney function while warfarin is not the risk factor associated with kidney function deterioration in nvaf patients without dialysis therapy. this work was supported by grants from the beijing municipal administration of hospitals clinical medicine development of special funding support (no. zy201302), the ministry of s&t program of chinese arrhythmia registration study (no. this work was supported by grants from the beijing municipal administration of hospitals clinical medicine development of special funding support (no. zy201302), the ministry of s&t program of chinese arrhythmia registration study (no. | background : warfarin is the most common oral anticoagulant to decrease the stroke risk associated with atrial fibrillation (af). there are very few prospective studies that have explored whether warfarin has an association with damage on renal function in chinese patients with nonvalvular af (nvaf). the aim of this study was to evaluate the effects of warfarin on renal function and study the factors associated with kidney dysfunction in chinese adult nvaf patients without dialysis therapy.methods:from january 2011 to december 2013, a total of 951 nvaf patients from 18 hospitals were enrolled. the estimated glomerular filtration rate (egfr) was calculated from baseline and follow - up serum creatinine levels. kaplan meier survival curves compared the survival of a 25% decline in egfr (hereafter, endpoint), while cox models estimated hazard ratios (hrs) and 95% confidence intervals for this event after adjustment for age, gender, and selected potential risk factors for renal dysfunction. cox regression analysis of the various clinical potential variables was performed to identify the predictors of a 25% decline in egfr.results:after a 58-month follow - up, 951 nvaf patients were divided by observation into warfarin (n = 655) and no anticoagulation groups (n = 296) and 120 (12.6%) patients experienced renal endpoint. kaplan meier survival curves showed that the survival period was not different in the two groups (2 = 0.178, log - rank p = 0.67), but patients with systolic blood pressure (sbp) < 140 mmhg have significant difference with patients with sbp 140 mmhg (2 = 4.903, log - rank p = 0.03). multivariate cox regression analysis revealed baseline egfr and sbp as independent predictors of the endpoint, with hrs of 1.00, and 1.02, respectively.conclusion:in patients with nvaf, egfr and sbp are associated with the deterioration of kidney function while warfarin is not the risk factor of the 25% decline in egfr.trial registration : chinese clinical trial registry (no. chictr - och-13003729) ; http://www.chictr.org.cn/showproj.aspx?proj = 5831. |
posterior reversible encephalopathy syndrome (pres) is a clinico - radiological entity associated with various conditions such as eclampsia, severe hypertension, and cytotoxic medications. clinically, the syndrome is usually reversible, but delayed diagnosis and failure to treat hypertension promptly may lead to ischemia, hemorrhage, and nonaneurysmal subarachnoid hemorrhage (sah) resulting in permanent damage to the affected brain tissue. radiologically, pres typically affects the cortex and subcortical white matter within the parietooccipital or posterior frontal lobes. the brainstem, thalamus, basal ganglia, and periventricular white matter are less commonly affected. we describe a patient with atypical pres involving the brainstem, thalamus, and periventricular white matter without cortical or subcortical edema of the parietooccipital lobe on magnetic resonance (mr) imaging, with rupture of a concomitant cerebral aneurysm. a 47-year - old woman with a 24-day history of hypertension followed by a headache was referred to our neurosurgery department. three days prior, she had been started on oral antihypertensive therapy with azilsartan 20 mg a day at a local hospital, and the patient 's headache improved gradually with the antihypertensive therapy. on arrival, fluid - attenuated inversion recovery (flair) and t2-weighted mr imaging revealed high - intensity lesions in the pons, bilateral thalamus, bilateral basal ganglia, and periventricular white matter. diffusion - weighted mr imaging detected no signal abnormalities in these regions, except in the bilateral basal ganglia, which exhibited low intensity [figure 1 ]. mr angiography revealed a saccular aneurysm located in the right internal carotid artery [figure 2 ]. she was not admitted to our hospital at this time because mr imaging did not reveal sah or acute ischemic lesions, and neurological examination revealed no deficits. two days later, she experienced severe headache and was admitted to our hospital. on arrival, flair revealed slightly high - density lesions in the right side of the sylvian cistern and basal cistern consistent with sah as well as high - intensity lesions in the pons, bilateral thalamus, bilateral basal ganglia, and periventricular white matter [figure 3 ]. two days after the onset of sah, she successfully underwent neck clipping of an aneurysm via a craniotomy under general anesthesia. intraoperatively, we did not identify local hemosiderin staining of the pial surface immediately adjacent to the aneurysm dome suggestive of a previous aneurysm rupture. her systolic and diastolic blood pressure were controlled with intravenous nicardipine and/or oral antihypertensive agents to < 150 and 100 mmhg, respectively. follow - up mr imaging performed 2 weeks later revealed diminution of the lesions [figure 4 ]. fluid - attenuated inversion recovery magnetic resonance images showing slightly high - density lesions in the right side of the sylvian cistern and the basal cistern consistent with subarachnoid hemorrhage (white arrows) and high - intensity lesions in the pons, bilateral thalamus, bilateral basal ganglia, and periventricular white matter (a) ; diffusion - weighted magnetic resonance images showing no signal abnormalities in the pons, bilateral thalamus, or periventricular white matter, but low intensity in the bilateral basal ganglia (b) magnetic resonance angiography showing a saccular aneurysm located in the right internal carotid artery fluid - attenuated inversion recovery magnetic resonance images showing a high - intensity lesion in the right sylvian cistern and the right side of the basal cistern consistent with subarachnoid hemorrhage (white arrows) follow - up magnetic resonance images performed 2 weeks later showing diminution of the lesions in the present case, flair mr imaging revealed high - intensity lesions in the pons, bilateral thalamus, and periventricular white matter, although diffusion - weighted mr imaging revealed no abnormal findings in those lesions. pres typically affects the cortex and subcortical white matter within the parietooccipital or posterior frontal lobes. the exact mechanisms underlying selective vulnerability of the brainstem, basal ganglia, and thalamus are not understood. it has been reported that mild hypertension induces edema predominantly in the supratentorial white matter, whereas severe hypertension induces vasogenic edema in the infratentorial structures, basal ganglia, and thalamus, and the mean arterial blood pressure of patients with severe hypertension typically exceeds 150 mmhg, which suggests that severe acceleration of hypertension is required for atypical pres. in this case, the patient 's mean arterial pressure blood pressure exceeded 150 mmhg before and after oral antihypertensive therapy. this syndrome is related to hypervolemia, hypertension, and hemodilution therapy (triple h) for cerebral vasospasm following sah. kuroda. reported a case involving a patient who developed pres, prior to induction of hypertensive therapy, 2 days after the onset of an sah due to a ruptured intracranial aneurysm. acute hypertension caused by a catecholamine surge at the time of aneurysm rupture stimulates a sympathetic storm, leading to an abrupt increase in blood pressure. furthermore, we did not intraoperatively identify local hemosiderin staining of the pial surface immediately adjacent to the aneurysm dome suggestive of a previous aneurysm rupture. thus, we speculated that sustained high blood pressure after onset of atypical pres can lead to concomitant aneurysm rupture. although pres is rarely associated with sah, to our knowledge, rupture of a concomitant cerebral aneurysm following pres has not been reported. we describe a patient with atypical pres with rupture of a concomitant cerebral aneurysm in the early stages of antihypertensive therapy for pres involving the brainstem, thalamus, and periventricular white matter without cortical or subcortical edema of the parietooccipital lobe on mr imaging. although the syndrome is generally a reversible phenomenon with antihypertensive therapy, failure to lower blood pressure and delayed diagnosis may have led to the concomitant aneurysm rupture in this patient. in the future treatment of hypertensive urgency with recurrence of symptoms and mean arterial blood pressure < 150 mmhg, it is advisable to immediately hospitalize the patient for aggressive blood pressure management, especially if pres is suspected based on clinical and radiological features. | although posterior reversible encephalopathy syndrome (pres) is rarely associated with subarachnoid hemorrhage, to our knowledge, rupture of a concomitant cerebral aneurysm following pres has not been reported. we describe a patient with atypical pres involving the brainstem, thalamus, and periventricular white matter without cortical or subcortical edema of the parietooccipital lobe on magnetic resonance imaging, with rupture of a concomitant cerebral aneurysm. preexisting extremely high blood pressure may trigger atypical pres, and failure to lower blood pressure may lead to a concomitant aneurysm rupture. in the future treatment of hypertensive urgency with a recurrence of symptoms and mean arterial blood pressure > 150 mmhg, it is advisable to immediately hospitalize the patient for aggressive blood pressure management, especially if pres is suspected based on clinical and radiological features. |
angiogenesis is an essential component of tumorigenesis, and it has been an important target for development of new anti - cancer drugs. in contrast, antivascular approach targets endothelial cells and pericytes of the already established tumor vasculature. the new class of tubulin - binding agents, named vascular disrupting agents (vdas), has been developed to target microtubules, not only in cancer cells, but also in the endothelium of tumor vessels. these vdas work by acting on the -subunit of endothelial tubulin, resulting in depolarization of microtubules and disorganization of actin and tubulin, which leads to a major change in endothelial cell shape, increased vascular permeability, increased interstitial pressure followed by inhibition of blood flow and vasoconstriction. its active drug (s-516) has a potent cytotoxicity against several cancer cells, including a p - glycoprotein overexpressing cell line (hct15). it induces cell cycle arrest at g2/m phase, and strong inhibition of tubulin polymerization. it has shown marked antitumor efficacy against both murine tumors (ct26 and 3ll) and human xenografts (hct116 and hct15) in mice. these promising preclinical data led to this phase i, firstin - human study of ckd-516 in patients with advanced solid tumors. the primary objective was to determine the maximum - tolerated dose (mtd) of ckd-516 when administered intravenously on day 1 and day 8 every 3 weeks. this was a phase i, first - in - human, dose - escalation study of ckd-516 in patients with advanced solid tumors conducted at seoul national university hospital and seoul national university bundang hospital. this study was approved by the institutional review boards of each hospital and was registered with the united states national library of medicine (clinicaltrials.gov) as nct01028859. the following inclusion criteria were used for patient selection : (1) histologically confirmed solid tumors refractory to standard therapy or for which there is no standard therapy ; (2) age, 20 to 75 years ; (3) eastern cooperative oncology group (ecog) performance status (ps), 2 ; (4) life expectancy of longer than 12 weeks ; (5) adequate organ function (adequate hematologic function) 1,500/l, platelet count 100,000 cells/l, hemoglobin 9.0 g / dl, serum creatinine 1.5upper limit of normal (uln), serum bilirubin 1.5uln, aspartate transaminase, alanine aminotransferase 3uln [5unl in case of liver metastasis ]) ; and (6) normal range of prothrombin time, activated partial thromboplastin time. the important exclusion criteria were (1) central nervous system metastasis ; (2) prior chemotherapy, radiation therapy, or surgery within 4 weeks prior to enrolling in the study ; (3) heart failure, angina pectoris, new york heart association (nyha) class iii, iv congestive heart failure within 6 months of study ; (4) uncontrolled hypertension (systolic blood pressure > 150 mm hg or diastolic blood pressure > 100 mm hg despite anti - hypertensive medication) ; and (5) under anticoagulation. ckd-516 was administered as a 30-minute intravenous infusion on day 1 and day 8 every 3 weeks. the starting dose was 1 mg / m / day, and dose was escalated using 3 + 3 design and modified fibonacci method. in each cohort, if a patient was withdrawn for any reason other than dose limiting toxicity (dlt) before completion of the 21-day administration period, the patient was replaced by another patient. response was evaluated every two cycles. however, whenever disease progression was suspected, the response evaluation was allowed at the investigator s discretion. patients in whom antitumor effect was assessed as stable disease (sd), partial response (pr), or complete response (cr) for overall response at the end of the second cycle of treatment were allowed to receive further treatment. treatment was to be continued until the subject experienced disease progression or unacceptable toxicity, withdrew consent, or required treatment with another therapeutic modality. dlt was defined as follows : (1) grade 4 neutropenia lasting for 7 days or with fever ; (2) grade 3 - 4 thrombocytopenia lasting for 7 days, or with bleeding or required platelets transfusion ; (3) grade 3 or 4 nausea / vomiting or diarrhea despite optimal use of antiemetic drugs or antidiarrheal drugs ; (4) other grade 3 or 4 non - hematological toxicity (except alopecia and anorexia) ; and (5) delay of > 2 weeks in administration of the next cycle due to inadequate recovery from toxicity of the first cycle. dlt was evaluated during the first cycle of treatment. the lowest dose at which dlt was observed in no more than two out of six patients in the first cycle of treatment was judged to be the mtd. blood was collected on day 1 before and at the end of infusion, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours after dosing. urine was collected on day 1, 0 - 4 hours, 4 - 8 hours, 8 - 12 hours, and 12 - 24 hours after dosing. plasma and urine concentrations of ckd-516 and its active metabolite s-516 were determined using a validated liquid chromatography - tandem mass spectrometry (lc - ms / ms) method with mosapride as an internal standard. briefly, 200 l of plasma or urine, methyl tert - butyl ether 1.5 ml, and mosapride 50 l at 10 ng / ml were mixed thoroughly and centrifuged at 5,000 g for 5 minutes. the organic layer was separated and evaporated ; the residue was reconstituted with 100 l of 80% methanol, diluted 2-fold with mobile phase, and injected into the lc - ms / ms system., 5 m ; phenomenex, torrance, ca) under the isocratic condition using 0.05% formic acid in 10 mm ammonium formate and acetonitrile (70:30, v / v). the multiple - reaction monitoring was based on m / z transition of 537 > 270 for ckd-516, 438 > 270 for s-516, and 422 > 198 for mosapride (is). the calibration ranges were linear with a lower limit of quantification of 0.5 ng / ml for ckd-516 and 0.2 ng / ml for s-516 in both human plasma and urine. the inter - assay precision and accuracy of the quality control (qc) samples were less than 9.54% and from 91.1% to 107%, respectively. each analytical batch had six qc samples of known value, i.e., two for low, intermediate, and high concentrations, respectively. the analytical results were accepted only if the concentrations of > four out of the six qc samples were determined within 15% of the known values. safety and tolerability were measured by adverse events (aes), vital sign, body weight, electrocardiogram (ecg), and laboratory tests. safety was assessed according to the national cancer institute common terminology criteria for adverse events (nci - ctcae) ver. pk parameters of ckd-516 and s-516 were calculated using the noncompartmental method with phoenix winnonlin 6.1 (pharsight, mountain view, ca). the pharmacokinetic linearity was assessed by linear regressions between dose and cmax or auclast and between log - transformed dose and log - transformed cmax or log - transformed auclast. tumor responses were assessed after the first cycle or earlier in patients with suspected progression. the following inclusion criteria were used for patient selection : (1) histologically confirmed solid tumors refractory to standard therapy or for which there is no standard therapy ; (2) age, 20 to 75 years ; (3) eastern cooperative oncology group (ecog) performance status (ps), 2 ; (4) life expectancy of longer than 12 weeks ; (5) adequate organ function (adequate hematologic function) 1,500/l, platelet count 100,000 cells/l, hemoglobin 9.0 g / dl, serum creatinine 1.5upper limit of normal (uln), serum bilirubin 1.5uln, aspartate transaminase, alanine aminotransferase 3uln [5unl in case of liver metastasis ]) ; and (6) normal range of prothrombin time, activated partial thromboplastin time. the important exclusion criteria were (1) central nervous system metastasis ; (2) prior chemotherapy, radiation therapy, or surgery within 4 weeks prior to enrolling in the study ; (3) heart failure, angina pectoris, new york heart association (nyha) class iii, iv congestive heart failure within 6 months of study ; (4) uncontrolled hypertension (systolic blood pressure > 150 mm hg or diastolic blood pressure > 100 mm hg despite anti - hypertensive medication) ; and (5) under anticoagulation. ckd-516 was administered as a 30-minute intravenous infusion on day 1 and day 8 every 3 weeks. the starting dose was 1 mg / m / day, and dose was escalated using 3 + 3 design and modified fibonacci method. in each cohort, if a patient was withdrawn for any reason other than dose limiting toxicity (dlt) before completion of the 21-day administration period, the patient was replaced by another patient. response was evaluated every two cycles. however, whenever disease progression was suspected, the response evaluation was allowed at the investigator s discretion. patients in whom antitumor effect was assessed as stable disease (sd), partial response (pr), or complete response (cr) for overall response at the end of the second cycle of treatment were allowed to receive further treatment. treatment was to be continued until the subject experienced disease progression or unacceptable toxicity, withdrew consent, or required treatment with another therapeutic modality. dlt was defined as follows : (1) grade 4 neutropenia lasting for 7 days or with fever ; (2) grade 3 - 4 thrombocytopenia lasting for 7 days, or with bleeding or required platelets transfusion ; (3) grade 3 or 4 nausea / vomiting or diarrhea despite optimal use of antiemetic drugs or antidiarrheal drugs ; (4) other grade 3 or 4 non - hematological toxicity (except alopecia and anorexia) ; and (5) delay of > 2 weeks in administration of the next cycle due to inadequate recovery from toxicity of the first cycle. the lowest dose at which dlt was observed in no more than two out of six patients in the first cycle of treatment was judged to be the mtd. blood was collected on day 1 before and at the end of infusion, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours after dosing. urine was collected on day 1, 0 - 4 hours, 4 - 8 hours, 8 - 12 hours, and 12 - 24 hours after dosing. plasma and urine concentrations of ckd-516 and its active metabolite s-516 were determined using a validated liquid chromatography - tandem mass spectrometry (lc - ms / ms) method with mosapride as an internal standard. briefly, 200 l of plasma or urine, methyl tert - butyl ether 1.5 ml, and mosapride 50 l at 10 ng / ml were mixed thoroughly and centrifuged at 5,000 g for 5 minutes. the organic layer was separated and evaporated ; the residue was reconstituted with 100 l of 80% methanol, diluted 2-fold with mobile phase, and injected into the lc - ms / ms system., 5 m ; phenomenex, torrance, ca) under the isocratic condition using 0.05% formic acid in 10 mm ammonium formate and acetonitrile (70:30, v / v). the multiple - reaction monitoring was based on m / z transition of 537 > 270 for ckd-516, 438 > 270 for s-516, and 422 > 198 for mosapride (is). the calibration ranges were linear with a lower limit of quantification of 0.5 ng / ml for ckd-516 and 0.2 ng / ml for s-516 in both human plasma and urine. the inter - assay precision and accuracy of the quality control (qc) samples were less than 9.54% and from 91.1% to 107%, respectively. each analytical batch had six qc samples of known value, i.e., two for low, intermediate, and high concentrations, respectively. the analytical results were accepted only if the concentrations of > four out of the six qc samples were determined within 15% of the known values. safety and tolerability were measured by adverse events (aes), vital sign, body weight, electrocardiogram (ecg), and laboratory tests. safety was assessed according to the national cancer institute common terminology criteria for adverse events (nci - ctcae) ver. pk parameters of ckd-516 and s-516 were calculated using the noncompartmental method with phoenix winnonlin 6.1 (pharsight, mountain view, ca). the pharmacokinetic linearity was assessed by linear regressions between dose and cmax or auclast and between log - transformed dose and log - transformed cmax or log - transformed auclast. tumor responses were assessed after the first cycle or earlier in patients with suspected progression. nine patients (39.1%) had ecog ps 0, 13 patients (56.5%) had ecog ps 1, and one patient had ecog ps 2. the most common tumors were colorectal cancer (n=11) and lung cancer (n=3). seventy - eight percent of patients had previously received more than three kinds of chemotherapies. the investigational products were administered at doses of up 12 mg / m / day. dlt was reported in two patients in the 12 mg cohort, which were grade 3 hypertension. one patient with dlt had hypertension, and the blood pressure was increased up to grade 3 at 1 hour (164/96 mm hg) and 4 hours (155/95 mm hg) after infusion of ckd-516. however, the blood pressure was normalized at 8 hours after infusion without any intervention. the other patient with dlt showed elevation of blood pressure up to grade 3 (175/95 mm hg) at 4 hours after infusion of ckd-516 and received anti - hypertensive medication (nicardipine), which led to the normalization of blood pressure 8 hours after infusion. there was no abnormality in troponin i, cpk - mb, and ecg in both dlt cases. the mtd of ckd-516 was determined as 12 mg / m / day. a total of 58 cycles of ckd-516 were administered to the patients. most commonly reported aes during all treatment periods were gastrointestinal ae, that is, diarrhea (34.8% ; 30.4% grade 1/2, 13.0% grade 3), nausea (21.7%, all grade 1/2), and vomiting (21.7%, all grade 1/2) (table 2). grade 1/2 anorexia and myalgia were 30.4% and 17.4%, respectively. abdominal pain occurred in 21.7% (grade 1/2, 21.7% ; grade 3, 4.3%). anemia occurred in 8.7% (grade 1/2, 8.7% ; grade 3, 4.3%). hypertension was observed two patients (two dlt cases [grade 3, 8.7% ], one patient [grade 2, 4.3% ]). the plasma concentration - time profiles of ckd-516 and its metabolite s-516 showed a similar trend at all dosing levels (table 3). cmax of ckd-516 were 45.8815.04 g / l (meanstandard deviation), 78.8939.99 g / l, 129.0255.50 g / l, 140.9317.56 g / l, 297.3389.29 g / l, 273.3293.37 g / l, and 646.75513.71 g / l at seven dosing levels, respectively. cmax of s-516 were 19.652.38 g / l, 37.523.72 g / l, 59.755.83 g / l, 128.837.12 g / l, 110.4317.30 g / l, 141.2027.74 g / l, and 222.753.04 g / l at seven dosing levels, respectively (fig. ckd-516 underwent almost complete biotransformation to s-516 with only < 1% of unchanged ckd-516 excreted in the urine. a good linear relationship between dose and pk parameters indicated the linear pharmacokinetic characteristics of ckd-516 and s-516 over all dose levels (fig. six patients were assessed as sd (26.1%) and 17 patients showed progressive disease (73.9%). median progression - free survival was 39 days (95% confidence interval, 37 to 41 days). three patients received the study treatment up to six cycles (1 osteosarcoma at 1 mg, 1 renal cell carcinoma at 3.3 mg, 1 colon cancer at 3.3 mg). the reasons for termination of treatment were disease progression (21 patients), no recovery of disease - related condition within 5 weeks (1 patient), and investigator s discretion (1 patient). nine patients (39.1%) had ecog ps 0, 13 patients (56.5%) had ecog ps 1, and one patient had ecog ps 2. the most common tumors were colorectal cancer (n=11) and lung cancer (n=3). seventy - eight percent of patients had previously received more than three kinds of chemotherapies. the investigational products were administered at doses of up 12 mg / m / day. dlt was reported in two patients in the 12 mg cohort, which were grade 3 hypertension. one patient with dlt had hypertension, and the blood pressure was increased up to grade 3 at 1 hour (164/96 mm hg) and 4 hours (155/95 mm hg) after infusion of ckd-516. however, the blood pressure was normalized at 8 hours after infusion without any intervention. the other patient with dlt showed elevation of blood pressure up to grade 3 (175/95 mm hg) at 4 hours after infusion of ckd-516 and received anti - hypertensive medication (nicardipine), which led to the normalization of blood pressure 8 hours after infusion. there was no abnormality in troponin i, cpk - mb, and ecg in both dlt cases. most commonly reported aes during all treatment periods were gastrointestinal ae, that is, diarrhea (34.8% ; 30.4% grade 1/2, 13.0% grade 3), nausea (21.7%, all grade 1/2), and vomiting (21.7%, all grade 1/2) (table 2). grade 1/2 anorexia and myalgia were 30.4% and 17.4%, respectively. abdominal pain occurred in 21.7% (grade 1/2, 21.7% ; grade 3, 4.3%). anemia occurred in 8.7% (grade 1/2, 8.7% ; grade 3, 4.3%). hypertension was observed two patients (two dlt cases [grade 3, 8.7% ], one patient [grade 2, 4.3% ]). the plasma concentration - time profiles of ckd-516 and its metabolite s-516 showed a similar trend at all dosing levels (table 3). cmax of ckd-516 were 45.8815.04 g / l (meanstandard deviation), 78.8939.99 g / l, 129.0255.50 g / l, 140.9317.56 g / l, 297.3389.29 g / l, 273.3293.37 g / l, and 646.75513.71 g / l at seven dosing levels, respectively. cmax of s-516 were 19.652.38 g / l, 37.523.72 g / l, 59.755.83 g / l, 128.837.12 g / l, 110.4317.30 g / l, 141.2027.74 g / l, and 222.753.04 g / l at seven dosing levels, respectively (fig. ckd-516 underwent almost complete biotransformation to s-516 with only < 1% of unchanged ckd-516 excreted in the urine. a good linear relationship between dose and pk parameters indicated the linear pharmacokinetic characteristics of ckd-516 and s-516 over all dose levels (fig. six patients were assessed as sd (26.1%) and 17 patients showed progressive disease (73.9%). median progression - free survival was 39 days (95% confidence interval, 37 to 41 days). three patients received the study treatment up to six cycles (1 osteosarcoma at 1 mg, 1 renal cell carcinoma at 3.3 mg, 1 colon cancer at 3.3 mg). the reasons for termination of treatment were disease progression (21 patients), no recovery of disease - related condition within 5 weeks (1 patient), and investigator s discretion (1 patient). this was the phase i, first - in - human study of novel vda, ckd-516, administered on d1 and d8 every 3 weeks in patients with advanced solid tumors. the result provides evidence of the tolerability of a vda in patients with solid tumor. the pk of ckd-516 and its metabolite (s-516) were demonstrated in this study, which showed the linear pk. one patient had colon cancer and previously received standard chemotherapies (xelox, folfiri, capecitabine). this patient had a history of hypertension without other cardiac disease and had received antihypertensive medication. the baseline blood pressure before infusion of ckd-516 was 125/82 mm hg, and was increased up to grade 3 at 1 hour (164/96 mm hg) and 4 hours (155/95 mm hg) after infusion of ckd-516, and normalized at 8 hours without any intervention. the other patient had ampulla of vater cancer with treatment history of gemcitabine, cisplatin, and 5-fluorouracil. after 1 hour of infusion, blood pressure was 111/75 mm hg, and elevated up to grade 3 (175/95 mm hg) at 4 hours and anti - hypertensive medication was administered, which led to the normalization of blood pressure at 8 hours. the most commonly reported dlts for other vdas under development are cardiac - related events including hypertension, corrected qt (qtc) prolongation, myocardial ischemia, atrial fibrillation, acute chest pain, acute reversible coronary ischemia, hypotension, troponin elevation or motor neuropathy or tumor pain [10 - 15 ]. other frequently reported aes are diarrhea, nausea, vomiting, fatigue, and headache. therefore, the findings in our study are consistent with those of other studies testing vdas. for the toxicity pattern, dose modification or cycle delay of subsequent treatment after the first cycle, a total of 58 cycles were delivered to all enrolled patients (10 cycles in 1.0 mg, 5 cycles in 2.0 mg, 16 cycles in 3.3 mg, 4 cycles in 5.0 mg, 6 cycles in 7.0 mg, 10 cycles in 9.0 mg, and 7 cycles in 12.0 mg). only two patients who showed dlts were treated with a reduced dose in subsequent cycles. gastrointestinal aes (diarrhea, nausea, and anorexia) were most common followed by myalgia and arthralgia. most were grade 1/2. during all subsequent treatment cycles, the vascular disrupting effect of ckd-516 was previously evaluated with intravoxel incoherent motion diffusionweighted magnetic resonance imaging (mri) or dynamic contrast - enhanced ultrasonography in a rabbit vx2 liver tumor model. in our study, to observe the vascular disrupting effect of ckd-516 by dynamic contrast - enhanced mri (dce - mri), 13 patients were evaluated by dce - mris taken at screening and 24 hours after the first infusion of ckd-516. a trend of decreased k and initial area under the curve (iauc) was observed. the reason for checking dce - mri at 24 hours after infusion of ckd-561 was based on previous animal data. the half - life of s-561, the active metabolite of ckd-561, was 85.1 minutes in dog. in an in vivo absorption experiment, the half - life of ckd-516 was 8.05 hours in dog. we hypothesized that the change of blood flow will take place within at least 3 to 4 times of half - life, around 24 hours. actually, the half - life of s-516 in our study ranged from 4 - 6 hours. we checked 5-hydroxyindoleacetic acid (5-hiaa) plasma concentration at pretreatment, 4 hours and 8 hours of ckd-516 infusion to determine whether the action mechanism of ckd-516 is associated with serotonin release, which was reported in other vdas. the levels of 5-hiaa of all patients were normal and there was no significant change of 5-hiaa levels after infusion of ckd-516, therefore, we concluded that the action mechanism of ckd-516 was not associated with serotonin release. in terms of antitumor efficacy three patients received ckd-516 up to six cycles (1 osteosarcoma, 1 renal cell carcinoma, 1 colon cancer). in our study, we used recist ver. 1.1 for evaluation of efficacy in accordance with other previous studies of vdas. based on limited data, selection of optimal tumor types for further development of vdas so far, there have been no biomarkers for prediction of the efficacy of vdas. based on the preclinical combination study of vdas with cytotoxic chemotherapies, now, a different dosing schedule of ckd-516 is under investigation (clinicaltrials.gov identifier : nct01560325). in this another phase i study, we are testing d1, d4, d8, and d11 infusion of ckd-516 out of a 21-day cycle. using the result of this phase in conclusion, this study confirms the feasibility of a novel vda, ckd-516, in patients with advanced solid tumor. ckd-516, at the mtd of 12 mg / m / day, administered on d1 and d8 every 3 weeks, is safe and well tolerated. | purposeckd-516 is a newly developed vascular disrupting agent. this phase i dose - escalation study of ckd-516 was conducted to determine maximum - tolerated dose (mtd), safety, pharmacokinetics, and preliminary antitumor efficacy in patients with advanced solid tumors.materials and methodspatients received ckd-516 intravenously on d1 and d8 every 3 weeks, in a standard 3 + 3 design. safety was evaluated by national cancer institute common terminology criteria for adverse events ver. 4.02 and response was assessed by response evaluation criteria in solid tumor ver. 1.1.resultstwenty-three patients were treated with ckd-516 at seven dosing levels : 1 mg / m2/day (n=3), 2 mg / m2/day (n=3), 3.3 mg / m2/day (n=3), 5 mg / m2/day (n=3), 7 mg / m2/day (n=3), 9 mg / m2/day (n=6), and 12 mg / m2/day (n=2). mean age was 54 and 56.5% of patients were male. two dose - limiting toxicities, which were both grade 3 hypertension, were observed in two patients at 12 mg / m2/day. the mtd was determined as 12 mg / m2/day. most common adverse events were gastrointestinal adverse events (diarrhea, 34.8% [30.4% grade 1/2, 13.0% grade 3 ] ; nausea, 21.7% [all grade 1/2 ] ; vomiting, 21.7% [all grade 1/2 ]), myalgia (17.4%, all grade 1/2), and abdominal pain (21.7% [21.7% grade 1/2, 4.3% grade 3 ]). the pharmacokinetic study showed the dose - linearity of all dosing levels. among 23 patients, six patients (26.1%) showed stable disease. median progression - free survival was 39 days (95% confidence interval, 37 to 41 days).conclusionthis study demonstrates feasibility of ckd-516, novel vascular disrupting agent, in patients with advanced solid tumor. mtd of ckd-516 was defined as 12 mg / m2/day on d1 and d8 every 3 weeks. |
obesity is a worldwide health burden, accompanied by a number of comorbidities including glucose intolerance, insulin resistance and type 2 diabetes. in this context, the myokine irisin, which is a cleavage product of the type i membrane protein fibronectin type iii domain - containing 5, has been hypothesized as a target to counteract obesity and type 2 diabetes. irisin is expressed in the muscle and the adipose tissue and has been associated with adiposity and body weight in animals and humans. however, the precise role and underlying mechanisms concerning irisin actions and signaling pathways remain incompletely understood. the aim of this research was to assess changes on circulating irisin concentrations in obese subjects presenting metabolic syndrome (mets) features after a treatment designed to lose weight and to analyze the potential relationships of this myokine with glucose homeostasis after dieting. this research reports the findings of the 8-week intervention period of the resmena randomized intervention trial (www.clinicaltrials.gov ; nct01087086), which was conducted following the consort 2010 criteria. a full list of inclusion criteria, as well as a complete description of the study methodology can be found in earlier publications. briefly, participants were randomized into two intervention groups, with the same energy restriction (30% e), but differing mainly in the carbohydrate / protein ratio and meal frequency : control group supplying 55% e from cho and 15% e from proteins within a 35 meals per day pattern, and resmena group providing 40% e from cho and 30% e from proteins within a 7 meals per day plan. ninety - six adults (mean age=50 years old ; range 2170 years old) with excessive body weight (mean body mass index=35.9 kg m ; range 26.949.4 kg m) suffering mets according to the international diabetes federation criteria completed the intervention period. all the participants gave a written informed consent to participate as approved by the ethics committee of the university of navarra (065/2009) and in accordance with the declaration of helsinki. participant 's dietary intake was assessed by means of 48-h weighed records at baseline and at the end of the intervention and further analyzed using the dial software (alce ingenieria, madrid, spain). subjects were asked to maintain their usual activity levels during the study, which was monitored at the beginning and endpoint with a validated 24-h physical activity questionnaire. overnight fasting plasma levels of glucose and triglycerides were measured in an autoanalyzer pentra c-200 (horiba abx, madrid, spain) with specific kits from this company. insulin concentrations were determined with an enzyme - linked immunosorbent assay kit (mercodia, uppsala, sweden) in a triturus autoanalyzer (grifols sa, barcelona, spain) and the homeostasis model (homeostatic model assessment - insulin resistance (homa - ir)) was applied to estimate insulin resistance. irisin plasma levels were determined using a commercial enzyme - linked immunosorbent assay kit following the manufacturer 's instructions (irisin elisa kit ek-06752 ; phoenix pharmaceuticals, inc., burlingame, ca, usa), on a spectrophotometric reader at a wavelength of 450 nm (versamax microplate reader, east falmouth, ma, usa). this test provided a range of detection of 0.0661024 ng ml and exhibited a coefficient of variation of 610% inter- and intra - assay. the samples were kept at 80 c and were analyzed immediately after the experiment was ended. the sample size of this secondary analysis was calculated for an =0.05 and a power of 80% based on the waist circumference reduction, as described elsewhere. normality distributions of the measured variables were determined according to the shapiro wilk test. irisin plasma levels were not normally distributed, but based on the sample size (n>60) a parametric test was performed. indeed, after analysis with a log transformation of irisin values the statistical outcomes were maintained. differences between baseline and endpoint values within groups were analyzed by a paired t - test. a multiple linear regression analysis was applied in order to assess the potential relationships among irisin with anthropometric and biochemical measurements (95% confidence interval). the median value of irisin baseline concentrations was considered as the cutoff for analyzing the effect of high- or low - irisin levels on glucose regulatory factors, as previously applied. this tool is based on the assignment of the studied population into two groups of disease risk. the association between irisin levels and carbohydrate intake was assessed using the parametric pearson correlation. specific statistical analyses (analysis of covariance) were performed after excluding outlier values in order to control the regression to the mean phenomenon. chicago, il, usa). an alpha level of 0.05 was set up for determining statistically significant differences. this research reports the findings of the 8-week intervention period of the resmena randomized intervention trial (www.clinicaltrials.gov ; nct01087086), which was conducted following the consort 2010 criteria. a full list of inclusion criteria, as well as a complete description of the study methodology can be found in earlier publications. briefly, participants were randomized into two intervention groups, with the same energy restriction (30% e), but differing mainly in the carbohydrate / protein ratio and meal frequency : control group supplying 55% e from cho and 15% e from proteins within a 35 meals per day pattern, and resmena group providing 40% e from cho and 30% e from proteins within a 7 meals per day plan. ninety - six adults (mean age=50 years old ; range 2170 years old) with excessive body weight (mean body mass index=35.9 kg m ; range 26.949.4 kg m) suffering mets according to the international diabetes federation criteria completed the intervention period. all the participants gave a written informed consent to participate as approved by the ethics committee of the university of navarra (065/2009) and in accordance with the declaration of helsinki. participant 's dietary intake was assessed by means of 48-h weighed records at baseline and at the end of the intervention and further analyzed using the dial software (alce ingenieria, madrid, spain). subjects were asked to maintain their usual activity levels during the study, which was monitored at the beginning and endpoint with a validated 24-h physical activity questionnaire. overnight fasting plasma levels of glucose and triglycerides were measured in an autoanalyzer pentra c-200 (horiba abx, madrid, spain) with specific kits from this company. insulin concentrations were determined with an enzyme - linked immunosorbent assay kit (mercodia, uppsala, sweden) in a triturus autoanalyzer (grifols sa, barcelona, spain) and the homeostasis model (homeostatic model assessment - insulin resistance (homa - ir)) was applied to estimate insulin resistance. irisin plasma levels were determined using a commercial enzyme - linked immunosorbent assay kit following the manufacturer 's instructions (irisin elisa kit ek-06752 ; phoenix pharmaceuticals, inc., burlingame, ca, usa), on a spectrophotometric reader at a wavelength of 450 nm (versamax microplate reader, east falmouth, ma, usa). this test provided a range of detection of 0.0661024 ng ml and exhibited a coefficient of variation of 610% inter- and intra - assay. the samples were kept at 80 c and were analyzed immediately after the experiment was ended. the sample size of this secondary analysis was calculated for an =0.05 and a power of 80% based on the waist circumference reduction, as described elsewhere. normality distributions of the measured variables were determined according to the shapiro wilk test. irisin plasma levels were not normally distributed, but based on the sample size (n>60) a parametric test was performed. indeed, after analysis with a log transformation of irisin values the statistical outcomes were maintained. differences between baseline and endpoint values within groups were analyzed by a paired t - test. a multiple linear regression analysis was applied in order to assess the potential relationships among irisin with anthropometric and biochemical measurements (95% confidence interval). the median value of irisin baseline concentrations was considered as the cutoff for analyzing the effect of high- or low - irisin levels on glucose regulatory factors, as previously applied. this tool is based on the assignment of the studied population into two groups of disease risk. the association between irisin levels and carbohydrate intake was assessed using the parametric pearson correlation. specific statistical analyses (analysis of covariance) were performed after excluding outlier values in order to control the regression to the mean phenomenon. at the beginning of the intervention, there were no differences between groups in any of the anthropometric and routine biochemical markers (p>0.05). after the intervention, an improvement (reduction) was observed on these measurements with apparently equal effectiveness between the two dietary treatments (p>0.05, table 1), except for adiponectin, which was increased in both groups, but without reaching statistical significance. changes in irisin concentrations were similar (p>0.05) in the control group (87.318.4 ng ml) as compared with the resmena group (59.811.8 ng ml), after following the energy - restricted treatment. considering the whole sample, participant 's mean body weight loss was 6.93.0 kg and irisin plasma concentrations diminished (figure 1a) in association with changes in body weight (r=0.21 ; p=0.046) and fat mass (r=0.22 ; p=0.037). as the main objective of this study was to evaluate the potential role of irisin on glucose homeostasis and given that some of the participants were diabetic, a preliminary analysis separating non - diabetic and diabetic participants was also performed. differences were found for glucose concentrations and homa index between both groups after the nutritional intervention with energy restriction, but similar outcomes were found concerning irisin concentrations (data not shown). similar values were found concerning physical activity assessments at the beginning and at the end of the intervention in both dietary groups. moreover, the regression analysis showed no relationships between physical activity factor and irisin levels changes (p=0.736). an association of circulating glucose (b=0.134, 95% confidence interval : 0.245 to 0.024 ; p=0.018) and irisin concentrations changes was found, irrespective of confounding factors : gender, age, diet, body weight loss and irisin baseline values. interestingly, after adjusting for gender, age and weight loss, participants belonging to the high - irisin group at baseline (> 308.0 ng ml) evidenced significantly greater reductions (figure 1b) on glucose (p=0.022), insulin (p=0.021), homa index (p=0.008) and triglycerides (p=0.006), compared with those belonging to the low - irisin group at baseline (< 308.0 ng ml). furthermore, the decrease in irisin concentrations was significantly greater (p<0.001) within the group with high - irisin values at baseline (126.615.9 ng ml) than within the lower irisenemia group (18.29.1 ng ml). after 8 weeks of nutritional intervention, irisin concentrations were positively correlated with carbohydrate intake (cereals, pulse, fruits and vegetables ; r=0.234, p=0.023 ; figure 1c). this study evidenced that irisin per se may exert an effect on the reduction of glucose, insulin and triglycerides concentrations after prescribing an 8-week nutritional intervention to obese subjects with mets traits. irisin is a recently discovered muscle - derived hormone, whose secretion is induced by exercise. this myokine has been shown to be able to increase energy expenditure, and therefore, it has been proposed to have a potential role in obesity and diabetes treatments. since discovery, a number of original studies have addressed various aspects of the biology of irisin. however, the regulation and specific role of irisin in human 's glucose metabolism remain still unclear. thus, the main objective of the current research was to investigate the potential relationships between irisin concentrations and glucose homeostasis, after dieting. the study was designed as a randomized controlled nutritional intervention comparing two energy - restricted dietary treatments. both control and resmena dietary strategies proved to be effective for improving mets disturbances by lowering anthropometric and biochemical markers, being these outcomes in agreement with other studies concerning hypocaloric diets. however, no differences between treatments were observed for any of the studied variables including irisin. for that reason, the sample was merged and considered as a whole for the subsequent analyses regarding irisin concentrations and its potential associations with glucose metabolism. first, changes on irisin concentrations after the 8 weeks of nutritional intervention were evaluated. this study evidenced that irisin plasma concentrations decreased after the energy restriction program and the subsequent weight loss, independently of the dietary group. this finding is in agreement with a previous study that reported a reduction in irisin levels after surgically induced weight markdown. then, the potential role of irisin on glucose homeostasis - related parameters was analyzed in order to reach the main objective of the research. the prime finding of the current investigation was that higher irisin concentrations at the beginning of the intervention were associated with greater reductions on glucose and insulin concentrations as well as on the homa index, independently of body weight loss. although this outcome should be carefully examined, similar results have been reported in children by al - daghri. where a crucial role for irisin in glucose homeostasis was suggested. on the other hand, those individuals with higher irisin concentrations at the beginning of the intervention also achieved higher beneficial effects regarding the lowering of triglycerides concentrations. this effect could be explained by the fact that triglycerides levels have been revealed to positively correlate with glucose levels. thus, the effects of irisin on the changes of glucose concentrations may have been subsequently reflected on triglycerides. in addition, taking into account that irisin has been evidenced to increase energy expenditure, the greater reduction observed in triglycerides according to the high - irisin levels at baseline may be also due to a higher utilization of triglycerides as energy substrate. taking together these outcomes, it can be suggested that irisin may be involved in the regulation of glucose homeostasis in obese subjects presenting mets features. thus, irisin could mean a physiological feedback to counteract potential glucose metabolism - related disturbances associated to an excessive body weight state. irisin would seem to be increased in unfavorable metabolic situations acting as a compensatory triggering mechanism. other authors have likewise claimed that the increase in irisin under obesity conditions may indicate a physiological adaptation to improve glucose tolerance, which is often impaired in obese subjects. indeed, this behavior has been observed predominantly in individuals with metabolic disease as it is the condition of our study population. however, other studies reported associations between plasma irisin levels and important metabolic factors in non - diabetic subjects, but not in individuals with type 2 diabetes. our suggested corollary would be that irisin is increased in metabolically altered situations and may diminish as a consequence of the weight loss, as irisin is then less ' needed to restore the altered metabolic state. thus, the theory about a possible irisin resistance appears similar to the well - known leptin insensitivity in obesity and can not be discarded as has been reported for leptinemia and insulinemia after dieting. the association between irisin concentrations and carbohydrate intake was related to the consumption of some sources of carbohydrates (cereals, pulse, fruits and vegetables). this outcome may be explained because the dietary modifications during the hypocaloric intervention evolved with shifts in carbohydrate consumption within the energy restriction. thus, irisin could be increased in response to the dietary pattern, depending on the carbohydrate content, in order to prevent / improve the rise on glucose, insulin or homa index values, linked to latter damage on multiple organs. this finding is interesting given that modifying the macronutrient distribution is a recurrent approach for treating obese and mets patients. the observed results appear to be irrespective to the physical activity, as patients in this study maintained the same physical activity level along the intervention. the statistical adjustments for sex did not revealed specific differences between males and females concerning the analyzed irisin outcomes. a limitation of this study is that it demonstrated an association but not evidenced causation. moreover, the methods to assess the dietary intake and physical activity were based on questionnaires, which could bias the results interpretation. also, some other relevant measurements in relation to glucose metabolism, such as ogtt or clamp - test would be appropriate. however, the design of the current trial based on a nutritional intervention involving a quite large human sample is indeed a valuable feature enabling pre- and post - test comparisons within subjects. an effect of regression to the mean could not be attributed since pertinent statistical procedures were performed in order to control this phenomenon. this research concerns the investigation of a potential role of irisin on impaired glucose homeostasis associated to obesity and, consequently, the metabolic interplay on glucose metabolism and insulin secretion control. | irisin is assumed to be a relevant link between muscle and weight maintenance as well as to mediate exercise benefits on health. the aim of this study was to assess the possible associations between irisin levels and glucose homeostasis in obese subjects with metabolic syndrome (mets) following an energy - restricted treatment. ninety - six adults with excessive body weight and mets features underwent a hypocaloric dietary pattern for 8 weeks, within the resmena randomized controlled trial (www.clinicaltrials.gov ; nct01087086). after the intervention, dietary restriction significantly reduced body weight and evidenced a dietary - induced decrease in circulating levels of irisin in parallel with improvements on glucose homeostasis markers. interestingly, participants with higher irisin values at baseline (above the median) showed a greater reduction on glucose (p=0.022) and insulin (p=0.021) concentrations as well as on the homeostasis model assessment index (p=0.008) and triglycerides (p=0.006) after the dietary intervention, compared with those presenting low - irisin baseline values (below the median). interestingly, a positive correlation between irisin and carbohydrate intake was found at the end of the experimental period. in conclusion, irisin appears to be involved in glucose metabolism regulation after a dietary - induced weight loss. |
bovine babesiosis is economically the most important tick - borne disease of cattle worldwide including areas of australia, africa, south and central america (bock. in addition, the united states is continuously under threat of reintroduction of the vector and the disease (bock. 2004). under natural conditions, babesia bovis transmitted by the tick rhipicephalus microplus, they multiply by merogony in erythrocytes while in ticks by sporogony (susan and asa 1999). the disease is the most prevalent in tropical and subtropical countries, affecting cattle industries causing a major economic impact worldwide (bse. costs due to babesiosis are the results of high mortality, ill - thrift, abortions, loss of milk / meat production and draft power and from control measures such as acaricide treatments, purchase of vaccines and therapeutics (bock. 2004). it was estimated that losses and control of babesiosis and anaplasmosis in kenya, zimbabwe, tanzania, south africa, china, india, indonesia and philippines cost 5.1, 5.4, 6.8, 21.6, 19.4, 57.2, 3.1 and 0.6 million us dollars annually, respectively (bock. kaufmann (1996) reported that the mortality rates in cattle infected by b. bovis without treatment could reach 7080%. the diagnosis of ruminant babesiosis is generally based upon the microscopic examination of giemsa - stained blood smears and clinical signs in acute cases. previous studies provide information on the relative susceptibility of various breeds of cattle to babesia infection (bock. 1997). in egypt, bovine babesiosis is caused mainly by b. bigemina and b. bovis and considered as the most important and endemic parasitic disease affecting cattle (nagati 1947, adham. bovine babesiosis has a significant impact on meat and milk production and consequently, on livestock management (adham. the rapidly changing patterns of demand for cattle and its products point to cattle production being an important and increasing component of the egyptian agriculture economy which required improving cattle health. egyptians farmers cross between the holstein - friesian breed and a native local breed known as baladi cattle breed (bos taurus) to improve production and disease resistance. water buffaloes (bubalus bubalis) represent an important source of various human needs, such as meat, horns, hides, milk and milk products, leather, land plowing, and transportation of people and crops (somparn. 2004). due to the fact that water buffaloes are raised together with cattle, among which bovine babesiosis is highly prevalent (iseki. 2010), they might be potential carriers for babesia parasites. babesia bovis infection was experimentally investigated in splenectomised buffaloes (mahmoud and abou - zeina 2008). yao. (1997) reported the clinical findings on buffaloes after experimental infection with cryopreserved b. bovis parasites. however, efforts to furnish information about natural infection with b. bovis in water buffaloes as well as crossbred cattle are necessary for better understanding of disease pattern under uncontrolled field conditions and subsequently, implementation the suitable policy for treatment and control. the objective of the present study was to investigate the natural b. bovis infection in water buffaloes in comparison to crossbred cattle under field conditions in egypt. blood samples were collected from the jugular vein into edta - containing tubes from 35 animals (23 cattle and 12 buffaloes) of both sexes and aged 25 years, and were originating from different villages : el - aslogy, shobk basta and tel basta around zagazig city, sharkia province. these animals were divided into 2 groups, the field - exposed (diseased) group comprised of 20 cattle and 9 water buffaloes which was examined at the veterinary teaching hospital, faculty of veterinary medicine, zagazig university, during the period from march to june 2008 and resulted to have persistent fever, anemia and anorexia. the control group (3 cows and 3 buffaloes) was carefully examined clinically and parasitologically and found healthy and free from external, internal, and hemoparasites. the common available foods for animals under the present study were mainly consisted of barseem (trifolium alexandrinum), rice or wheat straw and concentrate mixture (12 kg / head / day). crossbred cattle were resulted from crossbreeding between the imported holstein - friesian breed cattle and egyptian baladi cattle breed (bos taurus). samples collection, handling and examination of cattle and buffaloes under the current study were done after approval of animals owners. animals were subjected to clinical and hematological examinations at veterinary teaching hospital, faculty of veterinary medicine, zagazig university. the filed - exposed group showed various degrees of bovine babesiosis such as high fever (> 40 c), anorexia, hemoglobinuria (bloody urine), anemia, and jaundice. the control group was examined thoroughly for presence of any abnormal clinical changes and external parasites, and was thoroughly examined by different laboratory techniques such as direct smear, flotation, sedimentation and barmen s techniques and blood film to confirm the absence of any internal parasites and/or hemoparasites (rosenberger 1990). thin blood films were prepared immediately after taking the blood samples directly from the ear vein in the field to allow these smears to dry by air then fixed by using methanol for about 35min, allow them to dry by air after fixation step then stained with giemsa stain diluted at 8% with distilled water for about 3045min. they were dried by air and examined on olympus microscope using oil immersion lens at x1000 magnification (kelly 1984). blood film was examined for b. bovis at 1/41/2 inch from the end of the film by visually scanning from one side of the film to other (cross sectional method) to give constant and representative examination. each blood film and at least twenty microscopic fields of each slide were examined twice before being considered negative. approximately 5ml of blood was taken from the jugular vein of all animals with a syringe containing edta. the blood samples were subjected to hematological parameters analysis (schalm. 1975, coles 1986), that is, red blood cell (rbc) and white blood cell (wbc) counts were made with improved neubauer hacmocytometers, hemoglobin concentration (hb) by sahli s haemoglobinometer and packed cell volume (pcv%) by microhematocrit tubes. the obtained data were statistical analyzed by mean of computer based statistical program, spss (borenstein. data were analyzed using student s t - test to compare the mean data between groups. blood samples were collected from the jugular vein into edta - containing tubes from 35 animals (23 cattle and 12 buffaloes) of both sexes and aged 25 years, and were originating from different villages : el - aslogy, shobk basta and tel basta around zagazig city, sharkia province. these animals were divided into 2 groups, the field - exposed (diseased) group comprised of 20 cattle and 9 water buffaloes which was examined at the veterinary teaching hospital, faculty of veterinary medicine, zagazig university, during the period from march to june 2008 and resulted to have persistent fever, anemia and anorexia. the control group (3 cows and 3 buffaloes) was carefully examined clinically and parasitologically and found healthy and free from external, internal, and hemoparasites. the common available foods for animals under the present study were mainly consisted of barseem (trifolium alexandrinum), rice or wheat straw and concentrate mixture (12 kg / head / day). crossbred cattle were resulted from crossbreeding between the imported holstein - friesian breed cattle and egyptian baladi cattle breed (bos taurus). samples collection, handling and examination of cattle and buffaloes under the current study were done after approval of animals owners. animals were subjected to clinical and hematological examinations at veterinary teaching hospital, faculty of veterinary medicine, zagazig university. the filed - exposed group showed various degrees of bovine babesiosis such as high fever (> 40 c), anorexia, hemoglobinuria (bloody urine), anemia, and jaundice. the control group was examined thoroughly for presence of any abnormal clinical changes and external parasites, and was thoroughly examined by different laboratory techniques such as direct smear, flotation, sedimentation and barmen s techniques and blood film to confirm the absence of any internal parasites and/or hemoparasites (rosenberger 1990). thin blood films were prepared immediately after taking the blood samples directly from the ear vein in the field to allow these smears to dry by air then fixed by using methanol for about 35min, allow them to dry by air after fixation step then stained with giemsa stain diluted at 8% with distilled water for about 3045min. they were dried by air and examined on olympus microscope using oil immersion lens at x1000 magnification (kelly 1984). blood film was examined for b. bovis at 1/41/2 inch from the end of the film by visually scanning from one side of the film to other (cross sectional method) to give constant and representative examination. each blood film and at least twenty microscopic fields of each slide were examined twice before being considered negative. approximately 5ml of blood was taken from the jugular vein of all animals with a syringe containing edta. 1975, coles 1986), that is, red blood cell (rbc) and white blood cell (wbc) counts were made with improved neubauer hacmocytometers, hemoglobin concentration (hb) by sahli s haemoglobinometer and packed cell volume (pcv%) by microhematocrit tubes. the obtained data were statistical analyzed by mean of computer based statistical program, spss (borenstein. 1997). data were analyzed using student s t - test to compare the mean data between groups. cattle infected with b. bovis showed typical clinical signs of babesiosis, table 1. briefly, highly rise in body temperature (4041.5 c), conjunctival and vaginal mucous membranes were anemic and the clinical severity was ranged from paleness in mild cases to severe yellow discoloration (icterus) in more progressive cases, dark brown to dark red (coffee - color) urine, hemoglobinuria was common sign in cattle with severe clinical manifestation and accelerated heart and respiratory rates. various degrees of tick infestations were present around groins, horns, inter - mandibular space, and ears. water buffaloes infected with b. bovis showed a milder form (less severe) of clinical signs of infection in comparison to the clinical signs appeared on b. bovis - infected cattle. these clinical signs were in the form of highly rise in body temperature (4041.5 c), and conjunctival and vaginal mucous membranes were mainly anemic and pale in color and loss of body condition. icterus, hemoglobinuria and nervous manifestations were not detected / observed in the affected buffaloes. giemsa - stained blood smears from b. bovis infected animals showed intra - erythrocytic piroplasms of b. bovis that were in the form of pyriform or pear - shaped, fig. blood smears from b. bovis progressive cases of cattle showed severe hemolytic anemia with abnormalities in cell size (anisocytosis) and cell shape (poikilocytosis) of erythrocytes, fig. 2. giemsa - stained blood smears from b. bovis uninfected cattle and buffaloes showed no parasites or erythrocytic changes. the control group resulted to be healthy on clinical and laboratory examination and free from external, internal and hemoparasites. the mean values of rbcs, hemoglobin amount, pcv %, wbcs, and differential leucocytic count are listed in table 2. briefly, the important findings can be summarized as follows ; there is a clear significant difference in the haematological parameters between b. bovis - infected buffaloes and b. bovis - infected cattle in comparison to control group at p - value (0.01) and (0.001), respectively. haematological changes for b. bovis - infected buffaloes were less significant than their changes for b. bovis - infected cattle. cattle infected with b. bovis showed typical clinical signs of babesiosis, table 1. briefly, highly rise in body temperature (4041.5 c), conjunctival and vaginal mucous membranes were anemic and the clinical severity was ranged from paleness in mild cases to severe yellow discoloration (icterus) in more progressive cases, dark brown to dark red (coffee - color) urine, hemoglobinuria was common sign in cattle with severe clinical manifestation and accelerated heart and respiratory rates. various degrees of tick infestations were present around groins, horns, inter - mandibular space, and ears. water buffaloes infected with b. bovis showed a milder form (less severe) of clinical signs of infection in comparison to the clinical signs appeared on b. bovis - infected cattle. these clinical signs were in the form of highly rise in body temperature (4041.5 c), and conjunctival and vaginal mucous membranes were mainly anemic and pale in color and loss of body condition. icterus, hemoglobinuria and nervous manifestations were not detected / observed in the affected buffaloes. giemsa - stained blood smears from b. bovis infected animals showed intra - erythrocytic piroplasms of b. bovis that were in the form of pyriform or pear - shaped, fig. blood smears from b. bovis progressive cases of cattle showed severe hemolytic anemia with abnormalities in cell size (anisocytosis) and cell shape (poikilocytosis) of erythrocytes, fig. 2. giemsa - stained blood smears from b. bovis uninfected cattle and buffaloes showed no parasites or erythrocytic changes. the control group resulted to be healthy on clinical and laboratory examination and free from external, internal and hemoparasites. the mean values of rbcs, hemoglobin amount, pcv %, wbcs, and differential leucocytic count are listed in table 2. briefly, the important findings can be summarized as follows ; there is a clear significant difference in the haematological parameters between b. bovis - infected buffaloes and b. bovis - infected cattle in comparison to control group at p - value (0.01) and (0.001), respectively. haematological changes for b. bovis - infected buffaloes were less significant than their changes for b. bovis - infected cattle. babesia bovis is one of the most important blood parasites affecting cattle and buffaloes and in its acute forms, it lowers the productive performance of the affected animals (talkhan. most of the previous studies described the clinical findings of b. bovis infection in cattle of different breeds. to the best of our knowledge, this is the first study which investigating thoroughly the clinical and hematological pictures of natural b. bovis infection in water buffaloes under uncontrolled field conditions. the reported clinical findings of b. bovis infection in cattle come in agreement with what was previously described by brown and torres (2008), georgi. the demonstrated high fever could be attributed as response to the effect of unspecific toxic substances produced during the metabolism of babesia on thermoregulatory (radostits. it was notable that water buffaloes identified as b. bovis - infected showed a milder form (less severe) of clinical signs of b. bovis infection in comparison to the clinical signs appeared on b. bovis - infected cattle. this variation was represented in appearance of icterus, hemoglobinuria and nervous manifestations in clinically infected cattle while they were not reported in infected buffaloes. this finding may propose that buffaloes may have more tolerance to clinical infection with b. bovis than cattle. tolerance means that the host is infected by the pathogen, but suffers little adverse effect (fao 2007). it could be argued that buffaloes may have acquired natural immunity / tolerance to some extent against b. bovis infection. genetic variations within the host between cattle and buffaloes may explain the variation in their susceptibility. (1997) who found that b. bovis produces acute, often fatal, infections in buffaloes. the proportion of buffaloes identified as b. bovis - infected was (31.1%) while the proportion of cattle identified as b. bovis - infected was (68.9%) within the same period of the study. this finding could suggest that water buffalos have more tendencies to be carriers (apparently healthy) than showing clinical manifestations. (2008) who noticed that water buffaloes seem to be unapparent carriers of the parasite. the marked anemia and hemoglobinuria in cattle could be attributed to the severe haemolytic process associated the presence of babesia piroplams inside the erythrocytes and destruction of large numbers of these erythrocytes by the parasite resulting in hemoglobinaemia and consequently hemoglobinuria (georgi. 1990, fujinaga 1981), the physical effect of parasite multiplication (wright 1981), the increase of phagocytosis of erythrocytes by activated macrophages (shoda. 2000, court. 2001), the production of an anti - erythrocyte antibody (goe s. 2007) and the increase in the erythrocytic membrane permeability (alkhalil. 2007). hematological findings showed a significant decrease in the rbcs, wbcs counts, hb concentrations and pcv% in the b. bovis - infected animals in comparison to the control group, these observations were similar to what were reported by col and uslu (2007) and durrani. hematological changes resulted from b. bovis infection in buffaloes are less significant than the hematological changes of b. bovis infection in cattle, table 2. this finding reflected clinically on b. bovis - infected buffaloes which showed a milder form of clinical picture of b. bovis infection than b. bovis - infected cattle. the hemolytic anemia due to the breakdown of erythrocytes membranes leading to release of hemoglobin and manifested by the presence of free hemoglobin resulting in the discoloration of the plasma (sowemimo - coker 2002). extensive lipid peroxidation in biological membranes causes disturbances of its structural integrity, loss of fluidity, decrease in membrane potential, and increased permeability to ions (gutteridge 1995). these changes lead to rupture of the membrane and release of cell contents (halliwell and chirico 1993). babesia parasite (alkhalil. 2007), and b. bovis (aikawa. 1985) dramatically alters the permeability of its host erythrocytes to various organic solutes. b. bovis infection is associated with impairment of blood parameters and subsequently, hematological examination may be a useful tool for confirmation the clinical diagnosis of bovine babesiosis. water buffaloes showed a milder form of b. bovis infection than cattle suggesting that buffaloes may be more tolerant to the clinical infection with b. bovis than cattle. hematological changes as a result of b. bovis infection in buffaloes are less significant than hematological changes of b. bovis infection in cattle. b. bovis infection might be associated with severe clinical and hematological changes especially in cattle, which might be of bad prognosis. with respect to the study population, future studies should consider a larger sample size for cattle and buffaloes for the robustness of the findings. recent molecular techniques such as pcr showed many advantages with regard to the sensitivity and specificity for detection and surveillance of hemoparasites (nayel. hence, it would be advisable for future studies to use such techniques for investigating the b. bovis infection in buffaloes. | backgroundthere is a little or no data available on the natural babesia bovis (b. bovis) infection in water buffaloes (bubalus bubalis) comparing to the available one for cattle. this study was conducted to investigate the natural b. bovis infection in water buffaloes in comparison to crossbred cattle under field conditions in egypt.methods:a total of 35 buffaloes and cattle were clinically and laboratory investigated from march to june 2008. twenty - nine buffaloes and cattle out of 35 were naturally infected with b. bovis and showed signs of bovine babesiosis. three cows and three buffaloes showed no clinical signs and were free from external, internal, and blood parasites served as control group.results:babesia bovis - infected cattle showed typical signs of bovine babesiosis while b. bovis - infected buffaloes showed a milder form (less severe) of the clinical signs. advanced cases of cattle showed dark brown to dark red (coffee - color) urine, hemoglobinuria and nervous manifestations while these manifestations were not detected in the infected buffaloes. hematological changes in both species however, these changes were less significant in buffaloes than those reported in cattle.conclusion:this paper documents the first description of natural b. bovis infection in water buffaloes which were found to be more likely to be tolerant than cattle to the natural clinical infection with b. bovis and its subsequent haematological changes. our finding may lead to a better understanding of the disease pattern of b. bovis infection under field conditions in buffaloes. |
the lac operon consists of a regulatory domain and three genes required for the uptake and catabolism of lactose. a regulatory protein, the laci repressor, can bind to the operator and prevent the rna polymerase from transcribing the three genes. induction of the lac operon occurs when the inducer molecule binds to the repressor. as a result, the repressor can not bind to the operator and transcription proceeds at a given rate. the probability for the inducer to bind to the repressor depends on the inducer concentration inside the cell. the induction process is thus helped by the permease encoded by one of the transcribed genes, which brings inducer into the cell. in this way, if the number of permeases is low, the inducer concentration inside the cell is low and the production of permeases remains low. in contrast, if the number of permeases is high, the inducer concentration is high and the production of permeases remains high. this heuristic argument is useful for understanding the presence of two phenotypes, but it does not actually explain why the cells remain in a given state, or what makes the cells switch from the uninduced to the induced state. one needs quantitative approaches to understand the dynamics of this process, how the intrinsic randomness of molecular events affects the system, and how induction depends on the molecular aspects of gene regulation. despite its apparent simplicity, the lac operon system displays much of the complexity and subtlety inherent to gene regulation. in principle, its detailed modeling should include, among many other cellular processes, transcription, translation, protein assembly, protein degradation, binding of different proteins to dna, and binding of small molecules to the dna - binding proteins. in addition, the lac operon system is not isolated from the rest of the cell. induction changes the growth rate of individual cells, which in turn also affects the cell population behavior. for instance, if a gratuitous inducer is used, induction will slow down cell growth. therefore, extrapolating directly from the molecular level all the way up to the cell population level requires additional information about cellular processes that is not readily available. moreover, most of the molecular details of the cell are not going to be relevant for the particular process under study. the first step of modeling is, therefore, to identify the relevant levels, their interactions, and the way one level is incorporated into another. 2 illustrates schematically the separation of the lac system into molecular, cellular, and population levels. the three genes lacz, lacy, and laca are cotranscribed as a polycistronic message from a single promoter p1. the gene lacz encodes for the -galactosidase, which can either break down lactose into -d - galactose and d - glucose or catalyze the conversion of lactose into allolactose, the actual inducer. the product of lacy is the -galactoside permease, which is in charge of the uptake of lactose inside the cell. the role of laca is not yet fully understood because its product, a galactoside acetyltransferase, is not required for lactose metabolism (wang., 2002). the lac repressor is encoded by laci, which is immediately upstream of the operon. repression is greatly enhanced by the additional simultaneous binding of the repressor to one of the auxiliary operator sites o2 and o3. some gratuitous inducers, such as tmg, also use the lactose permease to enter the cell ; but in contrast to lactose, they bind themselves to the repressor and are not metabolized by the cell. in this case, it is possible to study the dynamics of induction by considering as variables only the internal inducer concentration, the nonfunctional permeases, and the functional permeases. uninduced cells (empty circles) have some probability to become induced (full circles). if uninduced cells grow faster, both types of cells could coexist ; if not, the entire population will eventually be induced. the molecular level explicitly includes the binding of the inducer to the repressor, changes in repressor conformation, binding of the repressor to the operator, binding of the rna polymerase to the promoter, initiation of transcription, production of mrna, translation of the message, protein folding, and so forth. almost all the quantitative aspects of the in vivo dynamics of these processes are unknown. the lack of information is typically filled out with assumptions based on parsimony. fortunately, not all the details are needed. at this level, what seems relevant is the production of permeases expressed as a function of the inducer concentration inside the cell. to obtain theoretically even a rough approximation of this function therefore, a more reasonable approach at the present stage of knowledge would be to extract this function directly from the experimental data. indeed, one can measure the rate of production of -galactosidase in mutant strains lacking the permease (herzenberg, 1958). in this case, external and internal inducer concentrations are both the same once equilibrium between the medium and the cytoplasm is reached. the other key piece of information is that the production of permeases is, to a good approximation, proportional to the production of -galactosidase, as both are produced from the same polycistronic mrna. the results obtained in this way could be used as an estimate for modeling the molecular level of wild - type cells. the core of the all - or - none process resides at the cellular level. some of the permeases produced will eventually go to the membrane and bring more inducer. novick and weiner (1959) inferred from experiments that only a few percent of the permeases integrate into the membrane and become functional. recent experiments, however, showed that the majority of the permeases integrate in the membrane (ito and akiyama, 1991), yet the question of how many are functional has not been addressed. despite intense studies on the permease (kaback., 2001), its in vivo functioning is still a challenging issue, which includes many open questions, such as the mechanisms of insertion into the membrane. the simplest assumption for modeling is that the produced permeases are inserted into the membrane and become functional with a constant probability rate. we believe this to be the weakest point of our model. in view of the all - or - none phenomenon, single - cell studies on the concentration and the functional state of the permeases would be extremely useful using techniques that are now available (thompson., 2002). when lactose is the sole carbon source, induction allows cells to grow. for gratuitous inducers, like the one used in novick and weiner (1957) experiments, this slowing down seems to be connected with the number of permeases in the membrane (koch, 1983). at this level the cellular level is integrated into the population level by considering the induced uninduced switching rates. these rates can be obtained by modeling at the cellular level by computing the probability for an uninduced cell to become induced and for an induced cell to become uninduced. the molecular level explicitly includes the binding of the inducer to the repressor, changes in repressor conformation, binding of the repressor to the operator, binding of the rna polymerase to the promoter, initiation of transcription, production of mrna, translation of the message, protein folding, and so forth. almost all the quantitative aspects of the in vivo dynamics of these processes are unknown. the lack of information is typically filled out with assumptions based on parsimony. fortunately, not all the details are needed. at this level, what seems relevant is the production of permeases expressed as a function of the inducer concentration inside the cell. to obtain theoretically even a rough approximation of this function, one would need detailed information about many molecular interactions. therefore, a more reasonable approach at the present stage of knowledge would be to extract this function directly from the experimental data. indeed, one can measure the rate of production of -galactosidase in mutant strains lacking the permease (herzenberg, 1958). in this case, external and internal inducer concentrations are both the same once equilibrium between the medium and the cytoplasm is reached. the other key piece of information is that the production of permeases is, to a good approximation, proportional to the production of -galactosidase, as both are produced from the same polycistronic mrna. the results obtained in this way could be used as an estimate for modeling the molecular level of wild - type cells. the core of the all - or - none process resides at the cellular level. some of the permeases produced will eventually go to the membrane and bring more inducer. novick and weiner (1959) inferred from experiments that only a few percent of the permeases integrate into the membrane and become functional. recent experiments, however, showed that the majority of the permeases integrate in the membrane (ito and akiyama, 1991), yet the question of how many are functional has not been addressed. despite intense studies on the permease (kaback., 2001), its in vivo functioning is still a challenging issue, which includes many open questions, such as the mechanisms of insertion into the membrane. the simplest assumption for modeling is that the produced permeases are inserted into the membrane and become functional with a constant probability rate. we believe this to be the weakest point of our model. in view of the all - or - none phenomenon, single - cell studies on the concentration and the functional state of the permeases would be extremely useful using techniques that are now available (thompson., 2002). when lactose is the sole carbon source, induction allows cells to grow. for gratuitous inducers, like the one used in novick and weiner (1957) experiments, this slowing down seems to be connected with the number of permeases in the membrane (koch, 1983). at this level the cellular level is integrated into the population level by considering the induced uninduced switching rates. these rates can be obtained by modeling at the cellular level by computing the probability for an uninduced cell to become induced and for an induced cell to become uninduced. the preceding discussion seems to indicate that three variables are relevant for the description of the functioning of the lac system. these are the concentrations of nonfunctional permease (y), of functional permease (yf), and of inducer inside the cell (i). another variable that we need to incorporate explicitly in the model is the concentration of -galactosidase (z), which is the quantity measured in the experiments. now, we are ready to model the dynamics of the induction process by writing down the phenomenological dynamical equations for these variables : \documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}\frac{dy}{dt}=f_{{\mathrm{1 } } } \left \left(i\right) \right -a_{{\mathrm{1}}}y{\mathrm{,}}\end{equation}\end{document}\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}\frac{dy_{f}}{dt}=b_{{\mathrm{1}}}y - a_{{\mathrm{2}}}y_{f}{\mathrm{,}}\end{equation}\end{document}\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}\frac{di}{dt}= \left \left[f_{{\mathrm{2 } } } \left \left(i_{ex}\right) \right -f_{{\mathrm{3 } } } \left \left(i\right) \right \right ] \right y_{f}+b_{{\mathrm{2}}}i_{ex}-a_{{\mathrm{3}}}i{\mathrm{,}}\end{equation}\end{document}\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}\frac{dz}{dt}=g\;f_{{\mathrm{1 } } } \left \left(i\right) \right -a_{{\mathrm{3}}}z{\mathrm{.}}\end{equation}\end{document } here, iex is the external inducer concentration ; g, b1, b2, a1, a2, and a3 are constants ; and f1, f2, and f3 are functions of their respective arguments. the molecular level description enters the equations through the specific form of f1, f2, and f3. f1(i) is the production rate of permeases as a function of the internal inducer concentration. it behaves like a quadratic polynomial for low inducer concentrations (f1[i ] c1 + c2i + c3i, with c1, c2, and c3 constants) and increases monotonically until it saturates for high concentrations. the functions f2(iex) and f3(i) account for the inducer transport by the permease in and out of the cell and are assumed to depend hyperbolically on their argument. with only these four equations one can explain the fact that there are inducer concentrations, iex, for which the cells remain induced, if they were previously induced, or uninduced, if they were uninduced. in mathematical terms, this happens because the equations have two stable solutions for such values of iex and the system thereby exhibits hysteresis. thus, the standard modeling approach can apparently explain the existence of the so - called maintenance concentration. there are many variations of this simple model. the first one, proposed already by novick and weiner (1957), was even simpler and explained to some extent the main features observed in the experiments (cohn and horibata, 1959a, b). in fact, subsequent, much more complex models, based on the standard biochemical reaction kinetics approach, did not provide any substantial additional insight. they basically showed that the observed behavior is also compatible with more intricate kinetics (chung and stephanopoulos, 1996). to fully understand the all - or - none phenomena, the standard approach is, however, not enough. one needs to take into account stochastic events to explain why, at some point, just by chance, a cell becomes induced. the classical approach is unable to explain the switch from the uninduced to the induced state. fortunately, it is possible to write down a stochastic counterpart of the previous equations. this is done by transforming the different rates (production, degradation, etc.) into probability transition rates and concentrations into numbers of molecules per cell. then, one can simulate the dynamical behavior of the four random variables governed by such stochastic equations on a computer (gillespie, 1977). fig. 3 a shows representative time courses of the -galactosidase content obtained from such computer simulations for cells placed under suboptimal induction conditions. at the single - cell level, there is a fast switch from the noninduced to the induced state. the time at which this transition happens is a result of the intrinsic stochastic nature of biochemical reactions and strongly varies from cell to cell (e.g., yellow, green, and blue lines in fig. the cell average exhibits a smooth behavior. in this case, the behavior of the single cell and the behavior of the cell average are thus completely different. as a consequence, classical reaction kinetics can not be used and has to be replaced by a stochastic approach. this type of approach started to be applied in the 1940s (delbrck, 1940) and was already well established in the late 1950s (montroll and shuler, 1958). only recently, however, has there been a renewed widespread effort to understand the role of stochasticity in cellular processes (rao., 2002). the thin (yellow, green, and blue) color lines correspond to representative time courses of -galactosidase content obtained from computer simulations for single cells at 7 m tmg. the observed differences in switching times from noninduced to induced states result from the stochastic behavior of the model. the dashed black line is the population -galactosidase content. to obtain the population results, we have considered in the simulations that induced cells representative time courses of the -galactosidase content obtained for induced (top) and uninduced (bottom) cells transferred to the maintenance concentration (5 m tmg) at time 0. note the semilogarithmic scale. (c) same as in panel a but now for cells at 500 m tmg. in this case, the cell average and population -galactosidase content are indistinguishable. the black line is the same as in panel c. red dots represent experimental values obtained by novick and weiner (1957). the dashed line is the same as the black line but shifted to the left 0.33 generations. it illustrates that the main differences between simulations and experimental results come from the early stages of induction. one should stress that even the stochastic approach is still unable to fully explain the experiments. in the simulations,, the production of -galactosidase for suboptimal inducer concentrations seems not to saturate at the maximum value, which is an indication of the coexistence of the induced and uninduced cells. as explained before, the reason for this is that the induced and uninduced cells grow at a different rate. therefore, we have to consider the dynamics of the cell population. only when this is taken properly into account are the simulations in agreement with experiments, as shown by the dashed line in fig. 3 a. the fact that fluctuations make cells switch from the uninduced to the induced state forces us to reconsider whether there really exists a maintenance concentration in the model. is there a range of inducer concentrations for which the cells do not switch at an appreciable rate from one state to another ? b shows the single - cell behavior for cells that were previously induced or uninduced at the expected maintenance concentration. indeed, in the simulations we performed for 1,000 cells, we recorded no single switching from one state to another ; for realistic values of probability rates such switching events would be too rare to be observed. the stochastic model seems to be thus compatible with the existence of the maintenance concentration. so far, we have pointed out just a few of the many limitations of the standard modeling approach and how to overcome them. considering stochastic and population effects greatly increases the complexity of modeling. in general, whether or not we should consider all of these effects depends not only on the given system but also on the particular conditions. for instance, an approach taking into account all three levels of description is not needed when the lac operon is induced at high inducer concentrations. in this case, the single - cell picture, the average over independent cells, and the population average all give very similar results, as can be seen in fig. 3 c. therefore, it should be possible here to use standard kinetic equations and avoid most of the hassle encountered beyond the standard approach. the main problem, however, is that there is no general a priori method to tell whether or not the standard modeling approach would be sufficient to describe the given system. in fig. there are some differences : the rise in -galactosidase activity is faster in the experiments than in the simulations. in addition, coming back to fig. 3 b, one can see that there is a small drop in -galactosidase content when cells are transferred from high to maintenance inducer concentrations. this drop is not present in the experiments (see fig. 3 in novick and weiner, 1957). one can not infer from the model whether these differences are a matter of details or of a more fundamental aspect of the lac system. the addition of more molecular details into a model (carrier and keasling, 1999) does not necessarily lead to better agreement with the experimental observations. the type of models and experiments that we have discussed can provide valuable information about the mechanistic structure of the lac operon. but, to really understand the functioning and underlying logics of cellular networks, one needs to consider them in their natural environment. only then is it possible to relate the network structure to the function it has acquired through evolution (savageau, 1977). in the case of the lac operon of e. coli, induction usually takes place in the mammalian digestive tract under anaerobic conditions (savageau, 1983), and the inducer is allolactose, a metabolic product of lactose, rather than gratuitous inducers, such as iptg or thiomethyl--d - galactoside (tmg). in addition, there can be other factors that can affect the induction process itself. for instance, recent genetic studies have uncovered a novel set of sugar efflux pumps in e. coli that surprisingly can pump lactose outside of the cell (liu. the example of the lac operon switch has been used here to illustrate the current state, applicability, and limitations of modeling of cellular processes. we have not tried to expose all the potential that modeling possesses ; there are now many published reviews advertising this aspect. rather, we have tried to use one of the simplest and best - studied examples to show the intricacy of modeling biological networks. first, standardized modeling methods can not be applied automatically even in a case as simple as the one we have described. one needs first to identify the relevant variables, adequate approximations, etc. adding more equations to include more details of interactions if more molecular details are considered, one can easily end up with huge sets of equations, but unless the relevant elements are identified, the model will remain useless. we have discussed, a four - equation model is able to explain the main results of the experiments of novick and weiner (1957), provided that fluctuations and population effects, which are usually overlooked, are taken into account. second, one of the main reasons for the success of models in matching the experimental results is that the experiments are kept under constant conditions and only a few variables are changed. third, networks are isolated neither in space nor in time. they form part of a unity that has been shaped through evolution. it is important not to disregard a priori any of the many complementary levels of description : molecular, cellular, physiological, population, intrapopulation, or evolutionary. in our opinion, because of these and similar reasons, productive modeling of biological systems, even in the post - genomic era, will still rely more on good intuition and skills of quantitative biologists than on the sheer power of computers. | we use the lac operon in escherichia coli as a prototype system to illustrate the current state, applicability, and limitations of modeling the dynamics of cellular networks. we integrate three different levels of description (molecular, cellular, and that of cell population) into a single model, which seems to capture many experimental aspects of the system. |
bacopa monniera l. (scrophulariaceae), commonly known as brahmi, is a creeping plant with bitter taste found in marshy areas in india. the leaf extract of b. monniera contains bioactive compounds bacosides a1a3 [13 ], bacopasaponins a a study claimed that bioactive compounds extracted from b. monniera improved acquisition, consolidation and retention of newly acquired behavior in rats. subsequent studies demonstrate that these compounds significantly attenuated the phenytoin, diazepam, hypobaric hypoxia, l - nna, scopolamine, mcpbg and d - galactose induced memory impairments in rats. b. monniera treatment modulates various neurotransmitters such as acetylcholine (ach), serotonin (5-hydroxytryptamine, 5-ht), gamma amino butyric acid (gaba), glutamate (glu) and dopamine (da) at different brain regions to enhance the cognitive functions [16, 1824 ]. it has been documented that variations in serotonin metabolism and transmission is often associated with changes in short and long - term memory [2528 ]. biosynthesis of serotonin is regulated by the rate - limiting enzyme tryptophan hydroxylase (tph) (ec. 1. 14. 16. 4), which converts l - tryptophan to 5-hydroxy - l - tryptophan using molecular oxygen, non - heme iron (ii) with tetrahydrobiopterin (bh4) as a cofactor [29, 30 ]. there are two forms of tph ; tph1 is responsible for most peripheral 5-ht, whereas, tph2 is neuronal specific [3134 ]. isoforms of tph (tph1, tph2) are highly homologous with an identity of 6871 % across the phyla, and possess similar three - dimensional structure with respect to the location of amino acid residues where the substrate binds or the active site geometry. tph2 holds different kinetic properties like higher specificity for l - tryptophan but a lower catalytic efficiency. it has higher molecular weight than tph1 because of the elongated n - terminal domain (approx. 56 kda compared to tph1 which is 51 kda) and also possess an additional phosphorylation site. studies indicate that up - regulation of tph expression leads to an enhancement of tph activity and 5-ht synthesis [37, 38 ]. recently, we found that the administration of b. monniera leaf extract in postnatal rats elevates the 5-ht level by up - regulating tryptophan hydroxylase-2 (tph2) and serotonin transporter (sert) expression. thus the elevated 5-ht level influences learning and memory by modulating other ach and glu, acting through the 5-ht3 receptor. based on the limitations, as a first attempt we adopted in silico approach to study the complex interactions. at this point, we used three different automatic molecular docking algorithms such as autodock, patchdock and hexdock to identify interactions between the complexes. through various interactions, molecular recognition occurs between protein and ligand, which is critical for identifying binding partners to the binding site. our in silico study showed the interaction of bacosides (a3, a) with tph, which is believed to be involved in the up - regulation of 5-ht synthesis. to uncover the structural basis of docking interactions between tph and bacosides, we sought to determine the complex formation. number of active compounds was identified for therapeutic targets based on the binding of ligand with protein, in which virtual screening and computational tools plays a major role [40, 41 ]. bacosides are non - polar glycosides ; lipid - mediated transportation by passive diffusion may facilitate transport across blood the general 3d molecular structure with combination of bacoside - tph showed that the bioactive compounds present in the b. monniera leaf extract interact with tph, which may up - regulate tph2 expression. in our study, we tested bacoside - tph interactions in three different algorithms developed based on combinations of various scoring functions. this would help us to identify the true hits and reduce the errors. after finding its interactions with bacoside a and a3, we have found out that both plugs into a hydrophobic pocket of tph. further, our analysis showed that all the three docking algorithms identified similar residues (tyr235, thr265, pro268, his272, glu317, phe318, ser337, glu340, ile 366) surrounding (within 3.0) bacoside a as best hit and interacts with different residues of tph. 1b) identified that thr265 (1.46 and 1.91) is the residue involved in hydrogen bond, where as autodock identified glu 317 (1.79) as an interacting residue (fig. 1). in addition, the estimated free energy for the complex is 9.29 kcal / mol with an inhibition constant (ki) of 156.17 nm. similarly, all the three docking algorithms showed tyr235, thr265, pro268, glu317, ser337 and cys364 residues (within 3.0) in the pocket of the tph surrounding bacoside a3. among them all the three docking algorithm identified different residues thr265 (1.91) (fig. bacoside a3 with tph complex showed docking score of 5472 with atomic contact energy 424.37. further, we noted 10.23 kcal / mol as a estimated free energy for bacoside a3-tph complex with the ki of 37.36 nm.fig. 13d molecular structure from combination of tph and bacoside a generated using hexdock (a), patchdock (b) and autodock (c) showing the interactions with key residuesfig. patchdock (b) and autodock (c) showing the interactions with key residues thr265, tyr235 and glu317 respectively 3d molecular structure from combination of tph and bacoside a generated using hexdock (a), patchdock (b) and autodock (c) showing the interactions with key residues tph and bacoside a complex 3d molecular structure generated using hexdoc (a), patchdock (b) and autodock (c) showing the interactions with key residues thr265, tyr235 and glu317 respectively in the present study, our in silico analysis showed that bacosides (a3, a) interact with tph. we found that the bacoside a3 showed higher interaction score when compared to the interaction of bacoside a with tph. the bioactive compounds : bacoside a and a3 formed hydrogen bond with tph at two key residues glu317 and tyr235. its note worthy to mention that tryptophan binds with leu124-asp139 and ile367-thr369 loop regions of tph which are involved in conformational change [44, 45 ]. interestingly, except glu317 (o of glu317 with h- of bacosides), the bacosides binding amino acids were not present at either the tryptophan or the co - factor bh4 (gly 234, leu 236, his 251, glu 273, glu 317) binding sites [44, 45 ]. in fact, we found that bacoside a3 and a formed hydrogen bond with glu317. although glu317 of tph bound with fe, it also interacts with bacoside a3/a at a different side chain. notably, bacoside a, a3 interact with thr265, which is in the line of active site channel of tph (residue 263269 and 363372) having high b - factor and less electron density. it appears that these regions are highly flexible and the interaction at this site might enhance the tph activity, possibly the reason for the observed elevated level of tph activity. further, our analysis showed that bacoside a3 interacts with tyr235 of tph, which is conserved in all known tph. specific mutants of tyr235 increase the km for substrate (l - trp) and lower km for cofactor (bh4). our in silico analysis showed that the bacosides (a3, a)-tph complex interact through hydrogen bond with specific sites. these specific sites are known to regulate the catalytic mechanism of tph [4446 ], bacosides interactions may facilitate tph activity and thus enhance the synthesis of serotonin. further study in bacosides interaction with different mutated tph may provide greater insight in understanding the effect of bacosides in serotonergic system for the potential applications. the pdb format of tph enzyme (pdb i d : 1mlw) was retrieved from protein data bank. likewise, the chemical structures of bioactive compounds bacoside a (compound i d : 53398644), bacoside a3 (compound i d : 44152167) were collected from database (http://pubchem.ncbi.nlm.nih.gov) and then converted as protein data using openbabel and docked with tph. different algorithms such as autodock, patchdock and hexdock was used based on complementary shape principles matching two molecules by searching complementary to each other. any type molecules (proteins, dna, peptides, drugs) can be docked in patchdock. a wide interface is ensured to include several matched local features of the docked molecules that have complementary characteristics. each candidate transformation is further evaluated by a scoring function that considers both geometric fit and atomic desolvation energy. finally, an rmsd (root mean square deviation) clustering is applied to the candidate solutions to discard redundant solutions. the generated tph and bacoside three - dimension (3d) molecule complex was further analyzed by the swiss - pdb viewer (ver 4.1). an algorithm has been used to identify the hydrophobic and hydrogen bond interaction, based on the distance between ligand protein atoms, which is below 3.0. | tryptophan hydroxylase (tph) catalyses l - tryptophan into 5-hydroxy - l - tryptophan, which is the first and rate - limiting step of serotonin (5-ht) biosynthesis. earlier, we found that tph2 up - regulated in the hippocampus of postnatal rats after the oral treatment of bacopa monniera leaf extract containing the active compound bacosides. however, the knowledge about the interactions between bacosides with tph is limited. in this study, we take advantage of in silico approach to understand the interaction of bacoside - tph complex using three different docking algorithms such as hexdock, patchdock and autodock. all these three algorithms showed that bacoside a and a3 well fit into the cavity consists of active sites. further, our analysis revealed that major active compounds bacoside a3 and a interact with different residues of tph through hydrogen bond. interestingly, tyr235, thr265 and glu317 are the key residues among them, but none of them are either at tryptophan or bh4 binding region. however, its note worthy to mention that tyr 235 is a catalytic sensitive residue, thr265 is present in the flexible loop region and glu317 is known to interacts with fe. interactions with these residues may critically regulate tph function and thus serotonin synthesis. our study suggested that the interaction of bacosides (a3/a) with tph might up - regulate its activity to elevate the biosynthesis of 5-ht, thereby enhances learning and memory formation. |
it is generally considered to be a benign proliferation of endothelial cells with secondary thrombosis and fibrin deposition or an excessive reaction to the normal reorganization process in a thrombus2,18). the lesion contains papillary structures composed of a single layer of swollen endothelial cells around a core of fibrous connective tissue and may be mistaken for angiosarcoma based on histopathological findings12). ipeh most commonly occurs in the skin and subcutaneous soft tissues, where it exhibits a benign clinical course. but, it can also occur in the nasal cavity, pharynx, larynx, internal auditory canal, labyrinthine, heart valve, breast, digestive tract, liver, kidney, cervix, uterus, female urethra, and pelvic veins12,17). ipeh associated with the central nervous system, with or without skull invasion, is rare9). a mass on the left frontal bone was incidentally detected in a 14-year - old male. after the operation, adjuvant radiotherapy consisted of craniospinal irradiation and chemotherapy had been given according to the m-051 protocol of the korean society of pediatric neuro - oncology. two years post - operatively, dilated or prominent vascular structures were detected around the left frontal bone marrow on gadolinium (gd)-enhanced axial t1-weighted magnetic resonance imaging (mri) (fig. six months later, a follow - up t2-weighted mri revealed a well - defined hyperintense nodular lesion in the same area with homogeneous enhancement (fig we presumed that the lesion was a benign bone marrow lesion, such as hemangioma, rather than metastasis. however, a routine follow - up axial gd - enhanced mri 7 months later demonstrated that the lesion was a well - circumscribed growing cystic mass with thin peripheral enhancement. prompted by this latest finding, we began to consider the possibility of a metastatic lesion. further growth of the rim enhanced cystic mass was evident upon examination 7 months later (fig. computed tomography showed a well - circumscribed, expansile osteolytic mass located on the inner table and diploic space of the frontal bone (fig. during the 20 months of follow - up, the lesion had grown slowly to a 2 cm - sized cystic osteolytic lesion, which was suggestive of a metastatic bone tumor. frontal craniectomy including complete excision of the mass with safety margin was done and cranioplasty with bone cement was applied. intra - operatively, the skull showed destructive changes of the inner table and diploic space, and the hemorrhagic content under the thin lining capsule (fig. pathologic findings showed both the capsule consisted of parallel lamellae of collagen and a vascular wall, vascular lumen, and papillary projection into the lumen associated with hemorrhage and calcification (fig. there was no evidence of cytologic atypia, abnormal mitosis, necrosis, or any solid spindle - cell areas. no post - operative complications occurred and no recurrence was evident at the 1-year follow - up. ipeh was described in 1923 for the first time, and is known by many eponyms in the literature, including masson 's vegetant intravascular hemangioendothelioma14), intravascular angioma - tosis18), masson 's pseudoangiosarcoma12), intracranial masson 's hemangioma4), and intravascular papillary endothelial hyperplasia22). it generally appears as a subcutaneous or intramuscular nodule on the finger, in the head and neck region, or in thrombosed vessels, principally the hemorrhoidal veins18,22). intracranial occurrence is very rare, with only a few cases being reported to involve the on central nervous system with or without skull invasion9). ipeh is generally considered to be a specific form of thrombus organization, rather than a primary intravascular tumor2,18). the primary type is an unusual reactive response of normal endothelial cells to thrombus formation, almost always in low flow venous channels. the lesion impairs the venous drainage system, which causes the congestion and hemorrhage24). the secondary type is associated with pre - existing cavernous hemangioma and hemorrhoidal veins complicated by thrombosis and the extravascular form develops in extravascular organized hematomas17). radiologically, the 18 previously described cases of intracranial ipehs were located on the cerebral hemisphere in nine patients, cavernous sinus and sellar in three patients, cerebellum in two patients, brain stem in one patient, posterior inferior cerebellar artery in one patient, within the internal auditory canal in one patient, and in the superior orbital fissure in one patient9). contrast enhancement has been documented in 61% of cases1,3 - 7,11,13,16,20) and associated hemorrhage has been described in 44.4% of cases5 - 9,19 - 21). underlying vascular malformations such as cavernous angioma, venous angioma, and arteriovenous malformation have been described in 22.2% of cases8,11,16,21). three cavernous sinus and sellar and two temporal ipehs (27.7%) involved the destructive invasion of adjacent bone1,11,20). the present location of the ipeh on the skull is rare. during 20 months of observation, a follow - up mri revealed a homogeneously enhanced mass, which would be consistent with thrombosis and endothelial proliferation. this lesion became a well - circumscribed cystic mass with thin peripheral enhancement, consistent with repeated hemorrhaging, which may have driven the development of the mass. during the period of examination, the lesion grew slowly to form a well - circumscribed, expansile osteolyic mass located on the inner table and diploic space of the frontal bone. histologically, it is characterized by papillary proliferations and thrombotic material within the vascular lumen associated with a normal endothelial lining. it must be differentiated from other malignancies such as angiosarcoma and kaposi 's sarcoma18). endothelial atypia, pleomorphism, necrosis, multiple layers of endothelium, and frequent mitoses are not supportive of a diagnosis of ipeh23). immunohistochemistry data adds to the confidence of the diagnosis, since ipeh typically is positive for factor viii - related antigen and cd34. recurrence has been noted in cases of subtotal resection1,11,15,19). because of the small number of reported cases, it is still difficult to draw conclusions as to the efficacy of adjuvant treatment. various doses of radiation have been used ; some have been effective1,11). likewise, the clinical outcome of chemotherapy remains vague19). we reported the first intracalvarial ipeh, which showed destructive radiologic development associated with hemorrhage. | intravascular papillary endothelial hyperplasia (ipeh) is a rare vascular benign lesion that rarely involves the central nervous system with or without skull invasion. we report a rare case of ipeh on the skull bone, which displayed destructive radiologic development associated with hemorrhage. a 14-year - old male presented with an incidentally detected a small enhancing, left frontal osteolytic lesion. previously, he underwent operation and received adjuvant chemoradiation therapy for cerebellar medulloblastoma. follow - up magnetic resonance imaging revealed a left frontal bone lesion, which expanded to an approximately 2 cm - sized well - circumscribed osteolytic lesion associated with hemorrhage for 20 months. frontal craniectomy and cranioplasty were performed. destructive change was detected on the inner table and diploic space of the skull. the mass had a cystic feature with hemorrhagic content without dural attachment. pathologic examination showed the capsule consisted of parallel collagen lamellae representing a vascular wall, vascular lumen, which was pathognomonic for ipeh. immunohistochemical staining revealed that the capsule was positive for cd34 and factor viii, which favor the final diagnosis of ipeh. this was the first case of intracalvarial ipeh. |
protein splicing is a post - translational autoprocessing event carried out by a class of proteins known as inteins. during this process, an intein domain excises itself from a larger precursor protein and ligates its n- and c - terminal flanking sequences (termed exteins) through a native peptide bond. most are cis - splicing inteins that are expressed as single polypeptide chains embedded within their host proteins. by contrast, the far less abundant trans - splicing inteins are transcribed and translated as two separate protomers that associate and fold into the canonical intein domain structure. the association of naturally split inteins is rapid compared with the subsequent protein splicing reaction. regardless of whether splicing occurs in cis or trans fashion, the mechanism of protein splicing is the same (figure 1). first, the n - extein / intein peptide bond is activated through an n - to - s acyl shift to form a linear thioester intermediate. next, this activated acyl group undergoes trans - thioesterification to form a branched thioester intermediate on the first residue of the c - extein, cys+1. in the last chemoenzymatic step, the c - terminal asn residue of the intein cyclizes, thereby resolving this branched intermediate (bi) into an excised intein and an n - extein / c - extein thioester adduct. finally, this transient thioester spontaneously rearranges to a native peptide bond to yield the spliced product, and the excised intein succinimide hydrolyzes to yield a free carboxylate. numbers 14 refer to the chemically distinct c - intein adducts that can be observed in the splicing assays described in this report (see figure 3 and figure s8 in the supporting information). for simplicity, only the -amino acid isomer of 4 is shown, but the ring in species 3 can open into an - or -amino acid form. it is noteworthy that while different families of inteins utilize subtle variations on this general biochemical mechanism (such as ser or thr nucleophiles rather than cys), the catalytic residues for protein splicing are always confined to the intein domain and the first c - extein residue. despite this fact, a growing body of experimental evidence indicates that the intein splicing efficiency is highly dependent on the identity of two or three local extein residues on either side of the splice junction. for example, introduction of non - native residues at the 3, 2, and 1 positions, located on the n - extein (figure 1), can alter the linear thioester formation efficiency or promote hydrolysis of this intermediate. mutation of the + 1, + 2, and + 3 residues, located on the c - extein (figure 1), can abolish or greatly diminish the splicing activity and even lead to premature asparagine cyclization before formation of the bi. for each intein family, this context - dependent activity is dictated by evolutionary pressures, as inteins are naturally embedded between highly conserved residues in a number of different endogenous host proteins. as a result, the chemical synthesis of larger and more complex peptides and proteins is an ongoing challenge, and inteins are being widely used to facilitate such syntheses. thus, the sensitivity of protein splicing to the local extein sequence (i.e., the residues immediately flanking the intein) has significant practical implications. all intein - based technologies are premised on a single notion : the chemical perturbations that an intein exerts on its endogenous host protein can be applied in a virtually traceless manner to any exogenous protein of interest. in reality, however, efficient and traceless synthesis of complex products is not always achieved. rather, current technologies often require either the incorporation of non - native residues surrounding the splice junction in the target molecule or sacrifices in reaction kinetics and product yield to obtain the desired native sequence. an improved understanding of the general splicing mechanism and its sensitivity to local extein sequences thus remains of central concern. of particular interest as protein engineering tools are the split dnae inteins, all of which endogenously generate the catalytic subunit of dna polymerase iii after protein trans - splicing (pts). until recently, many split intein - based technologies relied on the founding member of this family, termed ssp, which derives its name from the model cyanobacterium that encodes it, synechocystis species pcc6803. however, ssp catalyzes pts in hours, which is too slow for many practical applications. with the discovery and characterization of new split dnae inteins, such as the now prevalent nostoc punctiforme (npu) intein, it is clear that several members of this family catalyze protein splicing with extraordinary efficiency (in minutes or less). thus, many intein technologies are now being developed and improved with these new tools, including in vitro and in vivo protein semisynthesis, segmental isotopic labeling, peptide cyclization, and the construction of novel biosensors. the dnae split intein family, however, is also plagued by poor tolerance for non - native local extein sequences. all split dnae inteins are naturally embedded within the local n - extein sequence aey (figure 1, residues 3, 2, and 1) and the c - extein sequence cfn (figure 1, residues + 1, + 2, and + 3). several reports indicate that dnae inteins can tolerate significant deviation from this native n - extein sequence. conversely, the presence of non - native c - extein residues can lead to dramatic reductions in splicing efficiency. for example, mutation of the canonical cfn sequence to sgv was shown to inhibit bi resolution for ssp, although the contributions of each c - extein mutation were not individually assessed. additionally, the identity of the + 2 c - extein residue has a dramatic impact on the splicing activity for all members of the dnae family, but it is not clear what step in the splicing pathway is modulated by this residue. despite the fact that c - extein - dependent splicing activity is well - documented for dnae inteins, little is known about the magnitude of this effect on the reaction kinetics or the physical basis of this phenomenon. we envisioned that a detailed understanding of how c - extein residues participate in the splicing reaction could help guide the practical use of split inteins and help lay the foundation for the design of more promiscuous engineered inteins. to this end, we performed a detailed structure activity analysis on the npu intein, employing semisynthesis to alter the c - extein moiety systematically, thereby providing the raw materials for a series of kinetic and structural analyses. this effort led to the finding that the + 2 residue in the c - extein plays a critical role in constraining the active site of the intein during bi resolution. the work also draws attention to a loop region in the intein structure that appears to sense the c - extein composition and thus might be a productive focus of engineering efforts geared toward increasing intein promiscuity. our efforts began with the construction of a library of c - intein fragments (intc) bearing a variety of model c - exteins ranging from a single cys residue with different capping groups to tripeptides with unique sequences (see table 1). for rapid generation of the desired constructs (17 proteins in all), we employed a semisynthetic approach utilizing expressed protein ligation (figure 2). specifically, the intc fragments of npu and ssp (termed npuc and sspc, respectively) were expressed in escherichia coli fused to the cis - splicing his6-tagged gyra intein and enriched over ni columns (figure s4). the crude fusion proteins were then reacted with either a large excess of a cysteine derivative (100 mm) to yield an intc cys adduct directly, or they were thiolyzed with 100 mm 2-mercaptoethanesulfonate (mes) in the presence of a 15 mm di- or tripeptide to yield intc peptide adducts (figure 2a, b). the desired product from each reaction was readily purified by reversed - phase high - performance liquid chromatography (rp - hplc) (figures 2c and s7), and its identity was confirmed by electrospray ionization mass spectrometry (esi - ms) (figure 2d and table s2 in the supporting information). importantly, this semisynthesis approach allowed for the modular assembly of constructs with natural amino acid mutations within the c - intein and effectively any functional groups in the c - extein side chains and backbone. (a) semisynthetic scheme (r = oh, och3, nh2, nhch3, or an additional one or two amino acids, as indicated in table 1). (b) rp - hplc analysis of a one - pot mes - thiolysis / ligation sequence to synthesize npuc npuc mes and ligation product accumulation are shown in the left panel, and cleavage of the npuc gyra - his6 fusion protein is shown in the right panel. (c) rp - hplc and (d) esi - ms analysis of npuc cf(och3) after purification. in (d), the raw and deconvoluted mass spectra are shown in the top and bottom panels, respectively (expected monoisotopic mass = 4387.28 da). to provide a rigorous assessment of c - extein effects on pts, we developed two complementary analytical approaches that allowed us to distinguish various chemical species along the reaction coordinate in a time - resolved fashion. first, n - intein (intn) proteins bearing a minimized n - extein tripeptide (aey intn) were generated recombinantly and purified (figures s5s7 and table s2). these constructs were mixed with their intc counterparts at 30 c, and aliquots were removed from the reaction solution at various time points and quenched by acidification to ph 12. importantly, all of the reactions were carried out at ph 7.2 in the absence of thiol - based reducing agents to prevent any undesired hydrolysis or thiolysis reactions that would convolute the kinetic analyses. the time - point aliquots were analyzed by rp - hplc, and for most of the reactions, the various intc - related species (14 in figure 1) and the spliced product (5 in figure 1) could be readily separated (figures 3a and s9). for reactions where sufficient separation between species 15 was not achieved by rp - hplc, the quenched aliquots at various time points were desalted and analyzed as complex mixtures by esi - ms (figures 3b and s10). because of the similarities in sequence composition, size, and net charge among species 14, the molecules showed similar levels of ionization, and thus, the rp - hplc and esi - ms analyses gave virtually identical results (compare panels a and b in figure 3 ; for quantitative analysis of the error between the two assays, see figure s13). importantly, in both assay formats, the starting material and linear intermediate were indistinguishable, so the data were fit to a simplified kinetic model that collapsed the first two catalytic steps into a single equilibrium reaction (figure 3c, d). the results of our kinetic analyses are summarized in table 1 and figure 4. (a, b) time - dependent rp - hplc (a) and esi - ms (b) analyses of the reaction between aey npun and npuc the deconvoluted mass spectra in (b) are normalized to the intensity of the largest peak at each time point. (c) simplified three - state kinetic model of protein splicing compatible with the analytical techniques presented herein. (d) quantified reaction progress data for reaction 1 fit to the kinetic model in (c). the product curve represents the combined peak areas of all three products of the pts process (the two excised intein species 3 and 4 and the spliced product 5). we initially carried out a series of control reactions to validate our assays. the splicing kinetics of the wild - type npu and ssp inteins were assessed in their native n- and c - extein contexts (table 1, reactions 1 and 2). the overall rate constants for spliced product formation (ksplice) were 1.36 10 and 1.46 10 s, respectively, consistent with the results of previous measurements using gel - based assays. these experiments also demonstrated that bi resolution (described by k3) is the slow step for ssp, whereas for the faster npu reaction, the initial and latter steps of pts are kinetically coupled. as additional controls, we independently mutated the first catalytic cysteine (cys1) and the c - terminal asparagine (asn137) in npu to alanine and analyzed the effect of these mutations on the splicing activity. as expected, the c1a mutation completely inhibited splicing, although basal levels of succinimide formation and thus c - extein cleavage were observed on a time scale of hours (table 1, reaction 3). this result is consistent with the notion that cyclization of the c - terminal asparagine is stimulated by bi formation, as shown previously for the gyra intein. additionally, the n137a mutation abolished splicing and c - extein cleavage but only modestly reduced the kinetics of the initial steps (table 1, reaction 4). k1, k2, and k3 were extracted from a global fit of all three normalized curves for one reaction to the analytical solutions for the differential rate equations that describe our kinetic model. ksplice was extracted by fitting the product formation curve to a standard first - order rate equation. the reported values are means standard deviations from three individually fit unique trials. in reactions 3 and 13, k3 represents the rate of succinimide formation and thus c - extein cleavage in the absence of bi formation. in reaction 4, mutation of the catalytic asparagine abolishes succinimide formation, thus the reaction does not progress past the bi. in reaction 5, although all of the catalytic residues were present, no bi resolution was observed during the course of the assay. the extremely slow bi resolution in reaction 15 led to roughly 1020% n - extein hydrolysis as a side reaction, preventing a global fit to our kinetic model. for this reaction, k1 and k2 were extracted from a two - state equilibrium kinetic model using only the pre - equilibrium phase of the reaction (first 10 min). k3 was assumed to be identical to ksplice, which was determined by fitting the product formation curve to a first - order rate equation. next, we employed our kinetic assays to determine the effect of c - extein composition on individual steps in the pts reaction (table 1, reactions 512). these experiments revealed that variation of the c - extein had only a small effect on the kinetics of bi formation (k1 and k2) but profoundly affected the bi resolution step (k3) and thus the overall splicing rate constant (ksplice) (figure 4a, b). a detailed comparison of these kinetic analyses revealed several important trends (figure 4c). first, the c - extein chain length had a substantial effect on the activity. cys+1 alone could not sustain bi resolution with an uncapped carboxylate, suggesting that a negative charge near the active site is undesirable (table 1, reaction 5). capping the + 1 residue as an amide or ester restored a basal level of splicing activity (table 1, reactions 68). interestingly, cys+1 capped with a methyl ester afforded a 4-fold rate increase over the methylamide analogue, possibly indicating an inhibitory role for this amide n h moiety or an anomalous non - native effect of this subtle perturbation (table 1, reactions 6 and 8). ultimately, the effect of c - extein chain length on bi resolution was more pronounced once the entire phe+2 residue was added (table 1, reaction 10), but three c - extein residues were required to recapitulate the highest reported rates for npu (table 1, reaction 1). c - extein contributions to splicing activity. (a) forward (k1) and reverse (k2) rate constants for bi formation from starting materials. (b) rate constant for bi resolution (k3) and overall rate constant for trans - splicing (ksplice). (c) scheme highlighting the key conclusions from the kinetic data. through our kinetic analyses, we also identified two specific functional groups that make major contributions to bi resolution. first, we found that the amide bond after phe+2 provided a 6-fold rate enhancement relative to a methyl ester (compare reactions 9 and 10). this result suggests that the amide n h group is involved in a hydrogen bond that facilitates bi resolution, perhaps by stabilizing a catalytically competent conformation. the second, more significant functional group is the phe+2 phenyl ring. while this residue is known to be important, as discussed above, the extent of its contribution to bi resolution was not previously known. our measurements indicate that the addition of the bulky phe side chain enhances the bi resolution kinetics 100-fold relative to ala (compare reactions 1 and 12). interestingly, the presence of the phe side chain also stimulated the basal rate of succiminide formation (i.e., c - extein cleavage) in the context of a c1a mutant of npun (compare reactions 3 and 13), implying that the phe side chain forms favorable interactions even in the absence of the bi. by contrast, the side chain of asn+3 does not contribute to the pts reaction (compare reactions 1 and 11). given the significant contribution of the phe+2 side chain to the splicing kinetics, we next sought to understand the structural origin of its involvement in split intein chemistry. most high - resolution structures of inteins, including the only published structure of npu, do not contain c - extein residues. one important exception to this is a crystal structure of ssp bearing five native n - extein residues (kfaey), three native c - extein residues (cfn), and mutations of the terminal intein residues (cys and asn) to ala. in this structure, the phe+2 side chain packs against a catalytic histidine that lies on a flexible loop (figure 5a). this histidine (his125 in npu) is completely conserved in the dnae family and has been implicated as a general acid or base in the bi resolution step of many inteins. mutation of his125 in npu to asn reduced the rate of bi resolution roughly 60-fold, similar to the f+2a mutation (table 1, reactions 14 and 12, respectively). the ssp structure suggests that phe+2 participates in pts by stabilizing his125 through a direct interaction. indeed, mutating both residues in npu had a nonadditive effect on the bi resolution kinetics (gcoupling = 1.07 kcal mol), indicating some cooperativity between phe+2 and his125 with respect to this step (table 1, reaction 15 ; see figure s14 for thermodynamic cycle analysis). (a) crystal structure of the ssp dnae intein (pdb entry 1zde), with the close packing of his125 and phe+2 highlighted in spheres. the n - intein and c - intein are shown as blue and red ribbons, respectively. (b) the active site of ssp bearing catalytic cys and asn mutations, native extein residues, and a coordinated zinc ion. important residues surrounding the c - intein / c - extein junction (orange / black junction) are shown as sticks, and the c - extein is shown in gray. key catalytic residues are shown in orange, and other noncatalytic residues highlighted in this study are shown in green. (c) composite h and n backbone chemical shift perturbations (i) for c, n - labeled npuc in complex with unlabeled npun upon changing the + 2 c - extein residue from phe to ala (see the supporting information for calculations). the mean value is shown as a dashed purple line, and one standard deviation above the mean is shown as a dashed orange line. residues in secondary structure elements are marked with boxes above the bars : solid blue boxes denote strands, and open pink boxes denote loops. c hsqc spectra of segmental labeled npun : npuc complexes containing either phe (black) or ala (red) as the + 2 c - extein residue. c correlations (c1 and c2) are marked with dashed boxes. to obtain a better understanding of the structural impact of the + 2 residue, we carried out solution nmr analyses of npu in cfn(nh2) and can(nh2) c - extein contexts. nmr constructs were prepared analogously to those used in the kinetic assays but with some additional provisions. specifically, the npun protein contained the native n - extein sequence (aey) and an inactivating c1a mutation but was neither c- nor n - labeled. the npuc constructs, which bore the n137a mutation, were c- and n - enriched in the recombinant intc portion but not in the synthetic c - extein region. the n- and c - inteins were mixed, and the complexes were purified to homogeneity by size - exclusion chromatography (figures s15 and s16). the use of this segmental labeling scheme meant that only the npuc residues (ile103ala137), which had identical chemical composition in the two complexes, would be visible in heteronuclear correlation experiments. this was expected to simplify assignment while still allowing the putative interaction between the + 2 residue and the catalytic his125 to be interrogated. the inactivating mutations (c1a and n137a) ensured that chemistry would not occur during data acquisition. with the exception of several residues in the loop containing the catalytic his125 residue, we were able to assign the majority of the npuc backbone resonances in the complexes using standard triple - resonance experiments (figure s17). most of the backbone resonances were unperturbed when the + 2 c - extein residue was changed from phe to ala (figure 5c). the only exceptions to this were the amide resonances from ile119 and gly120, which showed a modest perturbation. these residues are located at the beginning of the loop containing the catalytic his residue and, in the ssp crystal structure, lie close to the c - intein / c - extein peptide bond that is ultimately attacked during bi resolution (figure 5b). the his125 backbone amide resonance was not itself sensitive to the nature of the + 2 c - extein residue. however, the aromatic side - chain protons of this residue did exhibit significant chemical shift perturbations upon mutation of the + 2 residue, suggesting an altered chemical environment for this side chain in the absence of the + 2 phenyl ring (figure 5d). together with our mutagenesis and kinetic data, these nmr studies lend support to the idea that the active - site conformation of npu is coupled to the identity of the c - extein + 2 residue. to gain additional insight into the interplay between c - extein residues and the npu active site, we carried out molecular dynamics (md) simulations of two wild - type intein complexes bearing either cfn(nh2) or can(nh2) as c - exteins (identical to the constructs in table 1, reactions 1 and 12). simulations were carried out in explicit solvent in 1 fs steps for 0.5 s. comparison of the two simulation trajectories afforded a more detailed picture of the coupling between the + 2 residue and the intein active site. one of the more striking results from the simulation was the effect of changing the + 2 c - extein on the dynamics of the his125 side chain. in the presence of phe+2 the his side chain primarily adopts a single rotameric state with only a brief excursion to an alternate rotamer (figure 6a and black trajectory in figure 6c). by contrast, with an ala+2 residue, his125 frequently switches among three side - chain rotamers and favors a different conformation than the one found with phe+2 (figure 6b and red trajectory in figure 6c). interestingly, the backbone and dihedral angles for his125 showed virtually no change as a function of c - extein composition (figure s19). these data are consistent with the fact that there were chemical shift perturbations for the his125 side chain but not the backbone. results of md simulations to probe + 2 amino acid - dependent active - site dynamics. (a, b) overlays of three representative frames from the md trajectories for (a) aey npun + npuc cfn(nh2) and (b) aey npun + npuc can(nh2), highlighting his125 rotameric states. (c) trajectory of the his125 side - chain 1 dihedral angle during the simulations. (d, e) representative frames highlighting the positioning of asn137 relative to the his125 loop in the (d) cfn and (e) can simulations. asn137 and cys+1 are shown as orange sticks, his125 is shown as an orange surface, and ile119 and gly120 are shown as green surfaces. (f) distance between the his125 and asn137 c atoms during the simulations. (g) distance between the ile119 and cys+1 amide nitrogens during the simulations. in (c), (f), and (g), data from the cfn and can simulations are shown in black and red, respectively, and the traces to the right of the trajectory graphs are histograms indicating the distribution of angles or distances sampled throughout the simulation. the second major consequence of the + 2 residue mutation was the overall positioning of the c - intein / c - extein junction (i.e asn137cys+1) relative to the his125 loop. in the simulation with cfn as the c - extein, asn137 remained buried in the groove above this loop, similar to the ssp structure (figure 6d). by contrast, in the can simulation, the entire strand bearing asn137 and the c - extein occupied space outside of this groove region (figure 6e). an important consequence of this difference is that the distance between asn137 and his125 (figure 6f) and that between ile119 and the scissile peptide bond (figure 6 g) were significantly shorter for the majority of the cfn simulation than for the can simulation. overall, these md simulations indicate that the presence of a sterically bulky amino acid at the + 2 position in the c - extein acts to constrain the motions of key catalytic residues, leading to a more compacted arrangement around the scissile peptide bond. in considering the mechanistic implications of these observations, it is important to emphasize that by necessity the simulations employed a linear precursor protein as the starting point. the use of a bi structure in the simulations would have been more desirable in view of the fact that our kinetic data revealed that formation of this intermediate stimulates cleavage of the peptide bond at the c - intein / c - extein junction (table 1, compare reactions 1 and 3). unfortunately, there is currently no high - resolution structural information on any intein in the bi state. thus, we were forced to extrapolate from the structures available. despite this caveat, the major conclusion from the simulation work is broadly consistent with our mutagenesis and kinetic data. in particular, we observed coupling between the + 2 residue and the catalytic his125 in both the simulations and the studies of the bi resolution kinetics. we further note that the phe side chain stimulates c - extein cleavage even in the absence of the bi (table 1, compare reactions 3 and 13), which argues that this bulky side chain augments catalysis even in the linear precursor. local c - extein residues appear to affect the structure and dynamics of residues surrounding the flexible his125 loop, thereby modulating the bi resolution kinetics. thus, it is conceivable that point mutations within the intein that alter the loop conformation or flexibility could also modulate the splicing activity and even the tolerance to non - native extein residues. in a previous directed - evolution study on an npun sspc chimera, we identified several mutations that make this intein more tolerant of the c - extein sequence sgv rather than cfn. intriguingly, one of these mutations was an asp - to - tyr mutation adjacent to his125 (asp124). we found that this mutation enhanced the rate of npu splicing by 50% in the presence of ala+2 (figure 7a ; compare reactions 12 and 17 in table 1). importantly, this mutation was still tolerated when phe+2 was present, suggesting that it increases the overall promiscuity toward c - exteins (compare reactions 1 and 16). the npu nmr structure and the ssp crystal structures indicate that asp124 packs against a -turn from the n - intein. because of this close packing, the bulky d124y mutation would require conformational rearrangement and possibly also rigidification of the catalytic his125 loop, which could modulate the activity. as predicted, in a 100 ns md simulation of npud124y with a can(nh2) c - extein, the his125 loop conformation was altered and the his125 rotamer dynamics constrained, and asn137 persistently remained above the his125 loop, similar to the npuwt cfn(nh2) simulation (figures 7b d, s21, and s22). this simulation suggests that the d124y mutation reduces the c - extein dependence by recapitulating the constraints on the active - site dynamics typically applied by phe+2, specifically the stabilization of his125 and the appropriate positioning of the c - intein / c - extein junction close to his125. (a) kinetic data showing rate enhancement for the bi resolution step with the d124y mutation in the can(nh2) c - extein context. solid lines correspond to best - fit kinetic curves for the wild - type intein (table 1, reaction 12), and dotted lines correspond to the d124y mutant (table 1, reaction 17). only the bi and product reaction curves are shown ; the starting material curve has been omitted for clarity. (b d) histograms showing the distributions of (b) his125 1 dihedral angles, (c) distances between the his125 and asn137 c atoms, and (d) distances between the ile119 and cys+1 amide nitrogens during the wild - type cfn (black), wild - type can (red), and d124y can (blue) simulations. in this work, we examined the molecular determinants for c - extein - dependent protein trans - splicing. this investigation was facilitated by the utilization of protein semisynthesis to generate inteins linked to a variety of c - exteins and the development of novel kinetic assays that provide information about individual steps along the pts reaction coordinate. through these studies, we not only extracted information on c - extein requirements but also gained additional mechanistic insights into split dnae intein splicing. specifically, our experiments confirmed that resolution of the branched intermediate is the slowest step for pts (k3) and also provided evidence supporting the notion that some dnae inteins have a highly activated n - terminal splice junction (k1/k2 > 2 for all npu constructs), consistent with our previous report. interestingly, this n - terminal activation appears to be roughly 10-fold slower and is significantly less efficient (k1/k2 = 0.67) for the ssp intein. additionally, we found that for npu, the rate of asn cyclization upon bi formation is 200-fold faster than its rate in the absence of the branched structure. stimulation of asn cyclization upon bi formation is also found in the cis - splicing gyra intein. we propose that this kinetic stimulation is a common feature of inteins, in effect creating a trigger that helps ensure the proper fidelity of the reaction by minimizing premature cleavage of the c - extein. lastly, it is particularly surprising that the h125n mutation does not completely abolish bi resolution but rather reduces its rate 60-fold. indeed, the splicing rate of this mutant is still higher than that of wild - type ssp. for many non - dnae inteins, this step requires two histidine residues, one analogous to his125 and another immediately preceding the c - terminal asn residue. in view of the lack of this penultimate histidine in the dnae inteins, his125 has been implicated as the sole general acid / base for bi resolution. our data suggest that while his125 is clearly important for bi resolution, other unidentified residues must also contribute to catalysis of this step. the current study improves our understanding of the relationship between c - extein composition and trans - splicing efficiency. the kinetic data indicate that the c - extein almost exclusively affects the bi resolution step. within the c - extein, we identified specific functional groups that contribute significantly to the splicing kinetics, in particular the phe+2 side chain. our nmr experiments and md simulations illustrate that this bulky functional group constrains active - site motions, forcing catalytic histidine and asparagine residues and the scissile peptide bond into close proximity. the need for a bulky side chain at the + 2 position is further highlighted by a recent genetic selection study on the npu intein showing that trp is also well - tolerated at this position. collectively, these data paint a picture of the npu active site that effectively extends beyond the intein domain itself to include the + 2 c - extein residue. during pts, the n - extein is transferred from the n - terminus of the intein onto a c - extein side chain, which creates a unique branched protein structure. as bi resolution is the slowest and often the rate - limiting step for many inteins, this structure is the most relevant to the overall activity. to date, all of the published high - resolution structural data on inteins involve either a precursor or product form of the intein. while these studies (including this report) have provided substantial insights into the structural basis for protein splicing, they can not examine interactions that are exclusively present in the bi. indeed, our kinetic analyses revealed several important functional groups in the c - extein that affect bi resolution (figure 4c), but only in the case of the phe+2 side chain could we postulate any kind of structural basis for this. thus, these results reinforce the need for high - resolution structural information on the bi in the pts reaction. the fullest deployment of split inteins in protein engineering ultimately requires a truly traceless trans - splicing system with no sequence requirements. while bulky hydrophobic residues other than phenylalanine are tolerated at the critical + 2 position for dnae inteins, thus alleviating some sequence constraints, these inteins are still only modestly promiscuous. our results suggest that the interplay between the c - extein and the his125 active - site loop has direct implications for the rational design of improved, more extein - tolerant split inteins. indeed, the d124y point mutation on this flexible loop increases the tolerance of npu for a + 2 alanine residue without affecting its activity in a native context. in a recent directed - evolution endeavor on a dnab family intein, a mutation at this position was also found to reduce c - extein sequence constraints. furthermore, we previously demonstrated that mutating other residues on this loop can generally enhance the activity of ssp and the npun these results collectively indicate that the conformational preferences of this loop are intimately linked with inadequate bi resolution both for intrinsically slow inteins and for efficient inteins in an exogenous c - extein context. thus, this loop is a hot spot on the intein structure that should be explicitly targeted in future engineering efforts for the design of more high - activity, broad - specificity inteins. semisynthetic intc extein proteins were generated through expressed protein ligation of a synthetic fragment, corresponding to the desired model c - extein, and a reactive recombinant fragment corresponding to the c - intein. model c - exteins were synthesized using standard solution - based or solid - phase protocols (see the supporting information for details). gyra - his6 fusion proteins, which were expressed in e. coli and purified using standard methods (figure s4). ligation reactions involved treatment of the purified intc gyra - his6 fusion protein with an excess of the model c - extein, usually in the presence of an additional thiol. semisynthetic products were purified by preparative rp - hplc and characterized by esi - ms (figure s7 and table s2). aey npun and aey sspn were expressed with an n - terminal his6-sumo tag in e. coli bl21(de3) cells from an iptg - inducible protein expression vector. the cells were lysed by sonication, and the protein was enriched over ni - nta resin in a ph 8.0 phosphate - buffered saline solution. the proteins were eluted from the ni column in the presence of 250 mm imidazole (figure s5), and the elutions were dialyzed to reduce the imidazole concentration to 5 mm. the dialyzed solutions were treated for 12 h at room temperature with his6-tagged ulp1, a sumo - specific protease, to yield the desired products. the proteolysis reactions were passed over ni - nta resin to deplete unreacted starting material, the cleaved his6-sumo tag, and ulp1 (figure s6). the proteins were further purified by size - exclusion chromatography on a superdex 75 column in splicing assay buffer (100 mm sodium phosphates, 150 mm nacl, 1 mm edta, ph 7.2) supplemented with 1 mm dithiothreitol. product identities were confirmed by esi - ms, and their purities were assessed by analytical rp - hplc (figure s7 and table s2). prior to any splicing assay, the n - intein solutions were dialyzed against splicing assay buffer (100 mm sodium phosphates, 150 mm nacl, 1 mm edta, ph 7.2) overnight at 4 c. it should be noted that thiols were omitted from this buffer since substantial n - extein cleavage was observed for reactions with a low k3. n - inteins and c - inteins were diluted to 15 and 10 m, respectively, and tris(2-carboxyethyl)phosphine (tcep) was added to each solution to a final concentration of 2 mm. splicing reactions were initiated by mixing equal volumes of n - inteins and c - inteins at 30 c. during the reaction, aliquots of the solution were removed and mixed 3:1 (v / v) with quenching solution (8 m guanidine hydrochloride and 4% trifluoroacetic acid). for rp - hplc analysis, 100 l of the quenched solutions were separated over a c18 analytical column, recording absorbance at 214 nm, and major peaks were collected and identified by esi - ms (figures 3a and s9 and table s3). for direct esi - ms analyses, 20 l of the quenched solutions were desalted using millipore c18 zip - tips, diluted, and loaded on the mass spectrometer by direct infusion. the complex mixture of multiply charged states of each species was deconvoluted into spectra depicting a well - defined mixture of singly charged species (figure 3b and s10 and table s3). peaks corresponding to species 15 in either the rp - hplc chromatogram or esi - ms spectrum were integrated and expressed as a fraction of total peak intensity for each time point. for the rp - hplc analyses, the product was expressed as the sum of the integrated intensities for species 35 to account for changes in relative extinction coefficients. for esi - ms analyses, the product was expressed as the sum of the integrated intensities of only species 3 and 4, since species 5 was not visible and the ionizabilities of 14 were assumed to be identical. the time - dependent reaction curves for all three states of the reaction (figures s11 and s12), starting material (1), branched intermediate (2), and products (3 and 4 or 35), were collectively fit to the analytical solution for the coupled differential equations describing our kinetic model (figure 3c ; also see the supporting information). from this global fit, we extracted the values for k1, k2, and k3 for each individual reaction. the value of ksplice was determined by fitting the product formation curves (3 and 4 or 35) to a first - order rate equation. reactions were repeated three or four times, and the means and standard deviations of all four kinetic parameters are reported in table 1. nmr experiments were carried out on uniformly n, c - labeled npuc ligated to an unlabeled c - extein [cfn(nh2) or can(nh2) ] in complex with unlabeled aey npun. experiments were run on 600 mhz (bruker or varian inova), 800 mhz, and 900 mhz bruker spectrometers. backbone resonance assignments of labeled npuc in complex with npun were achieved using triple - resonance experiments with standard pulse sequences. the side - chain carbons, c2 and c1, of his125 were resolved with a standard c, h aromatic heteronuclear single - quantum correlation (hsqc) experiment. standard pulse sequences were used for the measurements of r1, r2, and n h nuclear overhauser effect (noe) rates. all - atom md simulations were performed on npu constructs at constant temperature and pressure (300 k and 1 atm) using the md suite amber 12. simulations contained explicit water molecules, and the net charge of the system was neutralized with sodium ions. the constructs were generated from the first representative solution nmr structure of npu (pdb entry 2keq). prior to the simulations, this structure was modified in silico using ucsf chimera to generate the constructs of interest, namely, (1) a wild - type split intein complex with canonical extein sequences [aey npun : npuc cfn(nh2) ], (2) a wild - type split intein complex with a mutant c - extein [aey npun : npuc can(nh2) ], and (3) a d124y mutant with the same mutant c - extein sequence [aey npun : npuc can(nh2) ]. for the wild - type cfn and can constructs, 500 ns long simulations were run, and a 100 ns long simulation was run for the d124y mutant. prior to the runs, a series of minimization, heating, and density equilibration steps were performed. | split inteins play an important role in modern protein semisynthesis techniques. these naturally occurring protein splicing domains can be used for in vitro and in vivo protein modification, peptide and protein cyclization, segmental isotopic labeling, and the construction of biosensors. the most well - characterized family of split inteins, the cyanobacterial dnae inteins, show particular promise, as many of these can splice proteins in less than 1 min. despite this fact, the activity of these inteins is context - dependent : certain peptide sequences surrounding their ligation junction (called local n- and c - exteins) are strongly preferred, while other sequences cause a dramatic reduction in the splicing kinetics and yield. these sequence constraints limit the utility of inteins, and thus, a more detailed understanding of their participation in protein splicing is needed. here we present a thorough kinetic analysis of the relationship between c - extein composition and split intein activity. the results of these experiments were used to guide structural and molecular dynamics studies, which revealed that the motions of catalytic residues are constrained by the second c - extein residue, likely forcing them into an active conformation that promotes rapid protein splicing. together, our structural and functional studies also highlight a key region of the intein structure that can be re - engineered to increase intein promiscuity. |
the frequency of urinary calculi has increased owing to the dietary habits and lifestyle of the current generation as well as a rise in global warming.. currently followed management procedures for the treatment of urinary stones include lithotripsy and surgical methods. the recurrence rate seems to be high regardless of new advances in medical treatment management methods. approximately 75% of these patients will have at least one recurrence without proper treatment and preventive measures. calcium phosphate is one of the significantly occurring urinary crystal sediments in most patients with urolithiasis. some studies have shown the presence of calcium phosphate in small amounts in patients with urethral and renal stone diseases. it was also reported that a small amount of calcium phosphate is involved in stone attachment. this evidence suggests that calcium phosphate plays a vital role in the formation of renal stones in the initial stages while the stones are attached to the renal papilla. worldwide, calcium oxalate (caox, cac2o4) is the most predominant urinary calculi. in the urinary tract, increased urinary supersaturation followed by the formation of solid crystalline particles is responsible for the beginning of crystallization of caox in the tract. despite extensive research in urology to date, there are as yet no effective, satisfactory drugs for dissolving or disintegrating urinary stones. today 's traditional herbal medicines aid in the development of potential therapeutic drugs in the form of either extracts alone, extracts in combination with other herbs, or drugs in the form of phytochemical compounds isolated from herbs. since the ancient era, the olive tree (olea europaea) has been known for its medicinal properties in the mediterranean region. some studies have reported that the leaves and extracts of olive trees have nutritional importance and can be used as alternative remedies for numerous illnesses. in the mediterranean basin region, the use of olive oil in traditional medicine for several thousands of years is associated with a low occurrence of heart disease and some types of cancer. olive oil is well known for its hypotensive, antioxidant, cardiovascular, hypoglycemic, and hepatoprotective effects. reports on the beneficial effect of the mediterranean diet for nephrolithiasis are rare, however. there is no previous literature regarding the antiurolithic activity of olive oil in vivo. with this in mind, the current study aimed to explore the antiurolithic activity of olive oil in an ethylene glycol (eg)-induced urolithiasis model and the role of olive oil in the prevention of renal stones. all experimental research analytical - grade reagents and chemicals were procured from sigma chemical co. inc. all bioassay kits (uric acid, urea, creatinine, oxalate, inorganic phosphate, and calcium) were purchased from biovision inc. olive oil (extra virgin) was purchased from sakaka olive market (al - jawf, saudi arabia). to determine the suitable safe olive oil dose, acute toxicity was assessed in mice according to the organisation for economic co - operation and development protocol. oral doses of 0.5, 0.75, 1.0, 1.3, and 1.7 ml / kg body weight of olive oil was supplemented to each group with ad libitum access to water and food. the animals were observed for general behavior and signs of toxicity for 1 hour, 4 hours, and over the period of 24 hours after drug administration. the animals were further observed for signs of toxicity, weight loss, and mortality for 14 days. adult male mice (swiss albino) weighing 20 to 25 g were obtained from the medical school animal husbandry department at al jouf university. the experimental animals were kept in 12-hour dark - light cycles at 25 with ad libitum access to food and water. the olive oil - supplemented normal controls (group iii) received standard pellets + oral olive oil (1.3 ml / kg body weight). groups iv and v were fed orally with olive oil at various doses along with eg (0.75%) in drinking water for 28 days. group vi received cystone (750 mg / kg) as a standard drug and 0.75% eg in drinking water during the experimental period (28 days). eg (0.75% in drinking water) was used in groups iv through vi to induce urolithiasis and to promote the development of calcium oxalate crystals in the kidney during the experimental period. at the end of the experiment (on day 28), all animals were kept in metabolic cages individually for the collection of 24-hour urine samples. 3n hcl was added to the urine samples, followed by a 10-minute centrifugation at 1,500 rpm. the supernatant and debris were discarded and the samples were kept at -20. a chemwell biochemistry automatic analyzer (awareness technology, inc., palm city, fl, usa) was used for oxalate, calcium, and inorganic phosphate measurement in urine. after they had been deprived of food for 12 hours overnight, the mice were anesthetized with diethyl ether. after cervical decapitation, blood samples were collected into nonanticoagulant - treated test tubes and allowed to clot at room temperature. the samples were subjected to serum urea, creatinine, and uric acid analysis with the aid of commercially available bioassay kits according to the manufacturer 's protocols by use of a chemwell biochemistry automatic analyzer. both kidney samples were collected immediately after the animals were sacrificed under anesthesia, followed by rinsing in cold 0.15 m kcl. after that, the right kidney was minced with scissors and then homogenized with 0.15 m kcl by use of a remi 's glass homogenizer. the supernatant sample was obtained from the homogenate by centrifugation for 15 minutes at 1,500 rpm. the supernatant was used for estimation of oxalates by use of commercially available bioassay kits according to the manufacturer 's protocols by use of the chemwell biochemistry automatic analyzer. meanstandard deviation was used to express the data. to compare the groups, one - way analysis of variance all experimental research analytical - grade reagents and chemicals were procured from sigma chemical co. inc. all bioassay kits (uric acid, urea, creatinine, oxalate, inorganic phosphate, and calcium) were purchased from biovision inc. olive oil (extra virgin) was purchased from sakaka olive market (al - jawf, saudi arabia). to determine the suitable safe olive oil dose, acute toxicity was assessed in mice according to the organisation for economic co - operation and development protocol. briefly oral doses of 0.5, 0.75, 1.0, 1.3, and 1.7 ml / kg body weight of olive oil was supplemented to each group with ad libitum access to water and food. the animals were observed for general behavior and signs of toxicity for 1 hour, 4 hours, and over the period of 24 hours after drug administration. the animals were further observed for signs of toxicity, weight loss, and mortality for 14 days. adult male mice (swiss albino) weighing 20 to 25 g were obtained from the medical school animal husbandry department at al jouf university. the experimental animals were kept in 12-hour dark - light cycles at 25 with ad libitum access to food and water. the olive oil - supplemented normal controls (group iii) received standard pellets + oral olive oil (1.3 ml / kg body weight). groups iv and v were fed orally with olive oil at various doses along with eg (0.75%) in drinking water for 28 days. group vi received cystone (750 mg / kg) as a standard drug and 0.75% eg in drinking water during the experimental period (28 days). eg (0.75% in drinking water) was used in groups iv through vi to induce urolithiasis and to promote the development of calcium oxalate crystals in the kidney during the experimental period. at the end of the experiment (on day 28), all animals were kept in metabolic cages individually for the collection of 24-hour urine samples. 3n hcl was added to the urine samples, followed by a 10-minute centrifugation at 1,500 rpm. the supernatant and debris were discarded and the samples were kept at -20. a chemwell biochemistry automatic analyzer (awareness technology, inc., palm city, fl, usa) was used for oxalate, calcium, and inorganic phosphate measurement in urine. after they had been deprived of food for 12 hours overnight, the mice were anesthetized with diethyl ether. after cervical decapitation, blood samples were collected into nonanticoagulant - treated test tubes and allowed to clot at room temperature. the samples were subjected to serum urea, creatinine, and uric acid analysis with the aid of commercially available bioassay kits according to the manufacturer 's protocols by use of a chemwell biochemistry automatic analyzer. both kidney samples were collected immediately after the animals were sacrificed under anesthesia, followed by rinsing in cold 0.15 m kcl. after that, the right kidney was minced with scissors and then homogenized with 0.15 m kcl by use of a remi 's glass homogenizer. the supernatant sample was obtained from the homogenate by centrifugation for 15 minutes at 1,500 rpm. the supernatant was used for estimation of oxalates by use of commercially available bioassay kits according to the manufacturer 's protocols by use of the chemwell biochemistry automatic analyzer. meanstandard deviation was used to express the data. to compare the groups, one - way analysis of variance was applied with the aid of spss ver. the study results showed that olive oil at doses of 1.3 and 1.7 ml / kg body weight orally was suitable for supplementation of the experimental animals. our results showed that serum urea, uric acid, and creatinine were significantly (p<0.05) higher in group ii animals than in group i (table 1). however, olive oil treatment (1.3 and 1.7 ml / kg body weight orally) significantly (p<0.05) lowered serum uric acid, creatinine, and urea levels in animals in groups iv and v compared with group ii. group vi animals showed significantly (p<0.05) lower levels of serum urea, uric acid, and creatinine than animals in groups ii, iv, and v. by contrast, group v animals (1.7 ml / kg body weight orally) showed significantly lower levels of serum urea, uric acid, and creatinine than did animals in group iv (1.3 ml / kg body weight orally), with a resultant significantly (p<0.05) greater decease compared with group ii in serum urea, uric acid, and creatinine in group v than in group iv. the study results revealed significantly (p<0.05) higher levels of urinary oxalate, calcium, and inorganic phosphate in group ii than in the other groups (table 2). however, supplementation with olive oil in groups iv and v resulted in significantly (p<0.05) decreased levels of oxalate, calcium, and inorganic phosphate in urine compared with group ii. cystone - treated animals (group vi) showed significantly (p<0.05) lower levels of urinary oxalate, calcium, and phosphate than in group ii animals (fig. in contrast, a similar type of effect was observed in group iv and v animals after administration of olive oil (1.3, 1.7 ml / kg body weight orally) in a dose - dependent manner. the kidney homogenate showed significantly (p<0.05) higher levels of oxalate, calcium, and inorganic phosphate in group ii animals than in the other groups (table 3). however, administration of olive oil to the animals in groups iv and v resulted in significantly lower (p<0.05) levels of oxalate, calcium, and inorganic phosphate in kidney homogenate compared with the eg - induced urolithiasis animal group (group ii). cystone - treated animals (group vi) exhibited significantly (p<0.05) lower levels of kidney oxalate, calcium, and phosphate than did group ii animals (fig. iv and v, the elevated levels of kidney homogenate parameters such as oxalate, calcium, and phosphate were restored after supplementation with olive oil (1.3 and 1.7 ml / kg body weight orally). the study results showed that olive oil at doses of 1.3 and 1.7 ml / kg body weight orally was suitable for supplementation of the experimental animals. our results showed that serum urea, uric acid, and creatinine were significantly (p<0.05) higher in group ii animals than in group i (table 1). however, olive oil treatment (1.3 and 1.7 ml / kg body weight orally) significantly (p<0.05) lowered serum uric acid, creatinine, and urea levels in animals in groups iv and v compared with group ii. group vi animals showed significantly (p<0.05) lower levels of serum urea, uric acid, and creatinine than animals in groups ii, iv, and v. by contrast, group v animals (1.7 ml / kg body weight orally) showed significantly lower levels of serum urea, uric acid, and creatinine than did animals in group iv (1.3 ml / kg body weight orally), with a resultant significantly (p<0.05) greater decease compared with group ii in serum urea, uric acid, and creatinine in group v than in group iv. the study results revealed significantly (p<0.05) higher levels of urinary oxalate, calcium, and inorganic phosphate in group ii than in the other groups (table 2). however, supplementation with olive oil in groups iv and v resulted in significantly (p<0.05) decreased levels of oxalate, calcium, and inorganic phosphate in urine compared with group ii. cystone - treated animals (group vi) showed significantly (p<0.05) lower levels of urinary oxalate, calcium, and phosphate than in group ii animals (fig., a similar type of effect was observed in group iv and v animals after administration of olive oil (1.3, 1.7 ml / kg body weight orally) in a dose - dependent manner. the kidney homogenate showed significantly (p<0.05) higher levels of oxalate, calcium, and inorganic phosphate in group ii animals than in the other groups (table 3). however, administration of olive oil to the animals in groups iv and v resulted in significantly lower (p<0.05) levels of oxalate, calcium, and inorganic phosphate in kidney homogenate compared with the eg - induced urolithiasis animal group (group ii). cystone - treated animals (group vi) exhibited significantly (p<0.05) lower levels of kidney oxalate, calcium, and phosphate than did group ii animals (fig. iv and v, the elevated levels of kidney homogenate parameters such as oxalate, calcium, and phosphate were restored after supplementation with olive oil (1.3 and 1.7 ml / kg body weight orally). men predominate over women in risk of stone formation, with an incidence in the range of 5% to 10% that is observed to peak between the ages of 40 and 50 years. based on the constituents (calcium) of the stone, renal stones are classified into calcium oxalate dehydrate, calcium oxalate monohydrate, and basic calcium phosphate stones, which commonly occur at a rate of 75% to 90%, followed by magnesium ammonium phosphate (struvite), uric acid, and cystine stones, respectively (10%15%, 3%10%, and 0.5%1%, respectively). generally, magnesium ammonium phosphate or calcium oxalate stones are more common among all types of stones. many medications and alternative remedies have been used to treat urinary stones in the past several years. generally, the majority of practice in traditional medicine is based on plants and their products, but the rationale behind their use is often not scientifically well established through clinical trials and systematic pharmacological studies. only a few herbal drugs however, the use of plant products in traditional medicine for renal stone therapy has assumed importance. previous studies showed that flavonoids may reduce renal apoptosis owing to their antioxidant and diuretic properties. use of synthetic drugs in the treatment of urolithiasis on a long - term basis could lead to adverse effects. extracorporeal shock wave lithotripsy is the standard procedure for the removal of renal stones in current renal stone management compared with open renal surgery. the potential role of plant extracts in the prevention of kidney stone formation is through inhibition of the growth of caox crystals induced by urinary macromolecules and excretion of the caox crystals attached to the surface of the epithelial cells along with the urine. eg - induced renal calcium oxalate in mice is used to mimic the formation of urinary stones in humans. therefore, in the present study, the eg - induced urolithiasis model was used to access the anti - urolithiasis effect of olive oil. in urolithiasis, obstruction of the urinary system due to the formation of calculi in the renal tissue leads to a decrease in the kidney glomerular filtration rate, which results in the accumulation of nitrogenous waste produces such as uric acid, serum urea nitrogen, and creatinine in the blood. in this study, elevated serum urea, uric acid, and creatinine levels were seen in mice with eg - induced urolithiasis, with marked renal damage that may have been due to the release of nitrogenous waste substances in the blood. however, curative treatment with olive oil (1.3, 1.7 ml / kg body weight orally) may causes dieresis and accelerate the preformed stone - dissolving process and also may prevent the formation of new renal stones in the urinary system. in the present study, hyperoxaluria resulted after the administration of 0.75% eg in group ii animals. a biochemical mechanism is associated with elevated concentrations of oxalate in the urine of group ii animals administered the 0.75% eg solution. the urinary excretion of oxalate in hyperoxaluria is comparatively more important in the formation of urinary stones than the urinary excretion of calcium. hyperoxaluria leads to the formation of calcium oxalate or apatite from the urine and the resultant development of crystals. it has been reported that hyperabsorption of calcium is due to defective renal tubular reabsorption. hyperoxaluria can cause the formation of renal stones in eg - fed animals owing to increased renal oxalate retention and excretion. our study results show that administration of olive oil (1.3, 1.7 ml / kg body weight orally) restored oxalate, inorganic phosphate, and calcium levels in urine similar to those in group vi (cystone - treated animals)., eg is metabolized to four main organic acids in vivo, glycooxalic acid, glycolaldehyde, glycolic acid, and oxalic acid, which is the initiative factor for lithiasis. the elevated levels of calcium, oxalate, and phosphate in the kidney homogenate supernatant of droups iv and v were also restored with olive oil supplementation in this study compared with the eg control group. the study results indicate that administration of olive oil (1.7 ml / kg body weight) reduced and prevented the growth of urinary stones. the current study revealed the antiurolithic effect of olive oil (1.7 ml / kg body weight) in an eg - induced renal calculi model. therefore, olive oil (1.7 ml / kg body weight) may prevent renal stone formation by inhibiting damage to the renal tubular membrane due to peroxidative stress induced by hyperoxaluria. the underlying biochemical mechanism of this potential effect of olive oil may be its antioxidant properties. | purposeat present, commercially available antiurolithic drugs have more adverse effects than potential therapeutic or preventive effects with chronic use. with this in mind, the present study was designed to assess the antiurolithic effect of olive oil in a mouse model of ethylene glycol (eg)-induced urolithiasis.materials and methodsadult albino mice were divided into 6 groups. group i was fed the vehicle only. group ii was supplemented with 0.75% eg alone in drinking water during the experimental period to initiate deposition of calcium oxalate in kidneys, which leads to urolithiasis in animals. groups iii (olive oil control group) through v were fed olive oil orally at various doses during the experimental period. group vi received cystone (750 mg / kg). groups iv vi additionally received 0.75% eg in drinking water ad libitum. spss ver.17.0 was used for statistical analysis.resultsthe study results showed significantly higher levels of serum urea, uric acid, and creatinine (p<0.05) in group ii than in groups iii vi and i. administration of olive oil at different doses restored the elevated serum parameters in groups iv and v compared with group ii. urine and kidney calcium, oxalate, and phosphate levels in groups iv vi were significantly lower (p<0.05) than in animals with eg - induced urolithiasis (group ii). group v mice showed a significant restoration effect on serum as well as urine and kidney parameters compared with group ii.conclusionssupplementation with olive oil (1.7 ml / kg body weight) reduced and prevented the growth of urinary stones, possibly by inhibiting renal tubular membrane damage due to peroxidative stress induced by hyperoxaluria. |
emerging evidence suggests that eating behavior and, consequently, body weight are determined by an interplay of top - down control processes and bottom - up impulses (appelhans, 2009 ; heatherton and wagner, 2011). specifically, studies have shown that individuals with both high impulsivity (as measured by poor motor response inhibition or high preference for immediate reward) and high susceptibility to food - related bottom - up impulses (as measured by implicit preference for food, self - reported food reward sensitivity, or approach bias toward food) exhibit higher intake of palatable foods in the laboratory (appelhans., 2011 ; hofmann. in other studies, similar interactive effects on outcome measures other than laboratory food intake were observed. for example, poor motor response inhibition in response to high - calorie (hc) food - cues was found in individuals reporting a combination of both high impulsivity and trait food craving (meule and kbler, 2014). in a study using neuroimaging, food - cue elicited activity in reward - related brain areas was predictive of higher body mass index (bmi), but only in individuals reporting low self - control (lawrence., 2012). despite these converging findings of studies using related, but a variety of methodologies, prospective studies reporting such interactive effects are rare. in one study, implicit preference for snack foods was measured with a single - category implicit association test, and response inhibition was measured with a stop - signal task. it was found that a combination of task performance in both measures prospectively predicted weight change : participants with both strong implicit preference for snack foods and low inhibitory control gained the most weight. as a measure of food - related bottom - up processes, a dot - probe task was used for assessing an attentional bias toward hc or low - calorie (lc) foods. attentional bias refers to selective attentional processing of certain stimuli (kemps and tiggemann, 2009 ; werthmann., 2015). in the current study, an attentional bias toward hc foods was represented by faster reaction times to dots appearing behind hc versus lc food - cues, indicating preferred attention allocation to such cues. as a measure of (low) top - down control, the barratt impulsiveness scale (bis) was used for assessing trait impulsivity. trait impulsivity can be defined as a predisposition toward rapid, unplanned reactions without regard to the negative consequences of these reactions (moeller., 2001). yet, impulsivity is a multifaceted construct and with the bis, for example, three facets can be differentiated representing attentional impulsivity (inability to focus attention or concentrate), motor impulsivity (acting without thinking), and non - planning impulsivity (lack of future orientation or forethought ; stanford., 2009). although both attentional bias toward hc food - cues and impulsivity have been implicated to play a role in the development of overweight and obesity (guerrieri., 2008 ; werthmann., 2015), prospective studies, which are necessary to determine causal influences, are rare (calitri., 2010 ; reinert., 2013 ; yokum., based on previous findings outlined above, it was expected that attentional bias toward hc food - cues and high impulsivity in combination would prospectively predict weight gain in female students. note, however, that the nature of interactions found across previous studies was inconsistent and, thus, this interactive effect may be additive (lawrence., 2012 ; meule and kbler, 2014) or non - additive (appelhans. this study adhered to the guidelines outlined in the declaration of helsinki as revised in 2008. participants were recruited via postings in the department of psychology at the university of wrzburg, germany, which advertised the study as an investigation on the cognitive processing of food pictures. in accordance with similar studies (e.g. nederkoorn., 2010), only women were recruited in order to avoid a possible confounding effect of sex as men and women differ in body mass, eating - related variables, and predictors of weight gain (holm - denoma., 2008). three participants reported to have a chronic disease (1x thyroid disease, 2x asthma). none of the participants reported a current mental disorder. at the beginning of the first semester, they performed a computerized dot - probe task, completed the short form of the bis (bis-15), and height and weight were measured for calculation of bmi (kg / m) in the laboratory. in accordance with similar studies (e.g. hodge., 1993), participants (n = 51, mage = 20.4 years, sd = 4.03) returned at the start of the second semester (after approximately 6 months) for a follow - up measurement of body weight (mean period between the two measurements was m = 164 days, sd = 9.82). twenty pictures were selected from the food.pics database (www.food-pics.sbg.ac.at), which contains information on calorie content, subjectively rated palatability, and physical features of the food pictures (blechert., 2014). ten pictures of hc (five pictures of savory foods, five pictures of sweet foods) and ten pictures of lc (five pictures of fruits, five pictures of vegetables / salad) foods were selected (figure 1(a)). food items of the two categories differed both in calories per 100 g (m = 321.15 kcal/100 g (sd = 126.43, range : 123539) vs m = 47.25 kcal/100 g (sd = 40.05, range : 9154), t(18) = 6.53, p.161). the interaction effect was still significant when including bmi as covariate (table 2). probing this interaction revealed that motor impulsivity scores were positively associated with weight gain in individuals with high attentional bias scores (b = 0.21, p =.027), but not in those with low attentional bias scores (b = 0.08, p =.318 ; figure 2). simple slopes probing the interaction of attentional bias motor impulsivity when predicting body mass index change. scores on motor impulsivity positively predicted weight gain in individuals with high attentional bias scores (i.e. those who exhibited an attentional bias toward high - calorie food - cues), but not in individuals with low attentional bias scores (i.e. those who exhibited an attentional bias toward low - calorie food - cues). the main aim of the current study was to investigate whether an attentional bias toward food - cues and impulsivity interactively predicted weight change in female students. results showed that weight gain was indeed predicted by an interaction between attentional bias toward hc foods and self - reported impulsivity, but only its motor impulsivity subscale. this result is in line with and extends findings from previous studies, in which related, but distinct food- and impulsivity - related measures were used. in the study by kakoschke. (2015a), for example, laboratory food intake was predicted by an interaction between approach bias for food and low motor response inhibition. although a similar interaction effect using a measure of attentional bias instead of approach bias in that study fell short of significance, plotting the interaction revealed that it was descriptively similar to the finding obtained for approach bias and to the interaction effect obtained in the current study. in the study by nederkoorn. (2010), weight gain was prospectively predicted by an interaction between implicit preference for food and low motor response inhibition. in accordance with this finding, the predictive power of impulsivity for weight gain could only be observed for self - reported motor impulsivity, but not for attentional and non - planning impulsivity. although results of studies using the bis are fairly inconsistent, it appears that attentional and motor impulsivity in particular are related to overeating, while there is at most a minor role for non - planning impulsivity (meule, 2013 ; meule and platte, 2015). in line with this, only attentional and motor impulsivity scores were positively correlated with attentional bias for food - cues in a study by hou. unexpectedly, however, only non - planning impulsivity was correlated with attentional bias scores in the current study. in a recent study, scores on non - planning impulsivity positively correlated with striatal brain activations during hc versus lc food choices (van der laan., 2015). thus, although non - planning impulsivity does not appear to be directly related to body mass, it may indeed be associated with a higher reactivity to hc food - cues (e.g. higher reward - related brain activation, biased attention allocation). therefore, future studies are needed that address which facets of impulsivity are related to measures of overeating and to body weight, and examine possible moderators and mediators of these relationships in particular. preliminary evidence, for example, suggests that the relationship between motor impulsivity and food intake may be mediated by levels of external eating (kakoschke., 2015b). it is important to note that there were no additive effects of attentional bias for hc foods and impulsivity when predicting weight change, but there was a crossover interaction. yet again, the nature of this interaction closely parallels the one found by nederkoorn., participants with strong implicit food preferences and effective response inhibition tended to lose weight over 1 year. similarly, participants with attentional bias for hc foods and low motor impulsivity tended to lose weight in the current study. the authors speculated that this may represent an overcorrection effect, whereby strong implicit preferences for tempting food cues may serve to remind people of the very self - regulatory goals they wish to attain [and ] given sufficient self - regulatory capacity in the form of behavioral inhibition, people may actually attain their self - regulatory goals this interpretation is also in line with the goal conflict model of eating behavior, which posits that successful restrained eaters (who have been found to score low on impulsivity ; van koningsbruggen., 2013) have formed a facilitative link from palatable food stimuli to eating control and even though palatable food stimuli also prime the eating enjoyment goal, the increased accessibility of the dieting goal helps them to inhibit eating enjoyment and to engage in healthy eating interpretation of results is limited by the non - representative sample of young women who primarily had normal weight. thus, future studies in other samples such as men and samples with a larger range in age and bmi are needed to confirm these relationships. moreover, impulsivity was assessed by self - report, which is vulnerable to bias. however, previous studies have found that scores on the bis are indeed related to response inhibition as measured by behavioral tasks, albeit correlations are rather small and inconsistent (aichert., 2012 ; lange and eggert, 2015 ; meule and kbler, 2014 ; meule., 2014). finally, future studies need to address whether weight gain is really the result of increased intake of foods presented in laboratory paradigms (i.e. the foods that participants showed attention allocation to in the current study or showed an implicit preference for in other studies). to conclude, the current study provides further evidence for the view that higher body weight is determined by an interaction between high automatic, motivational drive toward palatable food stimuli (in this case, automatic allocation of attention toward these stimuli) and low deliberative control capacity (in this case, high self - reported motor impulsivity). | strong bottom - up impulses and weak top - down control may interactively lead to overeating and, consequently, weight gain. in the present study, female university freshmen were tested at the start of the first semester and again at the start of the second semester. attentional bias toward high- or low - calorie food - cues was assessed using a dot - probe paradigm and participants completed the barratt impulsiveness scale. attentional bias and motor impulsivity interactively predicted change in body mass index : motor impulsivity positively predicted weight gain only when participants showed an attentional bias toward high - calorie food - cues. attentional and non - planning impulsivity were unrelated to weight change. results support findings showing that weight gain is prospectively predicted by a combination of weak top - down control (i.e. high impulsivity) and strong bottom - up impulses (i.e. high automatic motivational drive toward high - calorie food stimuli). they also highlight the fact that only specific aspects of impulsivity are relevant in eating and weight regulation. |
a pubmed, medine search was carried out with the key words : non - vital, young permanent tooth, and triple antibiotic paste. case reports, and in vitro, in vivo animal studies and investigations on tap were included for review. modern concept of medicine emphasizes prevention and reversal of the diseases. only when these attempts fail, we would take on the unfavorable approaches, i.e., surgical intervention and restoration with artificial prostheses. the success of non - surgical endodontic treatment method is based on appropriate cleaning, shaping, asepsis, and filling of the root canal. several case reports have been published on non - surgical management of tooth with a non - vital pulp and persisting sinus tract using tap. the systemic administration of antibiotics relies on patient compliance with the dosing regimens followed by absorption through the gastrointestinal tract and distribution via the circulatory system to bring the drug to the infected site. hence, the infected area requires a normal blood supply which is no longer the case for teeth with necrotic pulps and for teeth without pulp tissue. therefore, local application of antibiotics within the root canal system may be a more effective mode for delivering the drug. microorganisms in dentinal tubules may constitute a reservoir from which root canal and surrounding tissue infection and re - infection may occur. demonstrated that dentin itself can have an inhibitory effect on the bactericidal activity of intracanal medicaments like calcium hydroxide. william windley observed a statistically significant reduction in bacteria, following the irrigation and antibiotic paste protocol. however, this dropped to 30% following the application of the tap for 2 weeks. dental trauma may tear the apical neurovascular bundle and cause pulp necrosis, resulting in arrested root formation in immature teeth. the traditional approach for treating cases of a necrotic immature permanent tooth was apexification with calcium hydroxide or mta, but it does not lead to a further thickening of dentinal walls or an increased root length. it is difficult to get an appropriate apical seal in teeth with open apices by using the conventional endodontic treatment methods. long - term use of calcium hydroxide has several disadvantages such as multiple treatment appointments, probable recontamination of the root canal system during treatment period, and increased brittleness of root dentin which increases the risk of future cervical root fractures. an alternative material of choice for apexification is mta and it has shown high success rates.[2224 ] regenerative endodontics is an emerging field focusing on replacing traumatized and diseased pulp tissue in these teeth. utilization of stem cells to regenerate the lost tissues may reverse tissues to their normal state. regenerative endodontics deals with the healing of impaired dental tissues, including dentin, pulp, cementum, and periodontal tissues. regeneration can occur from vital pulp cells remaining at the apical end of the root canal, the multipotent dental pulp stem cells, the stem cells in the periodontal ligament, and stem cells from apical papilla or in bone marrow. revascularization of the pulp space in a tooth with necrotic infected pulp tissue and apical periodontitis has been thought to be impossible. nygaard - ostby and hjortdal successfully regenerated pulps after vital pulp removal in immature teeth, but were unsuccessful when the pulp space was infected. thus, if the canal is effectively disinfected, revascularization should occur similar to that in an avulsed immature tooth. once the canal infection is controlled, it resembles the avulsed tooth that has a necrotic but sterile pulp space. the blood clot is then introduced so as to mimic the scaffold that is in place with the ischemic necrotic pulp in the avulsed tooth. in this situation, the necrotic uninfected pulp acts as a scaffold for the ingrowth of new tissue from the periapical area. the absence of bacteria is critical for successful revascularization because the new tissue will stop at the level at which it meets bacteria in the canal space. the clinical effectiveness of the tap in the disinfection of immature teeth with apical periodontitis has been reported. calcium hydroxide can cause necrosis of the surrounding tissue, destroying remnant vital tissues that have the potential to differentiate into new pulp. when the canal is properly disinfected, the inflammatory process reverses and the tissues may proliferate. revascularization of immature teeth with apical periodontitis depends mainly on : (a) disinfection of the canal ; (b) placement of a matrix in the canal for tissue ingrowth ; and (c) a bacterial tight seal of the access opening. since the infection of the root canal system is considered to be polymicrobial, a combination of drugs would be needed to treat the diverse flora. thus, the recommended protocol combines the use of metronidazole, ciprofloxacin, and minocycline. raison bose compared tap, calcium hydroxide, and formocresol as intracanal medicaments in non - vital young permanent tooth. the triple antibiotic group showed the highest percentage increase in the dentin wall thickness compared with the other two groups. tap contains both bactericidal (metronidazole, ciprofloxacin) and bacteriostatic (minocycline) agents to allow for successful revascularization. in apexification, the canal is temporarily filled until a hard tissue barrier forms at the apex. because the canal space is filled, there is no space available for vital tissue to proliferate into the root canal and the possibility of revascularization is eliminated. investigations have proved that topical doxycycline and minocycline can improve radiographic and histological evidence of revascularization in immature avulsed permanent teeth. several reports have recently demonstrated the potential for revascularization after infection if a sterile environment is created. after disinfection, the canal should be filled with a resorbable matrix to encourage the ingrowth of new tissue. tap was proved to be biocompatible. tetracycline inhibits collagenases and matrix metalloproteinases, is not cyt otoxic, and increases the level of interleukin-10, an anti - inflammatory cytokine. reynolds. used dentin bonding agent and composite resin before placement of the triple antibiotic dressing to prevent discoloration, but the discoloration was only reduced. the discoloration by the tetracycline family is thought to be a photo - initiated reaction. it should be limited to the root canal because of the potential risk of tooth discoloration. success of the endodontic treatment relies upon the elimination of bacteria from the root canal. microorganisms in the periapical region can cause re - infection and failure. from the existing literature, it is clear that tap can be effectively used for sterilization of canals and healing of periapical pathology. the effectiveness of tap in managing non - vital young permanent tooth is based on the availability of viable stem cells. development of resistant bacterial strains and tooth discoloration are the possible drawbacks of this technique. | the success of the endodontic treatment depends on the microbial suppression in the root canal and periapical region. endodontic instrumentation alone can not achieve a sterile condition. with the advent of non - instrumentation endodontic treatment and lesion sterilization and tissue repair, local application of antibiotics has been investigated. triple antibiotic paste (tap) containing metronidazole, ciprofloxacin, and minocycline has been reported to be a successful regimen in controlling the root canal pathogen and in managing non - vital young permanent tooth. this paper reviews the existing literature on biocompatibility, efficiency, drawbacks of tap in endodontic therapy and pulp revascularization. |
non - small - cell lung cancer (nsclc) accounts for approximately 8085% of all lung cancer. the prognosis of nsclc is poor and it is estimated that the 5-year survival rate for nsclc is less than 15%. many nsclc patients are diagnosed at late stage, missing the chance for surgical resection. for these patients, however, the response rate to platinum - based regimen of advanced nsclc was less than 30%. epidemiological studies reveal the genetic factor, for example, gene polymorphism of certain genes, contributing to the different chemotherapy response in nsclc patients [4, 5 ]. however, the efficacy and reliability of these candidate remain to be further determined. osteopontin (opn) is a multifunctional cytokine involved in cell survival, migration, and adhesion which is associated with tumorigenesis, progression, and metastasis. opn is also implicated in tumorigenesis and has been proposed as a cancer marker [711 ]. clinical study showed that high opn expression is associated with poor survival of patients with stage i non - small - cell lung cancer, suggesting that opn could be a candidate target for cancer therapy [13, 14 ]. overexpression of opn is associated with more aggressive phenotypes in human non - small - cell lung cancer. several single nucleotide polymorphisms (snps) in the human opn encoding gene have been identified. several polymorphisms in the human opn encoding gene have been identified in different populations, of which the 156gg / g, 443c / t, and 66t / g polymorphisms were mostly studied. based on the increased expression of opn in the nsclc tissue, we postulate that the opn gene polymorphism is related to the chemotherapy response in nsclc patients. in this study, we enrolled inoperable nsclc (stages iiib and iv) receiving platinum - based chemotherapy to test this hypothesis. a total of 497 patients with inoperable advanced stage of nsclc, namely, stages iii b and iv nsclc, confirmed cytologically or histologically were enrolled into this study. to avoid the potential influence of poor clinical conditions on chemotherapy response, other eligibility criteria included normal blood chemistries (hemoglobin > 9 g / dl, neutrophil count > 1500/mm, and platelet count > 100 000/mm), hepatic function (bilirubin 50 ml / s), and normal electrocardiogram at the beginning of treatment. the study was approved by the ethics committees of our hospital and written informed consent was obtained from each participant. patient responses to treatment were determined by the world health organization criteria, which classify the response into four categories : complete response (cr), partial response (pr), stable disease (sd), and progressive disease (pd). cr and pr were combined as well responders, and sd and pd were grouped as poor responders. the chemotherapy response was assessed by two independent oncologists who were blind to our study. overall survival (os) and progression free survival (pfs) os was calculated from the date of chemotherapy to the date of last follow - up or death from any cause. pfs was defined as the interval between the date of chemotherapy and the date of confirmed relapse. dna was extracted from peripheral whole blood using a qiagen dna isolation kit (qiagen, valencia, ca, usa). the single nucleotide polymorphisms on the promoter region of opn gene were determined using taqman 5 allelic discrimination assay. it was performed using a commercially available kit assays - on - demandtm snp genotyping products (applied biosystems, foster city, ca)., 10 ng of sample dna in 25 l of reaction solution contains 12.5 l of the 2 taqman universal pcr mix (applied biosystems), and 1.25 l of predeveloped assay reagent from the snp genotyping product contains two primers and two mcb - taqman probes. reaction condition consisted of preincubation at 50c for 2 min, at 95c for 10 min, and followed by 40 cycles of 95c for 15 s and 60c for 1 min. amplifications were performed in an abi prism 7500 sequence detection system (applied biosystems). to estimate the deviation of frequency of gene alleles in tested population multivariate logistic regression analysis was used to determine the influence of opn polymorphisms on the chemotherapy response, controlling potential confounding conventional risk factors. a forward stepwise (likelihood ratio) among all patients that received platinum - based chemotherapy, 311 were chemotherapy responder (cr + pr) and 430 were nonresponder (sd + pd). nonresponders had higher prevalence of smokers, patients with stage iv and with poor differentiation than responders (all p 0.05). genotype frequencies of opn gene polymorphisms in chemotherapy responder and nonresponders were found to be fit in the hardy - weinberg equilibrium (all p > 0.05). table 2 shows the genotypes and allele frequency of 443c > t was significantly different between the responders and nonresponders. responders had a markedly higher frequency of 443tt genotype than responders (40.71% versus 19.09%, p gg and 66t > g were not significantly different between well responders and poor responders (all p > 0.05). the associations between opn genotype and os of nsclc patients were studied by log - rank test. there were no significant differences in os based on the opn genotypes at 156/g > gg and 66t > g locus (all log - rank p > 0.05, data not shown). in contrast, the median os time for patients with 443cc, 443ct, and 443tt genotype carriers was significantly different (table 3). the kaplan - meier curves for the effect of 443cc, 443ct, and 443tt genotype on os are shown in figure 1. next, the multivariate cox proportional hazards regression models were used to estimate the hazard ratios (hr) for os, with adjustment for age, sex, smoking status, histology, stage, and response status. table 4 shows that the tumor differentiation (well + moderate versus poor), chemotherapy response (well response versus poor response), and 443c > t gene polymorphism (cc versus tt) were closely correlated to poor nsclc prognosis. it is very important to select the patients who response to chemotherapy to achieve better clinical outcome and to avoid the side effect of chemotherapy. in this study we reported that opn gene polymorphisms influence the treatment response and clinical outcomes of advanced nsclc patients receiving platinum - based chemotherapy. we found that the 443c > t polymorphisms significantly associated with the chemotherapy response and predict prognosis of nsclc patient receiving chemotherapy. the 443tt genotype carriers tend to have better prognosis of nsclc. to the best of our knowledge, this is the first study regarding the opn genetic polymorphism and treatment response in nsclc. opn overexpressed in the nsclc tumor tissues has been reported significantly correlated with tnm stages and lymph metastasis. there are several polymorphic sites in the regulatory element of the opn promoter, and snps at nucleotide 443 are frequently detected and reported involved in regulation of opn expression in normal cells [1820 ]. a recent study on melanoma metastases found that those homozygous for the 443c allele expressed significantly higher levels of opn mrna compared to those that were either heterozygous (ct) or homozygous for the 443 t allele. the opn promoter polymorphisms at locus 443 significantly affect the metastasis and prognosis of human hepatocellular carcinoma. the functional 443t / c osteopontin promoter polymorphism influences osteopontin gene expression in melanoma cells via binding of c - myb transcription factor. opn 443c > t genetic polymorphism and tumor opn expression are associated with the risk and clinical features of papillary thyroid cancer in a chinese cohort [22, 23 ]. a previous study examined the possibility that snp in the promoter region of the opn at nt 443 is a marker predicting the therapeutic efficacy of pegylated interferon- (peg - ifn - alpha2b-) ribavirin combination therapy in egyptian patients with chronic hepatitis c. serum opn protein level and t / t homozygotes of snp at 443 were significant predictors for response. snp in the promoter region of opn at nt 443 and serum opn protein level are predictors of response to the efficacy of peg - ifn - alpha2b - ribavirin therapy in patients with chronic hepatitis c [25, 26 ]. another study reported that snps in the promoter region of opn may be useful as a marker to predict the efficacy of ifn - based therapies in patients with chronic hepatitis c [27, 28 ]. opn 156g / gg did not affect the treatment response to anti - tnf therapy in patients with rheumatoid arthritis. in this study, we reported that the opn gene polymorphism at 443c > t is also associated with the chemotherapy in nsclc patients. a recent study showed that the survival rates for patients with the 443cc genotype were significantly lower than the survival rates of the other two genotypes (443ct and 443tt) [30, 31 ]. in that study, these data suggest that opn gene polymorphism is a potential predictive marker of survival in lung cancer patients. we found that the 443c > t polymorphisms significantly associated with the chemotherapy response and predict prognosis of nsclc patient receiving chemotherapy. there are limitations in the present study ; one of them is because all of the subjects are chinese individuals, and the results should be interpreted with caution and need to be confirmed in larger and ethnically divergent population samples. on the other hand, the number of patients in the current study is relatively small, so the large - population research is needed to make stronger conclusion about the association between bone metastasis formation and 433 polymorphisms. | background. osteopontin (opn) is associated with prognosis of patients with non - small - cell lung cancer (nsclc). however, little is known about the association between opn gene polymorphism and the chemotherapy response in nsclc patients. methods. a total of 497 patients with inoperable advanced stage of nsclc (stages iii b and iv nsclc) were enrolled. all patients had received platinum - based chemotherapy. opn gene polymorphisms at 156 gg / g, 443 c / t, and 66t / g were determined. results. the genotypes and allele frequency of 443c > t were significantly different between the responders and nonresponders. responders had a markedly higher frequency of 443tt genotype than responders (40.71% versus 19.09%, p t gene polymorphisms were closely correlated to poor nsclc prognosis. conclusion. opn 443c > t gene polymorphism may be used as a molecular marker to predict the treatment response to chemotherapy in advanced nsclc patients. |
in recent years, the evolution of endodontics has broken several paradigms, driven by technological and techniques advances in all its phases of execution. however, despite all these technical and scientific developments, some concepts have not changed. the main objectives of root canal treatment continue to be the elimination of or reduction in the number of microorganisms within the root canal space, and the prevention of possible infection or reinfection. with this in mind, there is a clear interest in improving the effectiveness of the root canal filling techniques. proof of this can be found in the great number of different systems recently developed to limit microbiological action capable of inducing or even causing resistance of a periapical lesion. the fillings failures observed in several studies, resulting from many different methodologies, have given rise to a global trend, evidenced by the literature, toward enhancing the ability of endodontic filling materials to project themselves into the dentinal tubules. these materials not only act as antibacterial agents, theoretically speaking, but can also optimize the quality of the seals provided by these penetrations. this hypothesis is based on the concept that tubular dentine cement penetration considerably increases the contact surface of the filling material with the root canal walls, thereby improving the seal. the possibility of improving the adhesiveness of the filling materials also has the ultimate goal of preventing leakage. however, these hypotheses have not yet been completely clarified in the literature, and few studies have been performed to investigate these possible correlations. based on the above, this study aimed to analyze possible correlations between tubular dentine cement penetration, adhesiveness and leakage in roots filled with gutta percha and an endodontic cement based on epoxy amine resin. after approval of the ethics committee (process 5314), sixty mandibular central incisor teeth with single straight canals, complete rhizogenesis, with no resorption or previous endodontic treatment, and free foraminal access, were provided by the university tooth bank and selected for this study. all this information was verified by clinical analysis and vestibulo - lingual and mesiodistal radiographs. the crowns were then removed using a low - speed steel cutting disc (isomet - buehler, lake bluff, il, usa), standardizing all roots at 13 mm in length. the accesses were performed using a tapered - tip bur 3082 (kg sorensen, barueri, sp, brazil). working length was established by subtracting 1 mm from the point where the file was visible at the apical foramen. the coronal and middle thirds of each canal were prepared using gates glidden drills (dentsply - maillefer, ballaigues, switzerland) sizes 4, 3 and 2, by placing each instrument 2 mm deeper than the previous one. the apical foramina were standardized using real length instrumentation of the teeth up to instrument 25 k - flexofile (dentsply - maillefer, ballaigues, switzerland), and the apical thirds were prepared with the profile 04 system (dentsply - maillefer, ballaigues, switzerland) up to size 35 at working length. the canals were irrigated between each instrumentation with 2 ml of freshly prepared 2.5% naocl plus a flush of 3 ml of 17% edta for 3 min. the prepared canals were filled using the lateral compaction technique. after drying with paper points, a size 20 file was used to place 10 l of an endodontic cement based on epoxy amine resin - ah plus (dentsply - detrey, konstanz, germany) - into the canal, using a counterclockwise rotation. the sealer was labeled with a 0.1% rhodamine b dye (sigma - aldrich, st. louis, mo, usa) for the purpose of further analysis by optical light microscopy. a prefitted size 35, 0.04-taper gutta - percha cone (dentsply - maillefer, ballaigues, switzerland) was used as the master cone, and two accessory cones were used in addition. the filled roots were stored at 37c and 100% humidity for 7 days, to allow the sealer to set. the root apex was connected to a luer - type metal needle by means of a plastic tube. the allowed leakage margin for the tested groups was quantified according to the movement of a small air bubble inside a 25 l micropipette (microcaps - fisher scientific, philadelphia, pa, usa). the inside of the pipette and the entire system was filled with distilled water and a pressure of 10 psi was applied. after making sure there was no leakage in the connections, the system was activated and balanced for 4 minutes. the volume of fluid was calculated by observing the air bubble displacements, expressed in l / min-.10 psi. each specimen was sectioned horizontally at 3, 6 and 8 mm from the apex, using a low - speed steel cutting disc (isomet - buehler, lake bluff, il, usa). three slices per root were created, resulting in a total of 180 slices. a standard polishing procedure using sic paper (200, 300, 400, 600) followed by 3 m diamond paste was employed on the coronally facing surface of each slice to produce a high - reflection surface (figures 1a and b), and each slice was observed in a high - resolution stereomicroscope to acquire images at 1048x1048 pixels, covering the entire root surface. for each image, the outer perimeter of the root and the inner perimeter of the root canal walls were outlined and measured using the axiovision software 4.11 (carl zeiss, jena, germany) (figures 1c and d). the total cross - sectional area of the canal wall was obtained for each section by subtracting the value of the outer perimeter from the inner perimeter. the absolute cement penetration values (figure 2) for each section were then converted into a percentage of cement penetration into dentinal tubules, by calculating the total cross - sectional area of the canal wall previously obtained in the high - resolution stereomicroscope. total cross - sectional area (a) and canal cross - sectional area (b). the outer (c) and the inner (d) perimeter of the root canal walls outlined and measured using axiovision software 4.11 (carl zeiss, jena, germany) absolute cement penetration values outlined and measured using axiovision software 4.11 (carl zeiss, jena, germany) firstly, the thickness of each slice of the 60 sections corresponding to the middle third was measured with a digital caliper (mitutoyo ip67-mitutoyo, neuss, germany). then, the specimens were submitted individually to push - out bond strength tests using a universal - testing machine (emic dl200mf, so jos dos pinhais, pr, brazil) at a speed of 0.5 mm / min up to bond failure using a 0.50-mm diameter stainless steel cylindrical plunger. the plunger tip was sized and positioned so that it contacted only the filling material. because of the convergence of the root canal sections, the push - out force was applied from apical to coronal. the bond strength expressed in mpa at failure was calculated by dividing the load in newtons by the area of the bonded interface. the area of the bonded interface was calculated according to the following formula : area=2r x h, where is kept constant at 3.14, and r and h are the radius and height measured in millimeters of the filling material that was pushed out. the results obtained were submitted to the pearson test with a significance level of 95% (p<0.05) to perform the correlation analysis among the variables of the study using the statistical software spss 11.5 (spss inc., after approval of the ethics committee (process 5314), sixty mandibular central incisor teeth with single straight canals, complete rhizogenesis, with no resorption or previous endodontic treatment, and free foraminal access, were provided by the university tooth bank and selected for this study. all this information was verified by clinical analysis and vestibulo - lingual and mesiodistal radiographs. the crowns were then removed using a low - speed steel cutting disc (isomet - buehler, lake bluff, il, usa), standardizing all roots at 13 mm in length. the accesses were performed using a tapered - tip bur 3082 (kg sorensen, barueri, sp, brazil). working length was established by subtracting 1 mm from the point where the file was visible at the apical foramen. the coronal and middle thirds of each canal were prepared using gates glidden drills (dentsply - maillefer, ballaigues, switzerland) sizes 4, 3 and 2, by placing each instrument 2 mm deeper than the previous one. the apical foramina were standardized using real length instrumentation of the teeth up to instrument 25 k - flexofile (dentsply - maillefer, ballaigues, switzerland), and the apical thirds were prepared with the profile 04 system (dentsply - maillefer, ballaigues, switzerland) up to size 35 at working length. the canals were irrigated between each instrumentation with 2 ml of freshly prepared 2.5% naocl plus a flush of 3 ml of 17% edta for 3 min. the prepared canals were filled using the lateral compaction technique. after drying with paper points, a size 20 file was used to place 10 l of an endodontic cement based on epoxy amine resin - ah plus (dentsply - detrey, konstanz, germany) - into the canal, using a counterclockwise rotation. the sealer was labeled with a 0.1% rhodamine b dye (sigma - aldrich, st. louis, mo, usa) for the purpose of further analysis by optical light microscopy. a prefitted size 35, 0.04-taper gutta - percha cone (dentsply - maillefer, ballaigues, switzerland) was used as the master cone, and two accessory cones were used in addition. the filled roots were stored at 37c and 100% humidity for 7 days, to allow the sealer to set. the root apex was connected to a luer - type metal needle by means of a plastic tube. the allowed leakage margin for the tested groups was quantified according to the movement of a small air bubble inside a 25 l micropipette (microcaps - fisher scientific, philadelphia, pa, usa). the inside of the pipette and the entire system was filled with distilled water and a pressure of 10 psi was applied. after making sure there was no leakage in the connections, the system was activated and balanced for 4 minutes. the volume of fluid was calculated by observing the air bubble displacements, expressed in l / min-.10 psi. each specimen was sectioned horizontally at 3, 6 and 8 mm from the apex, using a low - speed steel cutting disc (isomet - buehler, lake bluff, il, usa). three slices per root were created, resulting in a total of 180 slices. a standard polishing procedure using sic paper (200, 300, 400, 600) followed by 3 m diamond paste was employed on the coronally facing surface of each slice to produce a high - reflection surface (figures 1a and b), and each slice was observed in a high - resolution stereomicroscope to acquire images at 1048x1048 pixels, covering the entire root surface. for each image, the outer perimeter of the root and the inner perimeter of the root canal walls were outlined and measured using the axiovision software 4.11 (carl zeiss, jena, germany) (figures 1c and d). the total cross - sectional area of the canal wall was obtained for each section by subtracting the value of the outer perimeter from the inner perimeter. the absolute cement penetration values (figure 2) for each section were then converted into a percentage of cement penetration into dentinal tubules, by calculating the total cross - sectional area of the canal wall previously obtained in the high - resolution stereomicroscope. total cross - sectional area (a) and canal cross - sectional area (b). the outer (c) and the inner (d) perimeter of the root canal walls outlined and measured using axiovision software 4.11 (carl zeiss, jena, germany) absolute cement penetration values outlined and measured using axiovision software 4.11 (carl zeiss, jena, germany) firstly, the thickness of each slice of the 60 sections corresponding to the middle third was measured with a digital caliper (mitutoyo ip67-mitutoyo, neuss, germany). then, the specimens were submitted individually to push - out bond strength tests using a universal - testing machine (emic dl200mf, so jos dos pinhais, pr, brazil) at a speed of 0.5 mm / min up to bond failure using a 0.50-mm diameter stainless steel cylindrical plunger. the plunger tip was sized and positioned so that it contacted only the filling material. because of the convergence of the root canal sections, the push - out force was applied from apical to coronal. the bond strength expressed in mpa at failure was calculated by dividing the load in newtons by the area of the bonded interface. the area of the bonded interface was calculated according to the following formula : area=2r x h, where is kept constant at 3.14, and r and h are the radius and height measured in millimeters of the filling material that was pushed out. the results obtained were submitted to the pearson test with a significance level of 95% (p<0.05) to perform the correlation analysis among the variables of the study using the statistical software spss 11.5 (spss inc., in isolated analyses of the three variables, the means and standard deviations, with a confidence interval of 95%, were the following : tubular dentine cement penetration (8.8754.540), adhesiveness (4.4412.683) and apical leakage (0.3180.215). the results obtained in the correlation analysis among these study variables were confronted and are shown in figures 3, 4 and 5, respectively. there was no statistically significant correlation between tubular dentine cement penetration and apical leakage (r : 0.08276), adhesiveness and tubular dentine cement penetration (r : -0.2412) and adhesiveness and apical leakage (r : 0.1340). representative image of the correlation analysis between tubular dentine cement penetration and leakage representative image of the correlation analysis between adhesiveness and tubular dentine cement penetration representative image of the correlation analysis between adhesiveness and leakage the hypothesis that tubular dentine cement penetration could improve sealability was firstly proposed in 1984. later these other studies were based on the original hypothesis that a better cement penetration into the dentinal tubules would somehow improve the quality of endodontic fillings. therefore, previous reports that tubular dentine cement penetration was one of the most important factors to be considered in choosing the filling material justify the importance of conducting comparative studies on this matter. most of the papers have analyzed the three variables studied in our paper - tubular dentine cement penetration, adhesiveness, and leakage, merely comparing different materials. only a few articles have tried to analyze possible correlations among these variables. comparing four endodontic cements (diaket, endomethasone, crcs and ketac endo) in relation to their projections into the dentinal tubules and their sealability using the same filling technique, sen,. (1996) showed that the best results were obtained by different materials in isolated analyses. as in our results, it was not possible to observe any correlation between these two variables. the methods used (scanning electron microscopy and dye leakage test) were different from those adopted in this study (optical microscopy and fluid filtration test), but the main methodological point was to use the same specimens to analyze different points, an approach considered essential for analyzing possible correlations among two or more variables. using a different protocol, another study investigated the dentine permeability obtained by two substances used in the final irrigation of root canals, namely, sodium hypochlorite and ethanol 95%. in addition, specimens from the different study groups were also submitted to the fluid transport test to analyze the sealability of their fillings. although a greater cement penetration and a lower level of leakage were observed in the group that used ethyl alcohol, this correlation was not verified statistically, thereby showing results similar to those of our study (r : 0.08276). after analyzing our results and most of the studies performed to date, it appears that no correlation exists between tubular dentine cement penetration and sealability, or else the research methods used were unable to detect any correlation. the majority of the leakage tests, including fluid filtration, showed leakage only when there was at least one void extending from the apical to the coronal thirds. a root canal filling that looks poorly condensed on the radiograph may contain many " cul - de - sac "- type voids and no leakage. on the other hand, very small " through - and - through "- type voids that are invisible on radiographs may be detected by the fluid filtration test and show considerable leakage rates. in this case, the leakage rates would probably not be smaller if a major amount of sealer penetrated into the dentinal tubules. it seems that a more plausible hypothesis would be that this variable may be improved when there is a better adaptation of the cement to the canal walls. addressing adhesiveness and cement penetration, a classic study correlating these variables was performed to compare the adhesiveness of two filling systems (gutta percha / kerr pulp canal sealer ewt and resilon / epiphany) and the authors found more favorable results for the resilon / epiphany system. however, an analysis of images by sem demonstrated no large cement penetrations into the dentinal tubules. in view of this finding, the authors suggested that there was no effective correlation between cement penetration and adhesiveness, a conclusion corroborated by the results of this study. our correlation results (r : -0.2412) and those mentioned above suggest that better adhesiveness is not related to a possible mechanical overlap provided by cement penetration or adhesive tags into the dentinal tubules. it seems legitimate to state that the establishment of a consistent hybrid layer in intratubular dentine plays a more important role in achieving better adhesiveness to the root dentine. nagas,.(2007) analyzed the adhesiveness and leakage in root fillings comparing different methods of photoactivation (quartz halogen light for 40 seconds, light - emitting diode for 20 seconds, and plasma arc for 6 seconds). it is worth highlighting that there were some important methodological differences in relation to the current study. the authors used different specimens to perform the push - out and leakage tests, and also carried out different statistical tests to analyze each variable individually. in the results related to adhesion however, when the leakage was analyzed separately, the differences between the two groups with the best results were not significant, suggesting a lack of correlation between these variables. this conclusion was demonstrated by the correlation results of our study as well (r : 0.1340). to date, in the only study that has shown a positive correlation between adhesiveness and leakage, there was an important methodological difference from those used in this research. the authors did not use gutta - percha or any solid material associated to the cement, which was used alone. considering that the fluid filtration test shows leakage only when there is at least one void extending from the apical to the coronal thirds, it is possible that the sealer alone may be able to improve the sealability observed by this methodology. this is an important experimental design feature, because standard correlation analysis posits that any random factor affects only one subject, and not others. this requirement is violated when two or more different experimental groups are created, as was the case in all the aforementioned studies. in fact, there was no rationale to justify the creation of two or three experimental groups when the main goal was restricted to verifying a potential cause - and - effect correlation. therefore, when attempting to verify a potential cause - and - effect correlation, a single sizeable group should be created. however, even when a single well - standardized group was used, assessed through updated and refined experimental models, a correlation could not be established among the variables. based on the experimental conditions and the results observed in this in vitro study, it is possible to prove that there are no correlations among tubular dentine cement penetration, adhesiveness and leakage. | objectiveto analyze possible correlations among tubular dentine cement penetration, adhesiveness and apical leakage in fillings performed with gutta percha and an endodontic cement based on epoxy amine resin. material and methodssixty similar, extracted human mandibular central incisors were irrigated, instrumented and filled following the same protocol. first, apical leakage was quantified by fluid filtration tests. then, these same specimens were sectioned for analysis of tubular dentine cement penetration and the middle thirds were submitted to push - out tests to analyze the adhesiveness of the fillings. resultsin brief, the means and standard deviations with a confidence interval of 95% were as follows : tubular dentine cement penetration (8.8754.540), adhesiveness (4.4412.683) and apical leakage (0.3180.215). the data were confronted using the pearson 's test (p>0.05), and it was possible to prove that there was no correlation between (1) tubular dentine cement penetration and apical leakage (r2 : 0.08276), (2) tubular dentine cement penetration and adhesiveness (r2 : -0.2412) and (3) adhesiveness and apical leakage (r2 : 0.1340). conclusionafter analysis of these data, it could be observed that there exists no correlation among the variables analyzed in this study. |
nordic walking is a physical activity consisting of walking with poles similar to ski poles. the poles are designed for the purpose of activating the upper body during walking. they are equipped with rubber or spike tips, and the walking itself resembles nordic - style skiing. beginners are asked to walk in an upright position, not leaning forward or backward. the head should be up and facing forward, and the poles are held close to the body. when the leading foot is moving forward, the opposite arm swings forward to waist height. the opposite pole strikes the ground level with the heel of the leading foot. the poles remain pointing diagonally backward and are pushed as far back as possible. the arm straightens and the hand opens, coming off the grip by the end of the arm swing. nordic walking is not expensive and can be performed throughout the year. but handling of the poles is difficult, the technique required to handle them can not be easily acquired by community - dwelling elderly individuals. an intriguing phenomenon is the extent to which the human motor system can transfer relevant strategies learned in one context to other similar situations. experimental paradigms using upper limb tasks show the ability of the nervous system to generalize learned motor skills to similar movements, contexts, or workspaces and even to the opposite limb1,2,3,4. such studies have yielded valuable insights into how the nervous system codes and generalizes motor skills. walking is a motor task that is highly flexible in its adaptation to different situations. extensive work has shown the ability of the locomotor system to adapt to changes in sensory input from the limbs5,6,7. in addition, the walking pattern is easily modified to accommodate different external constraints, for example, navigating over or around obstacles8 or different slopes of the walking surface9, 10. therefore, it is important for elderly individuals to train with movements in the backward direction to prevent falling forward. nordic backward walking (nbw) is a very difficult form of walking for beginners, and we do not currently know the effect of repetition of nbw on movement in the backward direction in the elderly. the aims of this study were : 1) to confirm the motor learning process of nbw in older adult community - dwelling volunteers and 2) to check the change in psychological condition resulting from nbw. we evaluated 19 older adult community - dwelling volunteers (11 female and 8 male ; mean age 644) (table 1table 1. general characteristics of participants (n=19)indexesmeansdage (years)644gender (male / femal)11/11functional reach test (cm)28.17.5bone density (g / cm)2.40.2mini - mental state examination (point)291). the volunteers (n=19) were included based on the following criteria : 1) self - report of good health and 2) absence of a history of psychiatric or neurological disorder. all participants gave informed consent, and the study was approved by the ethics committee of kansai university of welfare sciences. we measured nbw consecutively five times and then measured it a sixth time one hour later. no participants reported fatigue, likely because of the short walking distance and limited number of trials. the primary variables of interest were walking speed, stride length, and visual analogue scale (vas). the vas was used as an index of sense of fear for walking backward. a paired t - test was used to compare the mean front and back scores. the alpha level for statistical significance all data were analyzed using the statistical package for the social sciences (spss) version 19. our results demonstrated that following repeated sessions of backward walking, the walking speed during nbw in the first, second, third, fourth, and fifth sessions were 73 m / min, 80 m / min, 86 m / min, 88 m / min, and 91 m / min, respectively. significant differences were only found between the first and second sessions and between the second and third sessions. the walking speed in the sixth session (86 m / min), measured one hour later, was decreased in comparison with that in the fifth session. the stride lengths during nbw in the first, second, third, fourth, and fifth sessions were 0.52 m, 0.56 m, 0.58 m, 0.59 m, and 0.59 m, respectively. significant differences were only found between the first and second sessions and between the second and third sessions. the stride length in the sixth session (0.56 m), measured one hour later, was decreased in comparison with that in the fifth session. the vas scores after nbw for the first, second, third, fourth, and fifth sessions were 4.03 cm, 3.46 cm, 2.95 cm, 2.47 cm, and 2.00 cm, respectively. in addition, the vas score for the sixth session (2.00 cm), measured one hour later, was increased compared with that for the fifth session (table 2table 2. motor learning during nordic backward walking (n=19)1st2nd3rd4th5th6thbackward walking speed (m / min)732180208621882491288623stride length (m)0.520.100.560.120.580.110.590.130.590.120.560.11visual analogue scale (cm)4.032.393.462.352.952.252.472.392.002.291.662.04values are presented as the averagesd. this study investigated the learning process in beginners learning how to perform nbw using nordic poles. our results demonstrated that following repeated sessions of backward walking, the walking speed during nbw showed significant differences only between the first and second sessions and between the second and third sessions. the walking speed in the sixth session, measured one hour later, was decreased in comparison with that in the fifth session. unlike the subjects studied by lake and cavanagh11, our subjects demonstrated a clear increase in efficiency, likely as a result of increased efficiency of motor unit recruitment due to practice. the differing results can most likely be explained by considering the differences in the type of exercise used. backward walking is truly a novel task, whereas even individuals who do not specifically train with forward running do engage in forward walking. therefore, the motor pathways used for any forward locomotion are at least somewhat trained, whereas those used for backward locomotion are untrained. significant differences in stride length during nbw were only found between the first and second sessions and between the second and third sessions. the stride length in the sixth session, measured one hour later, was decreased in comparison with that in the fifth session. specifically, the increase was significant between the fourth and sixth week of training. schwane.12 suggested that a novel activity may require increasingly greater motor unit recruitment in order to complete a task. although we did not specifically examine muscle activity, backward walking requires greater sustained emg activity of the quadriceps than forward walking13, 14. thus, we believe our findings are the first to demonstrate that an individual may perform backward walking more efficiently after repeated sessions of backward walking training. significant differences in vas score after nbw were found for each session. in addition, the stride length in the sixth session, measured one hour later, was decreased in comparison with that in the fifth session. if the energy cost decreases as a person learns a new motor skill, as suggested by schwane.12, the prescribed exercise workload, per the regression equations reported by myatt.15 and clarkson.16, may not have the intended training effect. our findings are consistent with the hypothesis that efficiency increases when a task is no longer novel. therefore, it is important for elderly individuals to train with backward movements to prevent falling forward. the skill of the subjects in performing nbw rose with every repetition, but they learned at a slow pace. also, their level of skill tended to decrease when they took a break between sessions. however, their sense of fear did not worsen even if they took a break. this finding suggests that nbw is indeed a novel task and that motor learning occurs as a result of practice, leading to a more efficient recruitment of motor units. therefore, nbw was a form of training that matched with the preferences of the elderly and was thought to be useful as a backward walking exercise for the elderly. nbw can be performed anywhere and anytime, and therefore, widespread clinical application of nbw is possible. | [purpose ] the aims of this study were : 1) to confirm the motion learning process of nordic backward walking (nbw) in older adult community - dwelling volunteers and, 2) to check the change in psychological condition resulting from nbw. this study examined whether the learning process and psychological condition become more efficient after repeated sessions of nbw. [methods ] the subjects were 19 community - dwelling elderly individuals between the ages of 64 and 78 years. [results ] significant differences in walking speed during nbw were only found between the first and second sessions and between the second and third sessions. the walking speed in the sixth session, measured one hour after the fifth session, was decreased in comparison with that in the fifth session. significant differences in stride length during nbw were only found between the first and second sessions and between the second and third sessions. the stride length in the sixth session, measured one hour after the fifth session, was decreased in comparison with that in the fifth session. significant differences in vas score (sense of fear) after nbw were found for each session. vas score for the sixth session, measured one hour after the fifth session, was decreased in comparison with that for the fifth session. [conclusions ] the findings in the present study suggest that nbw is indeed a novel task and that motor learning occurs as a result of practice, leading to a more efficient recruitment of motor units. |
breast cancer is the most common tumor in women ; one in eight women will be affected in their lifetime. common sites of breast cancer metastasis include lungs, liver, bones, soft tissue, brain, and adrenal glands. gastrointestinal (gi) tract metastases from breast origin are considered rare in clinical practice ; however, the occurrence in autopsy series varied from 8% to 35% [3, 4 ]. most series report a greater propensity for lobular carcinoma to metastasize to the gi tract ; reports on this subject in the literature are poor and mostly limited to case reports. since metastases to gi from breast cancer are uncommon and peculiar, the main problem is to recognize them in patients affected by breast cancer and with gi symptoms, like nausea and vomiting, diarrhoea, and abdominal pain. these symptoms may be considered treatment related or secondary to other gi diseases, or to peritoneal carcinomatosis, and this can delay the definite diagnosis and treatment of gi involvement by breast cancer. we report here a series of 5 patients affected by gi metastases from breast cancer, and we reviewed the related literature. a retrospective analysis was conducted on the records of the patients with breast cancer admitted and treated at the oncology - hematology department, hospital of piacenza, italy, from january 2007 to december 2011 ; five patients with gastrointestinal metastases from breast cancer were identified, and they form the basis of this report. the medical records were reviewed for presenting symptoms, concurrent metastatic sites, treatment, and followup. pathology of the gi involvement as well as the primary tumor was reviewed by an expert pathologist in breast cancer. histologic grading, estrogen (er) and progesterone (pr) receptor expression, epidermal growth factor receptor-2 (her2) status, and proliferation rate (ki-67) were done in the sample of primary carcinoma and in the gi metastasis. original breast cancer presentations were staged according to the 2006 american joint committee on cancer staging system. a computerized literature search through medline, cancerlit, and embase was performed applying the words : breast cancer, gastrointestinal metastases, gastrointestinal tract, and palliative surgery. during the 5-year study period, 980 patients were diagnosed with breast cancer at our institution. five of these 980 patients (0.5%) had metastatic disease to the gi tract. patients ' characteristics are described below and summarized in table 1 ; the results of literature review are reported in table 2. a 47-year - old female underwent at another institution left breast mastectomy in march 1985 for a ductal infiltrating carcinoma (dic) pt2a n0/12. a complete staging was performed, with abdominal ultrasound, chest radiography, and bone scan ; no metastases were found. the histological biopsy of the mass and histological revision of the previous mastectomy showed er > 95% pr 0% ki-67 10%. she underwent local radiotherapy, with complete response, and endocrine therapy with anastrozole 1 mg / day. in september 2007 the patient was admitted to our department, since she developed fatigue and anemia : laboratory tests showed anemia, haemoglobin 7 g / dl, and the fecal occult blood test was positive ; an esophagogastroduodenoscopy (egds) and a colonescopy were performed : the first one was unremarkable, and the second one discovered the presence of a 4 mm tubule - villous adenoma with mild dysplasia of the right colon, that was removed. the patient was transfused with 3 units of red blood cells, an enteroscopy with videocapsule was performed ; an ulcerated and stenosing neoplasm, localized between the duodenum and the jejunum, was diagnosed. subsequently, a chest - abdominal computed tomography (ct) scan was performed, which revealed absence of lung and liver metastases. the patient underwent surgery, with jejunum resection, and the histological examination showed jejunum metastasis from breast cancer, er 70% pr 0% her2 negative, like the primary tumor. anastrozole was stopped and a treatment with chemotherapy : 5-fluorouracil, epirubicin, and cyclophosphamide (fec regimen), was started, and continued for 6 cycles. reevaluation ct scan performed in july 2008 resulted negative. from september 2009 endocrine therapy with exemestane the patient is alive and is treated with weekly paclitaxel, since she showed subcutaneous thoracic relapse in july 2011. a 40-year - old woman in may 2008 developed left breast cancer, ductal infiltrating carcinoma with signet ring cells g2 er 90% pr 35% ki-67 15% her2 negative, diagnosed with tru - cut biopsy ; in addition, ultrasound showed 2 pathologic lymph nodes in the left axilla ; at this time a colonscopy, performed for rectal bleeding, showed a substenosis 7 cm from the anal margin, biopsies diagnosed metastasis from breast cancer, moderately differentiated (g2) carcinoma, with partial aspects of signet ring cells, er 85% pr 30% her2 negative. chest x - ray, abdominal ultrasound, and bone scan showed absence of metastases. she was treated with epirubicin (75 mg / m) and docetaxel (75 mg / m) days 121, 6 cycles, with stable disease. in november 2008 endocrine therapy with tamoxifen 20 mg / day and lh - rh analogue 11.25 mg every 3 months was started, and symptomatic radiotherapy on the rectum was planned. ten months later, patient showed progression in the rectum and liver and was treated with liposomal doxorubicin (50 mg day 121) and carboplatin (auc 5 = 300 mg day 121). after 3 cycles she showed disease progression, and chemotherapy with oxaliplatin, folinic acid and 5-fluorouracil was initiated. patient is now well, with stable disease, and is treated with endocrine therapy, tamoxifene 20 mg / day. a 53-year - old woman in 1987 underwent left mastectomy for breast cancer at another institute, and then no other therapy was performed. in october 2007, patient was admitted to our department, since she had pain in the right chest, a bone scan was performed, and multiple bone metastases were detected. the reviewed histological specimen of the previous mastectomy showed dic g2 er > 80% pr > 80% ki-67 10% her2 negative. a ct scan of chest, abdomen, and pelvis confirmed multiple bone lesions without other metastases. the patient was treated with bisphosphonates and letrozole 2.5 mg / day. since the patient had dyspepsia, in march 2008, egds with biopsies was performed revealing the presence, in the fundus, of carcinoma with solid - chord structure, whom morphology and immunophenotype (g2, er 70% pr 60% her2 negative) were coherent with origin from the breast cancer. in april 2008, weekly chemotherapy with epirubicin and cyclophosphamide was started. after 2 months of treatment, she showed disease progression and underwent a chemotherapy with capecitabine and vinorelbine, but the patient died in august 2008 with disease progression. a 57-year - old women underwent, in january 2007, a quadrantectomy of the right breast and axillary dissection for lobular infiltrating carcinoma (lic) pt2 n3a (17/20) er 80% pr 85% ki-67 10% her2 negative. staging detected bone metastases. epirubicin (75 mg / m) and docetaxel (75 mg / m), days 121, chemotherapy was performed for 6 cycles, with partial response. after 1 year she had disease progression in the bone, so a new chemotherapy regimen with liposomal doxorubicin 50 mg / m days 121 was initiated. after 4 cycles, since tumour markers cea and ca 15 - 3 were increasing, liposomal doxorubicin was stopped after four cycles and endocrine therapy with fulvestrant 250 mg 1 fl i.m. per month was initiated. in november 2008, she showed persistent inappetence, nausea, and vomiting, an egds with biopsies revealed the presence in the cardia of metastasis from breast cancer (er 70%, pr 65%, ki-67 15%, her2 negative). in january 2009 capecitabine 1200 mg / m bid days 1 - 14q21 was initiated, but after 2 cycles the treatment, was stopped for haematological, gastrointestinal toxicity and disease progression. a 63-year - old women underwent in 1998 a radical mastectomy of the right breast and axillary dissection for lobular infiltrating carcinoma (lic) pt2 n2 er 50% pr 0% ki-67 10% her2 negative. a complete staging was performed, with abdominal ultrasound, chest radiography, and bone scan ; no metastases were found. epirubicine (90 mg / m) and cyclophosphamide (900 mg / m), days 121, chemotherapy was performed for 6 cycles as adjuvant therapy followed by endocrine therapy with tamoxifen 20 mg / die for five years. twelve years later, in october 2010, patient was hospitalised for severe fatigue, inappetence, nausea, anorexia, and bone pain. a total body ct scan and magnetic resonance imaging (mri) of the abdomen showed liver metastases and omogeneous thickening of the gastric wall. egds with biopsies was performed revealing the presence of carcinoma with solid - chord structure, whom morphology and immunophenotype were coherent with origin from the breast (er 40% pr negative and her2 negative)., we found 206 patients reported with gi tract involvement from breast cancer, from 1943 to june 2012. each part of the gi tract has been reported involved, from the tongue to the anum [186 ]. site of metastases, presentation 's symptoms (a patient could have more symptoms or more sites of metastasis), and other clinical data are reported in table 2.125 patients had metastases to the stomach (60%), 57 presenting with linitis plastica, 27 with obstruction / stenosis, 12 with ulcer erosion / bleeding, 11 with dyspepsia, 10 with pain as the main symptom, 2 with polypoid lesions, 2 with perforation, and in 4 cases presentations ' symptoms were not reported [1, 2, 2748].24 patients had metastases to the esophagus (12%), 20 presenting with stricture, 3 with achalasia, and 1 with nonspecific dysmotility [2, 826].23 patients had metastases to the colon (11%), 16 presenting with obstruction / stenosis, 2 with multiple strictures / diffuse infiltration, 2 with polypoid lesions, 1 with linitis plastica, 1 with asymptomatic abdominal mass, and in 1 case presentations ' symptoms were not reported [1, 2, 47, 53, 54, 6172].16 patients had metastases to the small intestine (8%), 12 presenting with obstruction / stenosis, 1 with multiple strictures / diffuse infiltration, 1 with peritonitis, 1 with bleeding, and in 1 case presentations ' symptoms were not reported [2, 4960].14 patients had metastases to the rectum (7%), 11 presenting with obstruction / stenosis, 1 with linitis plastica, 1 with abdominal pain, 1 with rectal bleeding [33, 39, 46, 56, 61, 7377].2 patients had metastases to the oropharynx (1%), 1 presenting with oropharyngeal dysphagia, and 1 with a tongue lesion [2, 7].2 patients had metastases to the anum (1%), 1 presenting with polypoid lesion, and in 1 case presentations ' symptoms were not reported [78, 79 ]. 125 patients had metastases to the stomach (60%), 57 presenting with linitis plastica, 27 with obstruction / stenosis, 12 with ulcer erosion / bleeding, 11 with dyspepsia, 10 with pain as the main symptom, 2 with polypoid lesions, 2 with perforation, and in 4 cases presentations ' symptoms were not reported [1, 2, 2748 ]. 24 patients had metastases to the esophagus (12%), 20 presenting with stricture, 3 with achalasia, and 1 with nonspecific dysmotility [2, 826 ]. 23 patients had metastases to the colon (11%), 16 presenting with obstruction / stenosis, 2 with multiple strictures / diffuse infiltration, 2 with polypoid lesions, 1 with linitis plastica, 1 with asymptomatic abdominal mass, and in 1 case presentations ' symptoms were not reported [1, 2, 47, 53, 54, 6172 ]. 16 patients had metastases to the small intestine (8%), 12 presenting with obstruction / stenosis, 1 with multiple strictures / diffuse infiltration, 1 with peritonitis, 1 with bleeding, and in 1 case presentations ' symptoms were not reported [2, 4960 ]. 14 patients had metastases to the rectum (7%), 11 presenting with obstruction / stenosis, 1 with linitis plastica, 1 with abdominal pain, 1 with rectal bleeding [33, 39, 46, 56, 61, 7377 ]. 2 patients had metastases to the oropharynx (1%), 1 presenting with oropharyngeal dysphagia, and 1 with a tongue lesion [2, 7 ]. 2 patients had metastases to the anum (1%), 1 presenting with polypoid lesion, and in 1 case presentations ' symptoms were not reported [78, 79 ]. in many reports data on treatment are lacking. the majority of the patients underwent chemotherapy and endocrine therapy, someone surgery, and few patients radiotherapy. in one case with esophageal involvement, esophageal dilatation was performed for stricture. in one case, breast cancer is the most common cancer in females and metastasizes frequently to lung, pleura, skin, bone, soft tissues, liver, surrenal gland and brain. the gi tract is an uncommonly reported site of metastatic disease. after reviewing the literature, except a series of 51 cases concerning a 20-year period, reports on this subject are poor and often limited to single case reports. spread to gi tract seems to be more frequent in lobular histology ; the reason is unknown, but some authors think that it could be related to a particular tropism of lobular cells [2, 80 ]. pectasides. reported, from 1995 to 2008, 8 cases of gastric metastases from breast cancer ; the majority of the tumors had lobular histology. it must be emphasized that this localization can simulate a primary gastrointestinal cancer, and any region of gi tract can be involved, from the tongue to the anum. the most common type of gastric involvement is as linitis plastica appearance due to diffuse intramural infiltration of the stomach by the tumor ; this is characterized by a narrowing of the stomach lumen, rigidity, and diminished peristalsis, as reported in our case 5. other appearance of gastric metastases includes nodular or polypoid lesions and ulcerated lesions ct scan can be useful for noninvasive assessment for estimation of the degree of tumor penetration through the gastrointestinal wall as demonstrated in our patient no. 5. with modern multiphase, multidetector spinal ct imaging, there is increased accuracy in the assessment of mural penetration [87, 88 ]. metastases to gi tract can be the first manifestation of breast cancer metastases, as well as it can represent relapse even after many years from the diagnosis of the primary tumor, as in our case 3. surgery is often needed for diagnosis and treatment of complications, like intestinal obstruction or bleeding. the most common site of gi involvement from breast cancer observed in our series, like in the literature, was the stomach (3 cases), while in 1 case the disease was localized to the jejunum, and in 1 to the rectum. colon involvement is quite common, while small intestine involvement is very rare and diagnosed very late, frequently at autopsy. this clinical presentation may be aspecific : abdominal pain and diarrhoea, intussusception or pain - simulating appendicitis ; bleeding may be the only clinical manifestation of jejunum involvement, like patient number 1 of our series. colon involvement from breast cancer can simulate a primary cancer of the colon or a crohn 's disease, both clinically and at imaging studies. oropharyngeal and esophageal involvement is rare but possible, presenting in general as dysphagia or pseudoachalasia [2, 825, 31 ]. a consistent finding in most reports is the overrepresentation of the infrequent lobular type of breast carcinoma in gi metastases [1, 61 ]. several clinical and autopsy series [5, 83, 84 ] have examined the differences in the pattern of metastases of lobular and ductal breast carcinoma. a significantly higher prevalence of gastrointestinal and peritoneal metastases with lobular breast cancer has been found frequently ; even in cases in which primary tumor is of mixed histology, it is the lobular component that tends to metastasize to the stomach [28, 33 ]. it has been suggested that ductal carcinomas tend to produce nodular stomach lesions, while lobular carcinomas tend to cause more diffuse disease. according to the data of the literature, in two of our 5 cases the histology was lic, two cases of dic histology, and in 1 case it was carcinoma with signet ring cells. the patients ' age varied, at the time of gi metastases diagnosis, from 40 to 75 years ; the time from diagnosis of the primitive tumor to gi metastases raised from 0 to 22 years in our series, like in the literature, and we found that a case of gastric metastasis from breast cancer occurred 30 years after surgery for primary tumor. in 4 out of 5 cases the diagnosis of gi relapse from breast cancer was made within few months (3 - 4) from symptoms ' appearance ; only in one case 18 months were needed to find gastric metastasis, and in this case a first egds with biopsies performed after symptoms ' appearance was negative ; this can be related to the submucosal infiltration of the metastatic tumor. it must be emphasised that endoscopy remains today the best diagnostic method to evaluate pathology of the upper gastrointestinal tract. however, we recall to the mind that in some conditions endoscopy can allow false negative results, that is, when the tumor is submucosal, and in these conditions endoscopy can be repeated or combined with endoscopic ultrasound (eus). reorientation of the ultrasound transducer on the echoendoscope allows for fine - needle aspiration capabilities, leading to increase of diagnostic accuracy in cancer diagnosis and staging. in one case (jejunum localization) all the 5 patients, at the time of gi relapse, had symptoms due to the site of relapse : inappetence, epigastralgia, dyspepsia, nausea, and vomiting (the patients affected by gastric relapse), and bleeding (the patient affected by localization to the jejunum and rectum). after reviewing the literature, data on treatment are fragmentary, and however patients underwent surgery when possible ; the majority of the patients was treated with chemotherapy, one of them with endocrine therapy (especially after chemotherapy as maintenance therapy), and very rarely radiotherapy was performed. in 4 of 5 cases of our series, patients underwent chemotherapy, and 2 of them underwent subsequent maintenance hormonal therapy, whole one received also radiotherapy for rectal bleeding. one patient was treated only with hormonal therapy because of medical conditions suboptimal contraindicating chemotherapy. survival among reported cases in this paper varied from early deaths in patients reporting massive gastrointestinal bleeding to survival extended up to 44 months from the diagnosis of gi metastases. in our series, according to the literature, the median overall survival after a diagnosis of gi metastasis is 16 months (range, 541 months), which is a little lower than the median survival of all women with metastatic disease secondary to breast cancer (range, 2436 months). in conclusion, gi metastases are possible, even uncommon, and oncologists must consider it for patients affected by breast cancer, who develop gi symptoms, even if diagnosis and treatment for the primary tumour have been performed many years before. adequate diagnostic procedures, like ct scan, endoscopy with biopsy, or eus endoscopy with biopsy, must be performed as soon as possible, without delaying, to obtain a definite cytohistologic diagnosis allowing an adequate treatment for the patients. | luminal gastrointestinal (gi) metastases from breast cancer are rare, reports are fragmentary and poor. the purposes of this study are to assess the gastrointestinal involvement from breast cancer in a retrospective study at a single institution and reviewing the related literature. between january 2007 and december 2011 a total of 980 patients with breast cancer were treated at our institution, patients ' records and report database were analysed. institutional review board approval was obtained for this study. a search of the literature using pubmed, cancerlit, embase, was performed. selected for the present review were papers published in english before june 2012. five of 980 patients (0.5%) showed gastrointestinal metastases from breast cancer, 3 patients had gastric involvement, 1 jejunum, and 1 rectum. reviewing the literature, 206 patients affected by gastrointestinal metastasis from breast cancer were identified : the most frequent site of metastasis was the stomach (60%). the majority of the patients underwent chemotherapy and endocrine therapy, someone surgery and radiotherapy. gi metastases from breast cancer are rare, but possible, and a very late recurrence can also occur. cyto - histological diagnosis is mandatory, to differentiate gi metastases from breast cancer to other diseases and to allow an adequate treatment. |
very low birth weight (vlbw) infants commonly receive one or more red blood cell (rbc) transfusions during their first months of life. in the majority of preterm infants, anemia is a result of multiple factors which include iatrogenic blood loss, inadequate rbc production, shorter rbc lifespan, insufficient iron stores, and rapid growth. recombinant human erythropoietin (r - huepo) therapy has been suggested as an alternative to rbc transfusion in the treatment of neonatal anemia. although meta - analysis of controlled clinical studies has shown some benefit in the prevention of rbc transfusions even in extremely low birth weight infants (1 week, hematocrit > 30%, phlebotomy loss averaging 20 kcal / kg / day and stable respiratory status defined as mean oxygen saturation > 90% in a fraction of inspired oxygen 0.40 for > 48 h regardless of ventilation. infants excluded were those who received rbc transfusions 3 days before study entry or during the study, those with an active, culture - proven infection, a major malformation, electroencephalographic - proven seizures, hypertension, intraventricular hemorrhage grade iii, immune hemolytic disease, necrotizing enterocolitis, or surgery. among the study participants who began the study, 3 infants with sepsis or sepsis - like episodes, 2 with necrotizing enterocolitis, 2 who received rbc transfusions were excluded. there were no statistical differences among the three study groups for common neonatal morbidities including respiratory distress syndrome (p = 0.65), bronchopulmonary dysplasia (oxygen dependency at 36 weeks of postconceptional age ; p = 0.31), patent ductus arteriosus (p = 0.60) and retinopathy of prematurity (p = 0.32). there were also no significant differences identified among the three study groups for birth weight or in age, body weight, or phlebotomy loss at the prior to and during the treatment period (table 1). the 21-day study consisted of a 3-day run - in baseline period during which all subjects received 9 mg / kg / day of iron polymaltose complex (ipc ; ferrum hausmann syrup, vifor international, st. study participants were randomized to receive 18 days of oral iron alone (group 1 : oral iron ; control group), r - huepo + oral iron (group 2 : epo + oral iron), or r - huepo + intravenous (iv) iron (group 3 : epo + iv iron) treatment beginning after a 3-day run - in period (table 2). hematological parameters and the iron status (stfr, ferritin) were determined on peripheral venous or arterial blood drawn on treatment days 3, 0, 4, 11, and 18. blood counts, reticulocyte counts, rbc and chr indices were performed on fresh whole blood samples by flow cytometry (technicon h.3 autoanalyzer, bayer austria gmbh, vienna, austria). ferritin was determined by a radioimmunoassay (bio - rad quantimmune ferritin irma, bio - rad diagnostics group, hercules, calif. transferrin was measured by an antigen - antibody turbidimetric method using rabbit human transferrin antiserum (boehringer mannheim, indianapolis, ind. the fi was calculated using the following equation : stfr [mg / l]/log ferritin [g / l ]. within - group comparisons were performed by paired - samples t test (two - tailed). between - group comparisons were performed by independent - samples t test and anova with bonferroni posthoc multiple comparisons test for normal distributed data or mann - whitney u test for non - parametric sample distribution. using the diagnostic plot to assess iron status relative to r - huepo and iron therapy, we retrospectively analyzed previously described data from a randomized controlled trial from clinically stable vlbw infants 1 week, hematocrit > 30%, phlebotomy loss averaging 20 kcal / kg / day and stable respiratory status defined as mean oxygen saturation > 90% in a fraction of inspired oxygen 0.40 for > 48 h regardless of ventilation. infants excluded were those who received rbc transfusions 3 days before study entry or during the study, those with an active, culture - proven infection, a major malformation, electroencephalographic - proven seizures, hypertension, intraventricular hemorrhage grade iii, immune hemolytic disease, necrotizing enterocolitis, or surgery. among the study participants who began the study, 3 infants with sepsis or sepsis - like episodes, 2 with necrotizing enterocolitis, 2 who received rbc transfusions were excluded. there were no statistical differences among the three study groups for common neonatal morbidities including respiratory distress syndrome (p = 0.65), bronchopulmonary dysplasia (oxygen dependency at 36 weeks of postconceptional age ; p = 0.31), patent ductus arteriosus (p = 0.60) and retinopathy of prematurity (p = 0.32). there were also no significant differences identified among the three study groups for birth weight or in age, body weight, or phlebotomy loss at the prior to and during the treatment period (table 1). the 21-day study consisted of a 3-day run - in baseline period during which all subjects received 9 mg / kg / day of iron polymaltose complex (ipc ; ferrum hausmann syrup, vifor international, st. study participants were randomized to receive 18 days of oral iron alone (group 1 : oral iron ; control group), r - huepo + oral iron (group 2 : epo + oral iron), or r - huepo + intravenous (iv) iron (group 3 : epo + iv iron) treatment beginning after a 3-day run - in period (table 2). hematological parameters and the iron status (stfr, ferritin) were determined on peripheral venous or arterial blood drawn on treatment days 3, 0, 4, 11, and 18. blood counts, reticulocyte counts, rbc and chr indices were performed on fresh whole blood samples by flow cytometry (technicon h.3 autoanalyzer, bayer austria gmbh, vienna, austria). ferritin was determined by a radioimmunoassay (bio - rad quantimmune ferritin irma, bio - rad diagnostics group, hercules, calif. transferrin was measured by an antigen - antibody turbidimetric method using rabbit human transferrin antiserum (boehringer mannheim, indianapolis, ind. the fi was calculated using the following equation : stfr [mg / l]/log ferritin [g / l ]. within - group comparisons were performed by paired - samples t test (two - tailed). between - group comparisons were performed by independent - samples t test and anova with bonferroni posthoc multiple comparisons test for normal distributed data or mann - whitney u test for non - parametric sample distribution. demographic and clinical features of the three study groups including gestational age and birth weight have been described previously and did not differ among the three groups at treatment day 3 or baseline. on day 0, none of the hematological or serological parameters demonstrated significance between group differences (table 3). as anticipated, subsequent hematological and serological parameters demonstrated significant differences among the groups according to their therapeutic assignment (fig. posttreatment changes in stfr concentration were significantly higher in group 2 (epo + oral iron) and group 3 (epo + iv iron) compared to group 1 (oral iron ; control group) (p < 0.01 ; fig. the stfr values increased significantly from baseline in both epo groups on days 11 and 18 compared to the control group. similarly, there was a posttreatment increase in the fi for both epo groups starting at day 11 (p < 0.05) compared to the control group (fig. a significant increase in chr occurred at day 18 (p < 0.05) in group 2, while in group 3 only a trend toward higher chr values was observed which did not quite achieve statistical significance (p = 0.059) (fig. 2c). beginning on treatment day 3 and continuing to day 18 at the end of treatment, serial iron status laboratory data for each group were plotted (chr vs. fi ; fig. as the study period progressed, the pattern of laboratory findings of group 1 infants differed significantly in the course of chr and fi in comparison to both r - huepo - treated groups. for group 1 infants, chr by the end of study declined into q4, the quadrant is indicative of functional iron - deficient anemia, e.g., for acd. in contrast, the two r - huepo - treated groups moved to the right, i.e. an increase in fi was observed. although the courses of group 2 and group 3 paralleled one another over time beginning with the initiation of r - huepo treatment, on day 18, group 2 infants (epo and oral iron) ended up in q2 (latent iron deficiency) while group 3 iv epo + iv iron - treated infants ended in q3 (iron - deficient anemia). the aim of the present study was to examine the clinical utility of using a diagnostic plot of thomas. to assess iron availability for erythropoiesis in vlbw infants with aop as an informative tool for clinicians. the data analyzed were derived from a previous study of vlbw premature infants treated with two different r - huepo and iron - dosing regimens used in the treatment of aop. this diagnostic plot utilizes two sensitive and specific parameters of the adequacy of iron for erythropoiesis for the x- and y - axes : fi and chr. the chr and the fi were used as the key hematological indicators for the intensity of the demand for iron and the adequacy of iron supply, respectively. the plot was used to identify the initial and subsequent erythropoietic and iron status of the three study groups that differed in the r - huepo and oral and parenteral iron supplementation each received. chr has been used in the diagnosis of different iron - deficient states and in the response to iron and/or r - huepo treatment. importantly, chr provides a direct assessment of the incorporation of iron into erythrocyte hemoglobin and thus serves as a direct indicator of the recent functional availability of iron for the erythron [13, 18 ]. stfr and zinc erythrocyte protoporphyrin were proposed as useful indicators for iron deficiency, because both parameters are directly associated with erythropoietic activity [10, 20, 21 ]. both have therefore been used to help identifying iron deficiency states in children [9, 22, 23 ]. the acute - phase reactant, ferritin, has primarily been used as an indicator of iron storage. in the absence of inflammatory disease, the stfr to ferritin ratio (fi) is inversely correlated with iron stores in various anemic states, including the acd and fid. in the previous study from which the present data were derived, we investigated the efficacy and safety of high doses of epo / iron therapy on the aop. in our application of the diagnostic plot of thomas. to the present data beginning on day 3, all three groups started in the upper left quadrant (q1) of the plot (fig. 3), i.e. indicating that baseline iron status of all three groups were adequate relative to their erythropoietic demand. we speculate that the control group became increasingly anemic as a result of multiple factors impacting iron status including : (1) low endogenous epo production, (2) diminished iron stores as a result of rapid growth, and (3) on going phlebotomy loss. we further speculated that the later two of these contributed to the decrease of chr observed in the control group despite receiving oral iron supplementation. without administration of epo, the course of the control group fell into the lower left quadrant (q4) of the diagnostic plot and remained there until the end of the treatment period on day 18. these results suggest that the group 1 control subjects receiving oral iron supplementation alone experienced both r - huepo and iron deficiency by the end of the study. this unanticipated finding that control group infants moved from their initial position in iron sufficient q1 to their final position at the end of the study in q4, characteristic of anemic conditions associated with fid / acd, is consistent with the findings of thomas.. on this point, we speculate that because the control group did not receive r - huepo treatment, iron was not incorporated in adequate amounts by reticulocytes. as a result, mean chr values for control subjects fell below the 28 pg cutoff value for q4. 200 l / l and a fi < 1.5, complete hemoglobinization did not occur. as such, treatment with r - huepo and additional iron supplementation might be considered as possible therapeutic considerations. in contrast to the control group, both r - huepo - treated groups had moved into quadrant q2 by treatment day 11. this can be interpreted as a successful response to the r - huepo therapy with a pronounced stimulation of erythropoiesis by the second week of treatment. no significant increase in chr was observed in the epo + iv iron group (p = 0.059) by day 18, while in the epo + oral iron group a slight, but significant, increase in chr was observed compared to the control group (p < 0.05). based on our diagnostic plot data and the recent review of iron supplementation in preterm infants by of ridley., it can be hypothesized that oral iron supplementation together with epo therapy is adequate clinical therapy for preterm infants during their period of aop which is in accordance with the considerations of ridley.. by day 18, the course of group 2 ended in q2, reflecting a latent iron deficiency. in contrast, the course of group 3 ended in q3, indicative of classic iron deficiency anemia. additional trials are needed to assess whether r - huepo administration might be stopped at this point and if iron therapy (oral and/or iv iron) alone is sufficient as a therapeutic intervention for patients entering q2 or q4 of the diagnostic plot. the diagnostic plot has served as a clinically useful tool in evaluating iron status of patients with renal anemia in which chr provides helpful information regarding fid in dialysis patients receiving erythropoiesis - stimulating agents. the diagnostic plot also appears helpful in monitoring iron therapy in cancer - related anemia. treatment of preterm infants with epo and iron is one therapeutic approach for aop exacerbated by phlebotomy (and therefore iron) loss. the diagnostic plot might also be helpful as a supporting diagnostic tool for clinicians seeking to identify the optimal r - huepo and iron - dosing regimens. the influence of supplementation with vitamin b12 and folic acid with or without r - huepo treatment and with iv iron to reduce the need for rbc transfusion is another example of a clinical situation that would be of interest to apply the diagnostic plot to determine if a positive effect in the stimulation of erythropoiesis is observed. a clinical study by worthington - white. indicated that premature infants require additionally vitamin b12 and folate, and it could be shown that the severity of anemia was reduced. in their study, infants treated with vitamin b12 alone or combined with folate had higher hemoglobin values than the untreated (p < 0.0005) or solely folate - treated (p < 0.01) groups. in future studies, it will be important to include untreated infants starting at birth when both ferritin and stfr levels are normally (i.e., in the absence of r - huepo and iv iron treatment and before receiving rbc transfusions) at their highest. our results indicate that the diagnostic plot developed by thomas. offers promise as a useful tool for assessing iron status in anemic preterm infants. in its reliance on laboratory determinations performed in most hospital laboratories, the diagnostic plot has the potential for providing a readily available, real - time indicator of iron status. this has particular relevance for anemic, rapidly growing low birth weight infants during treatment with erythropoiesis - stimulating factors which result in different degrees of iron demand for hemoglobin synthesis. additional studies carried out in a robust spectrum of clinical circumstances and other neonatal patient groups are needed before the clinical utility of this new diagnostic procedure can be unequivocally relied on. | backgroundin the early weeks of life, very low birth weight (vlbw) infants experience intense laboratory blood sampling leading to clinically significant anemia and the need for red blood cell transfusion. although controversial, treatment with recombinant human erythropoietin (epo) and iron has been recommended to stimulate erythropoiesis ; optimal dosing of epo and iron is still uncertain. objectives : to assess the validity of a four - quadrant diagnostic plot of iron availability (ferritin index) versus iron demand for erythropoiesis (reticulocyte hemoglobin content, chr) for differentiating iron status in anemic vlbw infants.methodsstudy subjects were enrolled in a previously reported randomized controlled trial of clinically stable vlbw infants < 31 weeks gestation and < 1,300 g at birth to receive 18 days of treatment with : group 1 : oral iron ; group 2 : epo + oral iron, and group 3 : epo + intravenous + oral iron.resultsat the end of treatment the ferritin index was significantly higher in both epo groups compared to the control group. by day 18, chr of the control group declined into the quadrant of the diagnostic plot characteristic of functional iron deficiency and anemia of chronic disease. both epo groups ended in the quadrants that are characteristic for latent iron deficiency and iron deficiency anemia, respectively.conclusionsthe diagnostic plot for differentiating anemia in vlbw infants may be an informative, clinically useful tool for iron status assessment under different physiologic and therapeutic erythropoietic states. larger additional studies in difficult patient populations are needed before the clinical utility of this diagnostic procedure can be unequivocally confirmed. |
alzheimer 's disease (ad) is a progressive neurological disorder resulting from the deposition of alzheimer -amyloid peptide (a) as senile plaques, the appearance of neurofibrillary tangles, and selective neuronal loss. the most abundant forms of a are 40 and 42 amino acid residues long and referred to as a40 and a42, respectively. the endocytic pathway has been implicated in the extracellular secretion of a40 and a42 [2, 3 ]. these peptides are derived from the endoproteolytic cleavage of the amyloid precursor protein (app) with -secretase, followed by -secretase. the endosomal contents can be either secreted from the cell [79 ] or transferred to the lysosome. the endosome has been found to be quite acidic (ph 6) with the recycled endosome slightly less acidic (ph 6.5) [11, 12 ]. exposure of a to endosomal ph conditions has been found to induce various conformational and oligomeric states [1315 ]. many oligomeric forms of a have been proposed and characterized as intermediates in the pathway to forming the amyloid fibre. some of these structures include trimers, pentamers, high molecular weight a-derived diffusible ligands (addls), protofibrils, and fibrils [1621 ]. here we present a method for generating a mixed population of a conformations using model endocytic conditions. using this method, we demonstrate that when a is exposed to endosomal conditions for an extended period of time, the ability of the peptide to bind and internalize into living rat adrenal pheochromocytoma (pc12) cells increases with time until it reaches a maximum beyond which its ability to interact with pc12 cells diminishes drastically. a40 and a42 were synthesized and purified as described previously. before cleavage from the resin, the fluorophore, n-(9-fluorenylmethoxycarbonyl)-n-tetramethylrhodamine-(5-carbonyl)-l - lysine (molecular probes, eugene, or) (abbreviated tmr), the crude peptides were purified by hplc using a superdex tricorn 10/300 gl peptide column (amersham biosciences, piscataway, nj) with 30 mm nh4oh running buffer. to maintain stock peptide solutions free from fibril seeds, solutions were stored at ph 10 and 4c immediately after chromatographic separation of monomeric peptides. a preparations were never lyophilized, as this process may allow for seeds to form. these solution conditions have been previously shown to maintain the monomeric state [16, 23, 24 ]. peptide purity and identity was confirmed using both maldi mass spectrometry and amino acid analysis. concentrations of stock peptide solutions were determined using amino acid analysis and confirmed by either tyrosine absorbance (275 nm, = 1390 cm m) or tmr absorbance for labelled peptides (550 nm, = 92000 cm m). at least three separate synthesized lots of a were used in this study and each displayed identical cell association rates when compared for quality assurance. oligomeric samples were prepared by diluting stock a samples to 30 m with 30 mm nh4oh and reducing to ph 6 with 0.2 m hcl and incubating for zero (a), 3 (a), 24 (a), or 90 hours (a) in the dark at 20c. hydrodynamic radius (rh) measurements were made at 20c with a dynapro dls instrument (protein solutions inc., peptide samples (30 m) were reduced to ph 6 using 0.2 mm hcl, centrifuged at 12000 g for 3 minutes and then rapidly added to a 1 cm path length cuvette and left in the instrument. dls data was collected at various time points over 90 hours. particle translational diffusion coefficients were calculated from decay curves of autocorrelation of light scattering data and converted to hydrodynamic radius (rh) with the stokes - einstein equation. histograms of intensity versus rh were calculated using dynamics data analysis software (protein solutions inc., piscataway, nj). a samples (30 m) were either not filtered or spin - filtered for 30 minutes at 14,000 g using 10, 30, 100 kda (amicon ultra cellulose kmwo), or 0.1 m (amicon pvdf) spin filters. absorbance at 550 nm was collected on a molecular devices spectramax m5 (molecular devices corp., sunnyvale, ca) and graphs were created normalizing the absorbance signal from each filtered sample to the corresponding unfiltered sample. fluorescence measurements were obtained using 200 l of 30 m a samples, within a 96-well plate, after addition of 5-fold molar excess of thioflavin - t and incubation at room temperature for 30 minutes. emission at 485 nm was collected using 440 nm excitation on a molecular devices spectramax m5 (molecular devices corp., pc12 cells were maintained in dmem / f12 containing 10% fetal bovine serum (hyclone, logan, ut) with 100 units / ml penicillin and 100 g / ml of streptomycin. to induce differentiation of pc12 cells and for cell imaging, they were plated at 2.2 10 cells / cm in lab - tech chambered cover glass chambers and suspended in phenol red free dmem / f12 containing n2 supplement and 10 ng / ml ngf. cells were differentiated for 72 hours before media was replaced and peptide treatments (final concentration 1.5 m) were performed. tmr - labelled a samples (6 m) were prepared in phenol red free dmem / f12 media containing n2 supplement and 10 ng / ml ngf. either fresh media or cultured supernatants, obtained from cell culture after 72 hours, were used. to avoid interference from cell culture components, molecular weights of tmr - labelled a were obtained by selective monitoring of tmr absorbance at 550 nm. sedimentation experiments were performed at 20c on a beckman xli analytical ultracentrifuge using an an50-ti rotor. molecular weights were calculated using beckman xli data analysis software in which absorbance versus radial position data were fitted to the sedimentation equilibrium equation using nonlinear least - squares fitting. three - dimensional stacks of fluorescence micrographs were taken at 20c with a confocal laser - scanning system consisting of an lsm 510 zeiss meta nlo confocal microscope with a c - apo 40x water immersion objective (numerical aperture 1.2) and hene laser with a 543 nm laser line. the displayed images were captured using zeiss lsm image version 4 and prepared using imagej version 1.37v and represent a single cross - section through the cells. differentiated pc12 cells were treated with 50 m monodansylcadaverine (mdc) for 20 minutes at 37c with 5% carbon dioxide. after inhibitor treatment, a (final concentration 1.5 m) was added to the media containing mdc and imaged after 4 hours at 37c. a (final concentration 1.5 m) was also added to differentiated pc12 cells media for 4 hours at either 37c or 4c prior to imaging. differentiated pc12 cells were first treated with a (final concentration 1.5 m) for 6 hours at 37c followed by treatment with 40 nm mitotracker deep red and 50 nm lysotracker green dnd-26 for 20 minutes. after treatment, the media was exchanged with fresh phenol red free dmem / f12 media containing n2 supplement and 10 ng / ml ngf with 1.5 m dapi for nuclear staining. cells were imaged using argon laser with 488 nm laser line for lysotracker, hene laser with 633 nm laser line for mitotracker, hene laser with 543 nm laser for tmr - labelled a, and a tunable chameleon laser at 730 nm for two - photon excitation of dapi. 2d histograms and correlation coefficients were determined using image j version 1.42q with colocalisation threshold plugin. differentiated pc12 cell media was replaced with media containing 0.6 to 20 m a, a, a, or 10 m melitin as a positive control for 48 hours. cell survival was quantified using the sulforhodamine b assay, and absorbance was measured at 560 nm using molecular devices spectramax m5 (molecular devices corp., sunnyvale, ca). lc50 values were determined as the concentration of a required to kill fifty percent of the cells from an absorbance versus a concentration plot. to study the interaction between a and live cells, we synthesized and fluorescently labelled a40/42. the synthesized a was maintained in solution from purification to storage and was never lyophilized, as these solution conditions are known to significantly reduce the formation of a aggregation seeds [16, 23, 24 ]. we covalently attached tetramethylrhodamine (tmr) to the n - terminus of a via a flexible glycine linker to generate tmr - a. the n - terminus of a is highly accessible even in the fibril state [21, 27, 28 ] and attaching a fluorescent label to this site has been shown to neither alter its amyloidogenic properties [16, 29, 30 ], nor its solubility behaviour [31, 32 ]. we have previously shown that treating cultured cells with 1.5 m monomeric tmr - a42 leads to the formation of visible aggregates on the surface of pc12 cells within one hour of treatment. we initiated our current study by investigating whether a aggregation could occur in cell culture media alone. using analytical ultracentrifugation, we measured the molecular weights of a40 and a42 present in phenol red free cell culture medium, both freshly prepared and conditioned media taken from differentiated pc12 cells 3 days postdifferentiation. following the addition of 6 m a to each medium and subsequent 24-hour incubation at room temperature, both media preparations were centrifuged to equilibrium at room temperature in an analytical ultracentrifuge in order to determine the molecular weight of a conformations. the molecular weights of a40 and a42 from both media preparations were measured to be approximately 4103 da and 4425 da, respectively. these values both correspond to the expected monomeric molecular weights of a, falling within the 95% confidence intervals of 36504570 da for a40 and 36504770 da for a42. thus, the aggregation of a seen by confocal microscopy apparently occurs only after interaction with the differentiated pc12 cells and not with cultured supernatants. it should be noted that the concentration previously used to treat cells (1.5 m) and the concentration used for ultracentrifugation (6 m) are considerably lower than the reported 20 to 50 m range required for in vitro aggregation [3335 ]. since a40 and a42 are generated through endoproteolytic cleavage [79 ], and the ph of the endosome and recycled cellular vesicles is equivalent to ph 6, we characterized the a conformations formed under endosome conditions. a (30 m) was reduced to ph 6, and hydrodynamic radius calculations were collected after zero (a), 3 (a), 24 (a), and 90 (a) hours using dynamic light scattering (figure 1). the average hydrodynamic radius of a40 was found to increase from 2.0 nm at time zero (a40) to 216 nm after 90 hours at ph 6 (a40). a more striking increase was found with a42, beginning with 1.7 nm at time zero (a42) to 451 nm after 90 hours at ph 6 (a42). for each of these samples, the development of increasingly higher ordered aggregates was observed over time and the samples that were treated for 90 hours contained particles over 1000 nm in radii. to further investigate the relative levels of peptide aggregation at endocytic ph, we filtered the a40 and a42 samples through various molecular weight cutoff (mwco) spin filters (figure 2). approximately 67% of a40, 60% of a40, and 55% of a40 were recovered through the 10 kda mwco spin filter and approximately 80% were recovered through the 100 kda mwco filter, except for a40 with only 60% recovered. in contrast, only 55% of a42, a42, and a42 were recovered through the 10 kda filter and approximately 60% were recovered through the 100 nm filter. these results indicate that the majority of peptide conformations present under these conditions were able to pass through a 10 kda molecular weight filter, but that just over 40% could not be recovered through the 100 nm filter for the a40, a42, a42, and a42 samples. we also used thioflavin - t to assess the time - dependence of the extent of amyloid fibril formation at endocytic ph. thioflavin - t is a dye known to shift its fluorescence from 430 nm to 490 nm upon binding specifically to the cross--structure of amyloids but not to monomeric or small oligomeric complexes [36, 37 ]. we observed enhanced thioflavin - t fluorescence at all time points (figure 3) ; however, thioflavin - t bound most strongly to a40, a42, and a42. the high thioflavin - t binding to a samples suggests that these late stage a conformations are the most aggregated. using confocal microscopy we have previously shown that monomeric a42 associates with cells more rapidly than a40, with significant staining observable after six hours of treatment. to determine whether the aggregation state of a affects cell association, we exposed each of the a40 and a42 samples to differentiated pc12 cells and monitored the kinetics of association by confocal microscopy. upon treating cells with a40, the cell surface association moreover, a40 was observed to significantly internalize into these cells after only 6 hours, whereas significant internalization of a40 was only visualized around 24 hours (figure 4). similarly, a42 internalized into differentiated pc12 cells after only one hour of treatment, whereas a42 treatment only became observable at 24 hours (figure 4). interestingly, when differentiated pc12 cells were treated with late - stage a40 or a42, very few cells underwent internalization or even exhibited cell surface interaction with these aggregated peptide forms (figure 4). to illustrate the contrast between these treatments, we collected the three - dimensional image slices through a cell from the base to the apex for the 6-hour treatment with a42, a42, and a42 (figure 5). a42 was only observed around the periphery of the cell, whereas some a42 was located inside the cell. by contrast, a42 treatment seemed to localize to extracellular regions and did not produce the punctate pattern as observed with the a42 sample. to quantify the frequency of cells that exhibited peptide internalization, we randomly selected five fields of view from at least three separate 6-hour treatment experiments and plotted the percentage of cells having internalized a (figure 6). was found to have a large number of aggregates with hydrodynamic radius over 1000 nm (figure 1) and were found to significantly bind thioflavin - t (figure 3), then these aggregates may favour self - association over cell association. to determine if the internalization of endocytic a is mediated through cellular processes, such as receptor - mediated endocytosis or through direct peptide - mediated processes like membrane pore formation, we monitored the effects of temperature and monodansylcadaverine (mdc) on internalization. mdc is a known inhibitor of receptor - mediated clathrin - dependent endocytosis [38, 39 ]. at physiological temperature (37c), after a 4-hour treatment of cells with a40 and a42, internalization was observed (figure 7). at 4c membrane vesicle formation when a40 or a42 association with differentiated pc12 cells was monitored at 4c, none of the cells were found to have internalized these peptides (figure 7). similarly, when differentiated pc12 cells were treated with mdc, neither a40 nor a42 were observed within the cells. these observations indicated that a was internalized through a cell - directed import mechanism, rather than an independent penetration route through the cell membrane. the location of deposited intracellular a40/42 was examined using intracellular organelle markers in differentiated pc12 cells. cells treated with a40 and a42 for 6 hours were visualized using lysotraker green dnd-26 for the lysosome, mitotracker deep red for the mitochondria, and dapi for nucleus staining (figure 8). the staining pattern of each cellular organelle marker in the same image plane as the a40 and a42 treatment was determined (figure 8) and quantified (figure 9). only the signal from the lysosome marker costained with both internalized a40 and a42. the red signal from the peptides costained well with green signal from the lysosomes, resulting in the yellow signal in the merged image. when quantified, 71% of the lysosome signal intensity colocalised with the a40 channel and 80% with the a42 channel (figure 9). whereas only 9% of the mitrochondria signal intensity colocalised with a40 channel and less than 1% with the a42 channel (figure 9). the intensity of both a40 and a42 fluorescence signals was found to increase in the lysosomes over time, which may reflect the accumulation of a40/42 in the lysosome. to determine the relative toxicity of these a conformations, a cytotoxicity assay was performed where differentiated pc12 cells were treated with various peptide concentrations for 48 hours (figure 10). we calculated the lethal concentration at which 50% of cells were killed (lc50), for each of the a conformations. we found that a40 was moderately more toxic than a40, with a lower lc50 value (figure 10). surprisingly, a40 was also found to have a lower lc50 value compared to a40. a42 was also found to be moderately more toxic than a42, whereas a42 had a much higher lc50 value compared to a42 (figure 10). we have developed a method to produce a collection of a conformations that differ in their extent of aggregation and investigated the interaction between these states of a40 and a42 and differentiated pc12 cells. others have described the methods to isolate individual soluble oligomeric forms of a, using various chemical reagents and protocols [4144 ]. in addition, purified oligomeric a molecules from cell culture [45, 46 ] or transgenic mice have also been monitored. these purified sources of oligomeric a offer great potential in understanding the progression of a aggregation ; however, they can not be directly visualized over the time course of their effects on cells during maturation from earlier to later conformations. since it is well established that a monomers can oligomerize under the physiological ph of the endosome [1315 ], and a is formed through endoproteolytic cleavage, we have utilized this condition to determine which conformations are present and how these mixed conformations interact with differentiated pc12 cells. it is known that the extent of oligomerization / fibrillation is very dependent on experimental conditions. have indicated that a can be induced to form fibrils via dilution from 100 mm naoh to neutral ph in the presence of 10 mm hepes/100 mm nacl buffer, while dilution into phosphate buffered saline results in oligomer formation. in our experiments, the peptide was diluted from 30 mm ammonium hydroxide (ph 10) to ph 6 with final condition of 1 mm ammonium chloride as the only additional chemical. we do note that the normal human physiological concentration of ammonium chloride in blood and cerebrospinal fluid is approximately 20 to 50 m [49, 50 ], and that hyperammonemia has been linking to alzheimer type ii astrocytosis [51, 52 ]. when extracellular monomeric a associates to the surface of cells, we speculate there are three possible outcomes : (1) it can act as a stable template to allow further a aggregation ; (2) the peptide can penetrate through the cell membrane depositing in the cytoplasm ; or (3) the peptide may be internalized into the cell within endocytic vesicles, which would result in a reduction in the surrounding ph. in the third case, the endocytic vesicles containing a can theoretically be recycled back to the cell surface or directed to the lysosome where a further reduction in ph would occur. we have previously visualized oligomeric a on the surface of neuronal cell lines, and here sought to determine first whether this aggregation occurred prior to cell surface deposition in the cell culture mediaor are aggregation and cell surface binding concomitant and linked processes.using ultracentrifugation analysis of a preparations either in freshly prepared cell culture media or in conditioned media removed from cultured cells after 3 days, we did not observe any conformation larger than the monomer. these results indicate that components in our cell media or secreted factors from cultured cells are not responsible for the observed a aggregation present on the surface of neuronal cells. after adding monomeric a to cells, we noted a maturation time for the visual appearance of a on the cell surface and in the cell interior (figure 4 ;). we postulate that the peptides on the cell surface might undergo a series of ph reductions through endocytic recycling, before becoming visible punctae on and inside the cell. since ph 6 is approximately endocytic ph, we characterized the kinetic effects of this condition on peptide conformations. exposing a40 for up to 24 hours at ph 6, we observed an increase in the average hydrodynamic radius from 2.0 nm (a40) to 183 nm (a40) (figure 1). in addition, approximately 60% of these peptides were recovered through a 10 kda mwco filter and 80% were recovered through a 100 kda mwco filter (figure 2), and only minor thioflavin - t binding was found (figure 3). following exposure of up to 24 hours at ph 6, a42 had an increase in the average hydrodynamic radius from 1.7 nm (a42) to 246 nm (a42) (figure 1), approximately 55% were recovered through 10 kda mwco filter and only 60% were recovered through 100 nm filter (figure 2) and more significant thioflavin - t binding was measured (figure 3). both a40 and a42 treated for 90 hours at ph 6 were observed to have large average hydrodynamic radius particles, with 40% of these samples being withheld by a 100 nm filter and both displayed significant thioflavin - t staining. also, these early - and late - stage a samples were observed to have an ensemble of a conformations (figure 1). the interactions of these early - and late - stage a conformations were monitored with differentiated pc12 cells. whereas late stage a40 and a42 exhibited very few interactions with cells (figures 4, 5) and internalized at a very low frequency (figure 6), early stage a40 and a42 interacted with cells rapidly (figure 4) and internalized into the majority of the cells present (figure 6). interestingly, a40 showed a more rapid cellular association and internalization compared to a40 (figure 4). similarly, a42 was found to internalize more rapidly than a42, indicating that a specific conformation may play a role in cell binding and internalization. furthermore, our model of endocytic a toxicity (figure 10) is in agreement with previous findings utilizing chemically - produced oligomers of a which showed that a fibrils were less toxic than soluble oligomers [41, 53 ]. our results with a are also in agreement with studies that have indicated that regions with large amyloid plaques do not correlate directly with regions of significant neuronal loss [54, 55 ]. for example, the peptide sequences corresponding to tat (4860), penetratin, and oligoarginine are known to internalize into live cells. cellular import of tat (4860) and penetratin was shown to be temperature dependent, indicating the possible role of endocytosis for internalization [56, 57 ]. in contrast, oligoarginine containing c - terminal tryptophan was shown to follow a nonendocytic pathway, independent of energy requirements and temperature. another type of internalization process has been studied in the bacterium clostridium septicum, whose alpha toxin contains functional domains responsible for oligomerization and cellular pore formation. using specific domains that bind cell surface receptors, alpha toxin monomers oligomerize to form pores in human cells and thus impose direct entry. by exposing a to endosomal conditions, we were able to observe internalization of a40/42, which was inhibited at 4c or with the endocytosis inhibitor mdc (figure 7). throughout the course of our confocal experiments, we did not observe any disruption of the cell membrane, which would have been expected if membrane channels or pores were being formed by a. thus, the structural conformation of a that was observed to internalize into cells seems to follow a cell - mediated import mechanism. our results with the various a conformations correlate directly with previous reports that indicate the deposition of a in the lysosome [15, 59, 60 ] and with the previous study that internalized a can persist undegraded for days. from our findings taken together with previous studies, we present the following model for the interaction of a with neuronal cells (figure 11). as monomeric a binds the cell surface, this event could lead to a conformational change allowing for the further catalytic aggregation of a. recently, it has been shown that oligomeric a binds to gm1 ganglioside and alters physical properties of the plasma membrane which stimulates the amyloidogenic processing pathway of app [62, 63 ]. a that is bound to the cell surface may become internalized and recycled, allowing for acidification and further aggregation, which in turn may stimulate additional a generation. once the surface a reach some specific structural form, they may be internalized and directed to the lysosome for potential degradation. however, the reduced ph of the lysosome may instead cause further aggregation and persistence of a, resulting in lysosomal overload and cell death. the recently reported high turnover rate of monomeric a [64, 65 ] and the formation of large aggregate pools of a may indicate that the human body uses these mechanisms to sequester and remove the toxic a oligomers from neuronal cells. with age, the turnover rate for monomeric a may slow down, which could then result in the initiation of our proposed mechanism and may contribute to the late onset of alzheimer 's disease. it is also possible that endocytosis of a and lysosomal targeting are mechanisms by which the cell clears a aggregates from the cell surface. also, certain large aggregates of a may just be too large for the cell to internalize, and they may represent the precursor for extracellular amyloid formation. our study shows that using a simple method to generate various a conformations, the rates of cellular interaction and targeting can be followed with live cell cultures. using this model, we found that early endocytic conformations, rather than highly aggregated late forms, serve as the major contributors to rapid cell internalization. the mechanism of internalization likely involves a cell surface receptor - mediated process instead of peptide - mediated direct entry, resulting in accumulation in the lysosome. this method allows for conformation - specific therapeutics and conditions to be screened with live cells, circumventing the need to purify specific a conformations. | the amyloid peptides, a40 and a42, are generated through endoproteolytic cleavage of the amyloid precursor protein. here we have developed a model to investigate the interaction of living cells with various forms of aggregated a40/42. after incubation at endosomal ph 6, we observed a variety of a conformations after 3 (a3), 24 (a24), and 90 hours (a90). both a4224 and a4024 were observed to rapidly bind and internalize into differentiated pc12 cells, leading to accumulation in the lysosome. in contrast, a40/4290 were both found to only weakly associate with cells, but were observed as the most aggregated using dynamic light scattering and thioflavin - t. internalization of a40/4224 was inhibited with treatment of monodansylcadaverine, an endocytosis inhibitor. these studies indicate that the ability of a40/42 to bind and internalize into living cells increases with degree of aggregation until it reaches a maximum beyond which its ability to interact with cells diminishes drastically. |
oxidative stress is high in patients treated in chronic hemodialysis (hd) program, as evidenced by increased lipid peroxidation, e.g. elevated malondialdehyde (mda), while low antioxidant, e.g. decreased glutathione peroxidase levels [1 - 9 ]. furthermore, level of mda is significantly correlated with the severity of kidney dysfunction, as well as with the duration of hd program. moreover, actually, miller. (2006) found mda level being the best marker for risk estimation of cardiovascular events in patients being on long - term hd. mda levels were also associated with the antioxidant paraoxonase-1 activity suggesting that patient with chronic kidney disease exhibits an oxidant - antioxidant imbalance related to high levels of atherosclerotic risk factors. in opposite to mda levels, studies measuring reduced glutathione (gsh), as another marker of oxidative stress gave conflicting result in hd patients. (2008), in opposite, elevated gsh levels in kidney failure. in comparison with healthy subjects, among these patients (1993) showed a significantly lower activity of the enzyme glutathione peroxidase reverting back glutathione to reduced form (gsh) and an inverse correlation between mda and gsh suggesting the existence of a mainly intracellular oxidizing stress. regarding the catalase, in hd patients, as compared to controls, some authors demonstrated decreased [4, 6 ], others increased activity, while atamer. although adiponectin as an adipose tissue derived cytokine having insulin sensitizing, anti - inflammatory, and anti - atherosclerotic properties, has a controversial role in chronic renal diseases. some studies carried out in kidney failure revealed a negative correlation between the serum adiponectin and levels of oxidative stress markers, similarly to patients without kidney failure. an elevated serum level of this has been demonstrated in hd patients, probably due to both inadequate clearance, and enhanced synthesis / release of se - selectin. in cases without kidney failure, miller. demonstrated that the level of se - selectin correlated positively with body mass index (bmi) even if data were adjusted for age, sex, race, smoking habits, blood pressure, serum levels of lipids and insulin. however, the relations of these factors in hd patients are far from optimally clarified yet. our aim with this present study was to test further the possible associations of oxidative stress markers with other characteristics, e.g. se - selectin, and adiponectin. the study was carried out in accordance with the declaration of helsinki (2000) of the world medical association and approved by the local ethics committee. eighteen of hdps had polycystic kidney disease, 13 diabetic nephropathy, 12 glomerulonephritis, 11 pyelonephritis, 8 ischemic renal disease, 4 idiopathic kidney atrophy, 2 nephrotic syndrome and 2 had earlier nephrectomy. none of the patients involved in the study had evidence of liver disease defined as alanine aminotransferase or aspartate aminotransferase > 1.5 times the upper limit of normal range, thyroid disorders, infectious diseases or significant inflammation for 3 months prior to study, malignancy, coronary artery disease, blood pressure > 160/100 mmhg, cerebral vascular disease, smoking, triglyceride levels higher than 4.5 mmol / l, alcoholism, drug dependence, pregnancy or lactation, anticoagulant, lipid lowering, glucocorticoid, oral contraceptive or sex hormone replacement medication. the form of hemodialysed treatment was hemodiafiltration in every patient, except two of them in whom high flux hemodialysis treatment alone could be applied. kt / v (dialysis efficiency) was calculated by the use of kt / v = - in (r 0.03 - 0.075 x uf / w) equation, where r = ratio of blood urea concentration measured after and before dialysis, uf = volume of ultrafiltrate (i), w = body weight after dialysis (kg). mean (lower / upper quartile) hd time of patients was 38 (16.7 / 62.7) months. blood specimens for investigations were obtained before the dialysis sessions. for routine automated laboratory analyses the insulin resistance index calculation was based on homeostasis model assessment (homa - ir). commercially available sandwich enzyme immunoassays were used for the determination of serum concentration of adiponectin, and se - selectin (quantikine, r&d systems, minneapolis, mn, usa), and colorimetric assay for the measurement of total peroxidase activity (oxystat, biomedica medizinprodukte gmbh & co kg, wien, austria). plasma levels of mda were estimated by the thiobarbituric acid reactive substances (tbars). for this basically the matkovics method was used : whole blood was precipitated with a mixture of tca and tba and boiled, and the supernatant was measured by photometric method at 532 nm expressing the results in nmol / ml. for the determination of blood gsh level, at first tca was added, and then dtnb (5,5-dithiobis 2 nitrobenzoic acid [serva 20735 ]) to the supernatant of a low - temperature centrifugation. photometric measurement was done at 412 nm expressing the results of gsh in nmol / ml. during the determination of catalase enzyme activity from blood the decrease of peroxide concentration spectrophotometry on perkin - elmer spectrophotometer) getting the results in bu / ml, where 1 bu means 1 g peroxide destruction per minute. data were expressed as means s.d. in case of normal distribution, and median (lower / upper quartile) in case of non - normal distribution. analyses of univariate correlations were done by pearson s test and multivariate correlations were assessed by backward multiple regression analyses. the study was carried out in accordance with the declaration of helsinki (2000) of the world medical association and approved by the local ethics committee. eighteen of hdps had polycystic kidney disease, 13 diabetic nephropathy, 12 glomerulonephritis, 11 pyelonephritis, 8 ischemic renal disease, 4 idiopathic kidney atrophy, 2 nephrotic syndrome and 2 had earlier nephrectomy. none of the patients involved in the study had evidence of liver disease defined as alanine aminotransferase or aspartate aminotransferase > 1.5 times the upper limit of normal range, thyroid disorders, infectious diseases or significant inflammation for 3 months prior to study, malignancy, coronary artery disease, blood pressure > 160/100 mmhg, cerebral vascular disease, smoking, triglyceride levels higher than 4.5 mmol / l, alcoholism, drug dependence, pregnancy or lactation, anticoagulant, lipid lowering, glucocorticoid, oral contraceptive or sex hormone replacement medication. the form of hemodialysed treatment was hemodiafiltration in every patient, except two of them in whom high flux hemodialysis treatment alone could be applied. kt / v (dialysis efficiency) was calculated by the use of kt / v = - in (r 0.03 - 0.075 x uf / w) equation, where r = ratio of blood urea concentration measured after and before dialysis, uf = volume of ultrafiltrate (i), w = body weight after dialysis (kg). mean (lower / upper quartile) hd time of patients was 38 (16.7 / 62.7) months. blood specimens for investigations were obtained before the dialysis sessions. for routine automated laboratory analyses cobas integra 700 autoanalyzer was used (roche, schwitzerland). the insulin resistance index calculation was based on homeostasis model assessment (homa - ir). commercially available sandwich enzyme immunoassays were used for the determination of serum concentration of adiponectin, and se - selectin (quantikine, r&d systems, minneapolis, mn, usa), and colorimetric assay for the measurement of total peroxidase activity (oxystat, biomedica medizinprodukte gmbh & co kg, wien, austria). plasma levels of mda were estimated by the thiobarbituric acid reactive substances (tbars). for this basically the matkovics method was used : whole blood was precipitated with a mixture of tca and tba and boiled, and the supernatant was measured by photometric method at 532 nm expressing the results in nmol / ml. for the determination of blood gsh level, at first tca was added, and then dtnb (5,5-dithiobis 2 nitrobenzoic acid [serva 20735 ]) to the supernatant of a low - temperature centrifugation. photometric measurement was done at 412 nm expressing the results of gsh in nmol / ml. during the determination of catalase enzyme activity from blood the decrease of peroxide concentration spectrophotometry on perkin - elmer spectrophotometer) getting the results in bu / ml, where 1 bu means 1 g peroxide destruction per minute. data were expressed as means s.d. in case of normal distribution, and median (lower / upper quartile) in case of non - normal distribution. analyses of univariate correlations were done by pearson s test and multivariate correlations were assessed by backward multiple regression analyses. the following parameters had to be transformed logarithmically : hd time, diastolic blood pressure (bp), plasma glucose, homa - ir, hdl - cholesterol (hdl - c), adiponectin, mda, gsh, catalase and total peroxidase. pearson s correlations of oxidative stress markers and se - selectin with selected variables having special interest are demonstrated in table 2 and 3, respectively. between the oxidative stress markers there was a weak internal association (actually, only catalase and mda were associated to each other s). of the relations of oxidative stress markers with clinical and/or laboratory parameters, it was the gsh that showed correlation : a negative one with bp values and a positive one with hdl - c level, as expected. interestingly, the level of se - selectin showed correlation only with the age, namely an inverse one. multivariate correlations. investigating the inverse connection between the age and level of se - selectin, the latter was a dependent variable in a multiple regression model in which anthropometric, blood pressure and other laboratory variables were included. during these analyses, only age turned out being significant independent predictor of se - selectin (table 4). we applied multiple biochemical tests to approach the oxidative stress in order to eliminate false results. moreover, the expected internal association between these markers helped us to exclude methodological troubles related pitfalls. despite these, we found no strong relationships between the oxidative stress and the other investigated cardiometabolic variables. reduced glutathione as an antioxidant, as expected, showed correlations with other cardiovascular risk factors, namely, negative ones with both systolic and diastolic bp values, while a positive one with hdl - c. very few studies evaluated the coordinated behavior of oxidative stress markers, se - selectin and adiponectin in hemodialysed individuals. we could not confirm lim.s observation related to a negative correlation between the levels of oxidative stress markers and serum adiponectin in patients with kidney failure. in our population no significant relation of adiponectin to any of the investigated oxidative variables could be demonstrated. a novel finding of our work is a negative and independent correlation between age and se - selectin level. this is in contrast with a study carried out in patients with preserved kidney function that demostrated a positive correlation between se - selectin and age. currently our observation can not be convincingly explained by the available information, but it may be related to a profoundly different patient population. our negative results might derive from the limited number of investigated subjects and low statistical power. however, the size of our study group (n=70) was typical for such a study and in accordance to the capacity of a single hemodialysis centre. in kidney failure and hemodialysis of the biochemical cardiovascular risk markers those related to oxidative stress, endothelial dysfunction, or altered adipokine homeostasis are not necessarily strongly associated. larger studies may be needed to confirm our novel observation, a negative and independent correlation of age to se - selectin level. | aims, subjects and methodsmarkers of oxidative stress and inflammatory activation of endothelium, as well as the adipose tissue secreted adipokines, e.g. adiponectin show altered pattern in renal failure. however, their internal relations have not been fully evaluated in this special patient population.in our cross sectional study, beside the routine clinical and biochemical parameters, plasma malondialdehyde, glutathione (gsh), catalase, total peroxidase, as well as serum e - selectin and adiponectin were measured in 70 hemodialysed (hd) patients.resultsgsh showed negative correlations with systolic and diastolic blood pressure (bp) values, while a positive one with hdl - cholesterol level, as expected. interestingly, the level of se - selectin was inversely correlated only with the age. in multiple regression analyses where anthropometric, bp and laboratory parameters were included and se - selectin was the dependent variable, the inverse association between the age and level of se - selectin turned out being an independent factor.conclusionsin hd kidney failure patients of the biochemical cardiovascular risk markers those related to oxidative stress, endothelial dysfunction, or altered adipokine homeostasis are not necessarily strongly associated. larger studies may be needed to confirm our novel observation, a negative and independent correlation of age to se - selectin level. |
a 37-year - old woman was referred to our institution for further management of a mass lesion located in the thoracic cavity, which was revealed on a chest computed tomography (ct) scan taken at another hospital. she complained of left - sided flank pain that was aggravated with deep inspiration for twelve months and began to experience dyspnea for one month. she had a history of pulmonary tuberculosis with a six - month course of anti - tuberculosis medication 20 years earlier. she did not have a history of smoking or environmental or occupational exposures to toxins. the initial chest radiograph taken at the other hospital 8 months previously demonstrated an ambiguous consolidation in the left lower lung without a definite mass lesion (fig. the patient did not receive treatment for the lesion but has been followed - up for eight months. a chest radiograph taken eight months after the initial visit revealed an irregularly shaped large mass occupying the left lower lung field and compressing the left lower lobe and diaphragm (fig. chest ct showed two irregularly shaped soft tissue masses adjacent to the parietal pleura in the left hemithorax, which contained dense calcifications without the invasion of the ribs (fig. 2). for correct diagnosis and proper treatment, surgical excision of the masses was performed through left thoracotomy via the 7th intercostal space. the intraoperative findings showed two hard and irregularly shaped masses with a hard osteoid surface. one was 8 cm in size and was located at the costophrenic angle, and the other was 4 cm in size, located adjacent to the inner surface of the shaft of the 6th rib. they had invaded the adjacent structures including the left lower lobe, chest wall, and the diaphragm, but had not invaded the adjacent ribs. these masses were radically excised involving the invaded organs to obtain adequate resection margins (fig. 3), and the defect of the diaphragm was reconstructed with a 2-mm - thick polytetrafluoroethylene (gore - tex wl gore and associates inc., the microscopic findings (h&e stain) showed the histologic appearance of the mass (fig. differences in nuclei size and shape were also observed in the h&e stain. in the immunohistochemistry stain, smooth muscle actin was detected. the microscopic findings of the two masses were the same, and therefore, these were considered seeding. the patient underwent four cycles of adjuvant chemotherapy with the regimen of high - dose methotrexate, cisplatin, and doxorubicin. currently, she is doing well without any evidence of recurrence for 14 months after surgery. extraskeletal osteosarcoma is a rare malignancy that accounts for approximately 1% to 2% of all soft tissue sarcomas and 4% of all osteosarcomas. extraskeletal osteosarcoma is defined as the malignant tumor originating from the soft tissues without involvement of the bone and composed of malignant cells of an osteoblastic phenotype producing an osseous matrix. it dominantly occurs in middle - aged patients in contrast to osteogenic sarcoma, which commonly occurs in younger patients under the age of 20 years, and the tumor can cause symptoms by its mass effect. approximately half the cases of this tumor occur in the lower extremity, and the other half frequently occur in the upper extremity, retroperitoneum, and the trunk. extraskeletal osteosarcoma rarely occurs in the pleural cavity, and a majority of these tumors arise from the diaphragm. this case is the first report on extraskeletal osteosarcoma that originated from the pleura in korea. radiologically, extraskeletal osteosarcoma often appears as a mass with soft - tissue opacity and a diverse degree of mineralization on the chest radiograph. in approximately 50% of the patients with this tumor, calcification or osteoid formation, the tumor is found to be separate from the adjacent osseous structures and has a pseudocapsule. however, differential diagnosis of extraskeletal osteosarcoma by a ct scan is generally difficult even though the ct scan can provide precise images, because calcification in the pleural cavity can be produced by other etiologies including benign and malignant diseases. the benign causes include trauma, infection, bleeding, calcifying fibrous pseudotumor, and calcified plaque by asbestos exposure, while the malignant causes include metastasis from osteogenic osteosarcoma, malignant pleural mesothelioma, parosteal osteosarcoma of the rib, and osseous paget s disease of the bone. therefore, comprehensive consideration including symptoms, occupational history, and medical history is required for correct diagnosis. in general, the most common site of metastasis by osteogenic osteosarcoma is the lung (up to 88% of osteoid osteosarcoma). therefore, the possibility of pulmonary metastasis should be considered first for patients who present with pleural osteosarcoma. in our case, however, primary osteogenic osteosarcoma occurring in the other organs was not found. the tumor has been known for a high - grade malignancy with poor prognosis (5-year survival rate : 37%), and it commonly metastasizes to the distant organs. radical resection of the tumor has been considered the appropriate initial treatment because the tumor is not particularly chemosensitive or radiosensitive. therefore, the role of chemotherapy in the treatment of the tumor is still being debated. regarding the efficacy of chemotherapy, a retrospective clinical analysis reported the clinical outcomes of the patients receiving adjuvant chemotherapy for extraskeletal osteosarcoma that occurred mainly at the lower extremities. the cited study reported favorable prognosis of adjuvant chemotherapy for extraskeletal osteosarcoma. in our study, the patient is doing well with adjuvant chemotherapy. in conclusion, this is an extremely rare case of extraskeletal osteosarcoma that originated from the parietal pleura. although the mass was initially considered to be a scar of previous pulmonary tuberculosis, as in this case, the possibility of primary malignant tumor should be considered in patients who present with pleural calcification. if the mass is assessed to be resectable, surgical resection of the tumor can be the standard option for correct diagnosis as well as proper treatment. | a 37-year - old woman was referred to our institution for further management of a mass lesion located in the thoracic cavity. the mass had grown by more than 10 cm over the course of a year and was initially considered to be a scar from previous pulmonary tuberculosis at another hospital. the patient had complained of left - sided flank pain for a year and experienced dyspnea for one month. chest radiography and chest computed tomography revealed an irregular - shaped mass in the left mid to lower pleural cavity. the mass was widely excised through left thoracotomy. pathologic examination of the biopsy specimen revealed a malignant spindle cell tumor, which consisted of components of osteosarcoma, pleomorphic sarcoma, and leiomyosarcoma. the patient underwent adjuvant chemotherapy and has been doing well without any evidence of recurrence for 14 months. |
noninvasive pressure support ventilation (psv), delivered via a nose- or a face - mask, is widely used because it reduces patient work of breathing (wob) [13 ]. this is critical for patients with impaired lung mechanics resulting from lung diseases such as copd or ards. the effectiveness of psv depends on the ability of the ventilator to adjust breath - by - breath inspiratory flow to match the patient s inspiratory demand. juban. reported that some copd patients recruit their expiratory muscles to terminate inspiration when the ventilator was still inflating the lungs. however, chiumello. observed that the breathing rate and tidal volume could be modified by regulating the flow - cycling criteria in patients recovering from acute lung injury (ali). breathing rate, tidal volume, and inspiratory time depend on the level of pressure support (ps) and the individual patient s lung mechanical properties (impedance and muscle effort). each psv breath is flow - cycled and termination criteria can be either a fixed flow value (e.g., 5 l / min), or a percentage of the peak inspiratory flow (e.g., 25%), referred to as conventional cycling. the ability to adjust flow - cycling criteria to use either conventional or automated regulation software algorithms is now available in many devices. patient - ventilator asynchrony, a misalignment between the timing of the ventilator cycle and the patient s respiratory cycle, is very common during assisted ventilation. high levels of asynchrony may prolong the time required for ventilator support and subsequently result in further complications and prolonged weaning from mechanical ventilation [1012 ]. approaches to auto - adjust triggering and cycling have been developed to minimize these problems. the aim of this bench study was to evaluate and compare the effects on breathing pattern and patient - ventilator cycling synchronization between automated cycling and conventional flow - cycling protocols during psv. the stimulator settings used in this study are consistent with the protocol published by ferreira. the series 1101 lung simulator (hans rudolph inc., shawnee, ks, usa) is a computerized lung simulator that consists of a piston that moves inside a cylinder. compliance, resistance, and inspiratory muscle pressure profile (negative pressure created by the respiratory muscles) may be set by the user. the simulator was adjusted to simulate a patient or a healthy adult placed in a semi - reclined position (incline 45). three combinations of inspiratory resistance (rins), expiratory resistance (rexp), respiratory compliance (crs), and breathing rate (br) were set to simulate lung mechanics in a patient with copd (rins=20 cmh2o / ls, rexp=20 cmh2o / ls, crs=50 ml / cmh2o, br=15 bpm), a patient with ards (rins=5 cmh2o / ls, rexp=5 cmh2o / ls, crs=25 ml / cmh2o, br=30 bpm), and normal adult lungs (rins=5 cmh2o / ls, rexp=5 cmh2o / ls, crs=100 ml / cmh2o, br=15 bpm) [1618 ]. the inspiratory times of the 3 types of respiratory mechanics (1.0 s for copd, 0.8 s for ards, and 1.5 s for normal adult) were chosen to assess the effect of inspiratory time on triggering and cycling synchronization. patient inspiratory effort was set at 5 cmh2o in the 3 patterns of respiratory mechanics, and pressure reduction generated 300 ms after the onset of an occluded inspiratory effort was 3.6 cmh2o. the simulator incorporates user - controlled leaks by a plateau exhalation valve (pev). for this experiment, the leak flow was maintained at 2528 l / min during a peak airway pressure of 20 cmh2o. an endotracheal tubes (i d, 22 mm) inserted through the mouth was used to direct gas coming from the facemask to the simulator. a medium - sized oronasal facemask without an exhalation port (bestfit ; curative medical inc., santa clara, ca, usa) was affixed tightly to the head of the mannequin with standard straps. a leak of < 12 l / min was measured at 20 cmh2o of positive pressure when the pev was removed. two bilevel ventilators were compared using the lung simulator with a system leak : v60 (respironics ; murrysville, pa, usa) and flexo st 30 (curative medical inc., santa clara, ca, usa). each ventilator was connected to the lung simulator by a standard disposable corrugated circuit (length, 2.0 m). humidifiers and heat and moisture exchangers were removed. both of the ventilators were set in psv mode, as follows : positive end - expiratory pressure (peep), 5 cm h2o ; pressure support (ps) level, 5 cmh2o (normal adult) and 15 cmh2o (copd and ards) ; back - up respiratory rate, 10 breaths / min ; maximal duration of the inspiratory phase, 4.0 s. the trigger sensitivity was set to be as sensitive as possible while avoiding auto - triggering. the inspiratory rise time was set to 100 ms. once the baseline pressure had stabilized, at least 5 min was allowed for the ventilator to synchronize with the simulator. if synchronization did not occur, changes in sensitivity, inspiratory effort, or both were made and recorded. if synchronization was not finally achieved, the ventilator was considered to be unable to provide assisted ventilation at the level of the leak. in all cases, failure to synchronize resulted in rapid auto - triggering or an inability to trigger. after stabilization, 10 representative breaths were collected with a sampling interval of 1 min. inspiratory triggering synchronization was assessed using the triggering delay (td), the triggering pressure - time product (ptpt). inspiratory time included ventilator insufflation time (ti vent), the time between the beginning of the simulated inspiratory effort and the end of the ventilator s insufflation, and ti simu, the simulated active inspiration time. cycling delay time (cdelay) was measured as the time from the end of the inspiratory effort of the simulator to the moment when the ventilator cycled from inspiration to expiration. a negative value reflects premature interruption of pressurization (premature cycling), and a positive value reflects a pressurization time that exceeds the patient s inspiratory effort (delayed cycling). the peak inspiratory flow (pif), the peak expiratory flow (pef), and the inspiratory tidal volume (vt insp) were monitored by the simulator. the expiratory tidal volume was measured by the ventilator (vt exp), and the leak volume (vt leak) was calculated from the difference of vt insp and vt exp. statistical analyses were carried out using the statistical software package, spss version 11.0 (spss ; chicago, il, usa). comparisons of variables at different cycling sensitivity settings were made using the t test. the stimulator settings used in this study are consistent with the protocol published by ferreira. the series 1101 lung simulator (hans rudolph inc., shawnee, ks, usa) is a computerized lung simulator that consists of a piston that moves inside a cylinder. compliance, resistance, and inspiratory muscle pressure profile (negative pressure created by the respiratory muscles) may be set by the user. the simulator was adjusted to simulate a patient or a healthy adult placed in a semi - reclined position (incline 45). three combinations of inspiratory resistance (rins), expiratory resistance (rexp), respiratory compliance (crs), and breathing rate (br) were set to simulate lung mechanics in a patient with copd (rins=20 cmh2o / ls, rexp=20 cmh2o / ls, crs=50 ml / cmh2o, br=15 bpm), a patient with ards (rins=5 cmh2o / ls, rexp=5 cmh2o / ls, crs=25 ml / cmh2o, br=30 bpm), and normal adult lungs (rins=5 cmh2o / ls, rexp=5 cmh2o / ls, crs=100 ml / cmh2o, br=15 bpm) [1618 ]. the inspiratory times of the 3 types of respiratory mechanics (1.0 s for copd, 0.8 s for ards, and 1.5 s for normal adult) were chosen to assess the effect of inspiratory time on triggering and cycling synchronization. patient inspiratory effort was set at 5 cmh2o in the 3 patterns of respiratory mechanics, and pressure reduction generated 300 ms after the onset of an occluded inspiratory effort was 3.6 cmh2o. the simulator incorporates user - controlled leaks by a plateau exhalation valve (pev). for this experiment, the leak flow was maintained at 2528 l / min during a peak airway pressure of 20 cmh2o. an endotracheal tubes (i d, 22 mm) inserted through the mouth was used to direct gas coming from the facemask to the simulator. a medium - sized oronasal facemask without an exhalation port (bestfit ; curative medical inc., santa clara, ca, usa) was affixed tightly to the head of the mannequin with standard straps. a leak of < 12 l / min was measured at 20 cmh2o of positive pressure when the pev was removed. two bilevel ventilators were compared using the lung simulator with a system leak : v60 (respironics ; murrysville, pa, usa) and flexo st 30 (curative medical inc., each ventilator was connected to the lung simulator by a standard disposable corrugated circuit (length, 2.0 m). humidifiers and heat and moisture exchangers were removed. both of the ventilators were set in psv mode, as follows : positive end - expiratory pressure (peep), 5 cm h2o ; pressure support (ps) level, 5 cmh2o (normal adult) and 15 cmh2o (copd and ards) ; back - up respiratory rate, 10 breaths / min ; maximal duration of the inspiratory phase, 4.0 s. the trigger sensitivity was set to be as sensitive as possible while avoiding auto - triggering. the inspiratory rise time was set to 100 ms once the baseline pressure had stabilized, at least 5 min was allowed for the ventilator to synchronize with the simulator. if synchronization did not occur, changes in sensitivity, inspiratory effort, or both were made and recorded. if synchronization was not finally achieved, the ventilator was considered to be unable to provide assisted ventilation at the level of the leak. in all cases, after stabilization, 10 representative breaths were collected with a sampling interval of 1 min. inspiratory triggering synchronization was assessed using the triggering delay (td), the triggering pressure - time product (ptpt). inspiratory time included ventilator insufflation time (ti vent), the time between the beginning of the simulated inspiratory effort and the end of the ventilator s insufflation, and ti simu, the simulated active inspiration time. cycling delay time (cdelay) was measured as the time from the end of the inspiratory effort of the simulator to the moment when the ventilator cycled from inspiration to expiration. a negative value reflects premature interruption of pressurization (premature cycling), and a positive value reflects a pressurization time that exceeds the patient s inspiratory effort (delayed cycling). the peak inspiratory flow (pif), the peak expiratory flow (pef), and the inspiratory tidal volume (vt insp) were monitored by the simulator. the expiratory tidal volume was measured by the ventilator (vt exp), and the leak volume (vt leak) was calculated from the difference of vt insp and vt exp. statistical analyses were carried out using the statistical software package, spss version 11.0 (spss ; chicago, il, usa). comparisons of variables at different cycling sensitivity settings were made using the t test. the respironics v60 and curative flexo st 30 ventilators were able to adapt to the system leak (2528 l / min) without requiring adjustment of the triggering settings. representative breathing cycles with auto - track setting in the 3 models are shown in figure 1. as shown in table 1, the vt leak differed under the cycling criteria in the copd model (p<0.001), which was the highest with the auto - trak cycling criteria (212.229.06 ml) and decreased with sensitivity level using conventional triggering (high : 106.427.35, moderate : 63.417.48, low : 25.2 v 7.16). the td was < 100 ms for the 2 ventilators in all respiratory mechanics models. for an inspiratory effort of 5 cmh2o, the ptpt was similar despite the modification of cycling criteria. higher values for pif were found during the auto - trak protocol than for conventional cycling criteria in copd and ards models (p<0.05 tables 1, 2). at 5 and 15 cmh2o of pressure support, the vt increased at lower cycling criteria sensitivity levels with conventional psv settings in all conditions. in the copd model, tinsp, pef and cdelay were also increased as a result of reducing the sensitivity level of the conventional cycling criteria (table 1). in the ards model, premature cycling was found during conventional protocols when cycling criteria was preset at the high and moderate levels. cycling delays only occurred at the lowest sensitivity level of the conventional cycling criteria (15.00.10 ms) and during auto - trak protocol (15.20.42 ms). in the normal adult model (table 3), tinsp was raised from 878.4019.02 ms to 1455.8020.95 ms after the cycling criteria was set at the lowest sensitivity level (p<0.05). premature cycling was also eliminated and a small delay in cycling (105.115.82) was found. premature cycling occurred during psv with the auto - trak system. according to the 3 respiratory mechanics models, the value of the inspiratory flow at the end of inspiration with the high, moderate, and low sensitivity levels for the cycling criteria during conventional protocols were approximately 35% pif, 15% pif, and 5% pif (figure 2). the td was < 100 ms for the 2 ventilators in all respiratory mechanics models. for an inspiratory effort of 5 cmh2o, the ptpt was similar despite the modification of cycling criteria. higher values for pif were found during the auto - trak protocol than for conventional cycling criteria in copd and ards models (p<0.05 tables 1, 2). at 5 and 15 cmh2o of pressure support, the vt increased at lower cycling criteria sensitivity levels with conventional psv settings in all conditions. in the copd model, tinsp, pef and cdelay were also increased as a result of reducing the sensitivity level of the conventional cycling criteria (table 1). in the ards model, similar outcomes premature cycling was found during conventional protocols when cycling criteria was preset at the high and moderate levels. cycling delays only occurred at the lowest sensitivity level of the conventional cycling criteria (15.00.10 ms) and during auto - trak protocol (15.20.42 ms). in the normal adult model (table 3), tinsp was raised from 878.4019.02 ms to 1455.8020.95 ms after the cycling criteria was set at the lowest sensitivity level (p<0.05). premature cycling was also eliminated and a small delay in cycling (105.115.82) was found. according to the 3 respiratory mechanics models, the value of the inspiratory flow at the end of inspiration with the high, moderate, and low sensitivity levels for the cycling criteria during conventional protocols were approximately 35% pif, 15% pif, and 5% pif (figure 2). during noninvasive positive pressure ventilation, air leak around the mask is unavoidable, and this can interfere with patient - ventilator synchrony and aggravate intolerance. asynchronous events include ineffective triggering, double - triggering, auto - triggering, premature cycling, and delay cycling. observed patient - ventilator asynchrony incidents in 60 patients with acute hypercapnic or non - hypercapnic respiratory failure during noninvasive psv. evaluated the effects on patient - ventilator synchrony with 2 different expiratory cycling mechanisms (flow - cycling and time - cycling) in the recovery of patients with acute lung injury. in the presence of air leak, the time - cycling mechanism provided a better patient - machine interaction than the flow - cycling mechanism (25% of pif) nevertheless, tikioka. found that in ards or ali patients the patient - ventilator interaction could be adjusted by modifying the conventional flow - cycling criteria. premature cycling with double - triggering appeared when the flow - cycling criteria sensitivity was set at a high level (35% of pif). delayed cycling was often observed with the lowest sensitivity flow - cycling level (1%). in the present study of simulated obstructive, restrictive, and normal respiratory mechanics, automated triggering and conventional cycling at different flow sensitivities independent of the cycling criteria, delayed cycling was always observed in the copd model. improved cycling synchrony was found with high - level cycling criteria, in which the cut - off point for inspiratory flow was about 35% of pif. however, the selection of high - level cycling criteria could result in severe premature cycling in restrictive and normal lung conditions. these data suggest that modification of cycling criteria may be able to improve patient - ventilator synchrony. optimal patient - ventilator synchrony, especially during non - invasive ventilation (niv), can be very difficult to achieve due to the presence of air leaks. although several intensive care units (icu) and transport ventilators are now designed specifically to provide niv, algorithms, inherent in the design of a mechanical ventilator, the absence or deficiency of leak compensation can increase the risk of asynchrony and lead to an increase in wob and patient discomfort.. found that different niv algorithms provided by icu ventilators could result in various cycling patterns. a recent study compared the performance and patient - ventilator synchrony between different kinds of ventilators during niv. by using a lung model that simulated obstructive disease with spontaneous breathing effort, the authors observed that auto - triggering and delayed cycling occurred with icu and transport ventilators. dedicated niv devices that use an niv algorithm demonstrated better performance and fewer asynchrony events. some dedicated niv machines, such as the respironics bipap vision, required no adjustment of triggering and cycling sensitivity because it can adapt to air leaks ranging from 0 to 37 l / min. poor performance was observed at the preset ps and peep levels in the respironics bipap vision and drager carina bi - level ventilators when the leakage was increased to 52 l / min. this is a pattern of the actual airflow of the patient, offset by 15 l / min and delayed by 300 ms. when the inspiratory flow of the patient crosses the shape signal, the ventilator terminates inspiration and cycles to exhalation. the auto - trak system improves patient - ventilator synchrony by adjusting to changing breathing patterns and dynamic leaks. the auto - adaptive triggering, cycling, and leak adjustments may help reduce the time that clinicians spend adjusting thresholds and re - fitting masks. previous studies have shown that a triggering delay times between 100 and 120 ms do not generate adverse clinical effects. vasconcelos. compared the auto - trak software algorithm with conventional settings in terms of patient - ventilator synchrony and discomfort in 12 healthy volunteers who underwent psv via an endotracheal tube (6 mm) positioned with a mouthpiece. the asynchrony index (ai) and discomfort scores were not significantly different between the 2 protocols. however, in the present study, the use of the auto - trak system was associated with a greater triggering delay (169 ms) in the copd model and premature cycling in the ards model. study are that the tests were performed by an icu ventilator with conventional settings and the performance of the auto - trak system was evaluated with a 6-mm endotracheal tube, not a mask. in the present bench model study, the use of automatic triggering and cycling systems with a facemask demonstrated different inspiratory termination characteristics than the conventional flow - cycling setting used for psv. the benefit of an automatic cycling setting is better patient - machine cycling synchronization in patients with respiratory failure, simplified ventilator management, and fewer errors caused by individual manipulations. in conclusion, the use of automatic triggering during psv has a better effect on patient - ventilator cycling synchrony, as evident by shorter triggering time delays and lower ptpt, than conventional flow - cycling settings in respiratory failure patients. the advantage of conventional flow - cycling criteria settings was found to be the avoidance of serious asynchronous events when respiratory mechanics are extremely unstable. | backgroundpressure support ventilation (psv) is a standard method for non - invasive home ventilation. a bench study was designed to compare the effectiveness of patient - ventilator inspiratory termination synchronization with automated and conventional triggering in various respiratory mechanics models.material/methodstwo ventilators, the respironics v60 and curative flexo st 30, connected to a hans rudolph series 1101 lung simulator, were evaluated using settings that simulate lung mechanics in patients with chronic obstructive pulmonary disease (copd), acute respiratory distress syndrome (ards), or normal lungs. ventilators were operated with automated (auto - trak) or conventional high-, moderate-, and low - sensitivity flow - cycling software algorithms, 5 cmh2o or 15 cmh2o pressure support, 5 cmh2o positive end - expiratory pressure (peep), and an air leak of 2528 l / min.resultsboth ventilators adapted to the system leak without requiring adjustment of triggering settings. in all simulated lung conditions, automated cycling resulted in shorter triggering delay times (< 100 ms) and lower triggering pressure - time product (ptpt) values. tidal volumes (vt) increased with lower conventional cycling sensitivity level. in the copd model, automated cycling had higher leak volumes and shorter cycling delay times than in conventional cycling. asynchronous events were rare. inspiratory time (tinsp), peak expiratory flow (pef), and cycling off delay time (cdelay) increased as a result of reduction in conventional cycling sensitivity level. in the ards and normal adult lung models, premature cycling was frequent at the high - sensitive cycling level.conclusionsoverall, the auto - trak protocol showed better patient - machine cycling synchronization than conventional triggering. this was evident by shorter triggering time delays and lower ptpt. |
the lower gastrointestinal tract of mammals is inhabited by a very dense and diverse bacterial microflora that exists in a state of mutualism with the host. a constant molecular dialogue between the microbiota and the host dysregulated immune responses to the commensal microbiota and accompanying intestinal inflammation occur directly as a loss of equilibrium between finely tuned pro- and anti - inflammatory mechanisms within the gastrointestinal tract [1, 2 ]. although the intestinal mucosa is populated by a number of specialized lymphocyte populations that mediate diverse immune responses to gut pathogens and commensals, these adaptive responses are elicited following sensing of microbial or environmental factors by innate immune cells. the importance of innate immune pathways in intestinal homeostasis is underscored by genome - wide association studies that have identified polymorphisms in many innate immunity genes that influence susceptibility to inflammatory bowel disease (ibd). the innate immune compartment in the gut encompasses many innate leucocyte populations, as well as several types of intestinal epithelial cells (iec), which act together to maintain a balanced immune response to the microbiota. here, we review the key roles of innate immune activation in the gut, including maintenance of homeostasis during steady state, restoration of the epithelium following insult or injury, and induction of protective and pathogenic inflammatory responses. the intestinal epithelium consists of a single layer of columnar epithelial cells that provide an effective physical barrier separating the vast bacterial load of the intestinal flora from cells of the host immune system. the continuous crypts and villi that make up the intestinal epithelium possess several physical, biochemical and immunological mechanisms ensuring intestinal homeostasis, i.e. mutualistic interactions with commensal microbes contrasted by protective immunity to invasive pathogens. actin - rich microvillar protrusions from the apical iec surface form a mechanical brush border, which, in combination with goblet cell - secreted mucins, comprise a sterile barrier that is impermeable to most intestinal microbes. the mucus layer of the intestine consists of an inner glycocalyx of membrane - anchored mucins, covered by an outer layer of secreted mucins, which, in addition to being a viscous barrier to microbes, forms a matrix loaded with high concentrations of iec - derived antimicrobial peptides and secretory iga. iec are permanently in contact with the intestinal lumen contents and, therefore, ideally located to undertake immunosurveillance of commensal and pathogenic populations within the intestinal microbiota. microbe - associated molecular pattern triggering of pattern recognition receptors (prr) classically drives a nuclear factor-b (nf-b)-dependent pro - inflammatory response and initiation of both innate and adaptive immune responses to the invading microbe. triggering of prr signalling within iec is critical for a broad spectrum of host - protective responses to pathogenic species in the intestine, including enhanced production of cytokines, chemokines and induction of b - cell class switch recombination for the production of secretory iga. in order to discriminate between commensal and invasive pathogenic bacteria, iec display compartmentalized expression of toll - like receptors (tlr), as well as differential regulation of transcription networks in response to tlr ligands. it is now well recognized that under steady state conditions, sensing of the microbiota and basal prr signaling in iec is important for intestinal homeostasis and constant renewal of the epithelial barrier. iec intrinsic tlr triggering maintains immune pressure on the commensal flora through production of broad - spectrum anti - microbial peptides (amp) that are able to limit both colonization and translocation of commensal and pathogenic species. in addition, following infection or insult, iec intrinsic prr signalling promotes epithelial renewal, restitution and tight junction fortification by driving expression of anti - apoptotic and proliferative genes. acute intestinal inflammation observed in the dextran sodium sulphate (dss) colitis model arises due to a loss of epithelial integrity and disruption of barrier function between the intestinal microbiota and lamina propria immune cells. this triggers a potent inflammatory response mediated primarily by innate leucocytes, followed by restitution and repair of the epithelial barrier. early studies using the dss colitis model found that genetic deficiency in tlr4 or myd88 resulted in increased iec apoptosis and more severe intestinal pathology, demonstrating a cell autonomous role for tlr signalling in iec proliferation and integrity. these protective functions are not only dependent on sensing of the microbiota by tlr because recent studies demonstrated that similar responses were elicited by activation of cytosolic nod - like receptor (nlr) signalling pathways in iec. thus, mice deficient in nlrp3, or the associated adaptor asc, or the inflammasome effector caspase-1, exhibited hyper - susceptibility to acute dss colitis, with increased histopathological damage, elevated chemokine levels and leucocyte infiltration that was accompanied by increased systemic dispersion of commensal bacteria [5, 6 ]. both nlrp3 and caspase-1 deficiency resulted in significantly decreased iec proliferation and turnover during acute colitis, and bone marrow chimera experiments demonstrated that genetic deficiency in nlrp3 in the intestinal epithelium was the major factor responsible for the exacerbated disease phenotype [5, 6 ]. the protective effects of nlr signalling in iec during dss colitis were associated with the production of interleukin (il)-18, a cytokine that is secreted following nlr - mediated activation of caspase-1 [5, 6 ]. consistent with these findings, a polymorphism in the human nlrp3 gene that leads to reduced nlrp3 expression was identified as a risk allele for crohn 's disease (cd). mutations in nod2 (card15), which encodes the cytosolic nlr nod2 that detects the bacterial peptidoglycan derivative muramyl dipeptide (mdp), have long been associated with susceptibility to cd, but the mechanisms responsible remain unclear. nod2 signalling in myeloid cells has been reported to attenuate inflammatory responses induced by tlr and may also limit il-1 secretion, suggesting that constitutive detection of mdp by myeloid cells may dampen pro - inflammatory responses in the gut. however, several lines of evidence suggest that intrinsic nod2 signals also play an important role in iec homeostasis. nod2 has been linked to the secretion of -defensins by paneth cells in the small intestine, and administration of mdp has been reported to protect against experimental colitis in mice. moreover, recent studies have identified a link between nod2 and autophagy, a conserved catabolic pathway in which cytoplasmic contents are targeted for degradation in lysosomal compartments. autophagy is upregulated in response to starvation, stress or infection and may play an important role in defence against intracellular bacteria. polymorphisms in autophagy genes, such as irgm and atg16l1, have been associated with susceptibility to cd, and it was recently reported that activation of nod2 could stimulate autophagy and that nod2 directly facilitated the recruitment of atg16l1 to sites of bacterial entry at the plasma membrane [9, 10 ]. furthermore, autophagy is closely linked to endoplasmic reticulum (er) stress and the consequent induction of the unfolded protein response. the highly secretory nature of many iec makes them pre - disposed to er stress, and polymorphisms in er stress genes are associated with ibd. taken together, these findings suggest that prr, er stress and autophagy circuits function in an integrated manner to regulate bacterial handling in the intestinal epithelium [2, 11 ]. nf-b signalling within both iec and haematopoietic cell compartments is fundamental to the maintenance of intestinal homeostasis. appropriate pro - inflammatory stimuli initiate signalling cascades ultimately resulting in ib kinase (ikk) complex - dependent activation and nuclear translocation of nf-b subunit heterodimers that drive gene transcription. selective disruption of homeostatic nf-b signalling within iec, by targeted ablation of the ikk regulatory subunit nemo (nemo) or of both ikk - catalytic subunits ikk and ikk, led to spontaneous iec apoptosis and severe intestinal inflammation. nemo mice exhibited reduced amp secretion from iec resulting in increased levels of commensal bacteria crossing the epithelial barrier to the lamina propria. in addition, iec in nemo mice were hypersensitive to tumor necrosis factor (tnf)--induced apoptosis and genetic ablation of tnfr1-ablated intestinal inflammation. similarly, mice rendered specifically deficient in rela or ikk in iec exhibited increased susceptibility to chemically induced colitis. recent evidence indicates that the nf-b target gene a20 (tnfaip3), which has been associated with cd, plays a key role in preventing excessive apoptosis of iec, as mice with selective ablation of a20 in iec exhibited increased susceptibility to dss colitis and to tnf--triggered apoptosis. another important anti - inflammatory circuit induced by nf-b signalling in iec microbiota sensing by iec elicits ikk-dependent production of tslp that plays a key role in conditioning local dendritic cell (dc) populations within the intestine that in turn regulate the type and magnitude of effector t - cell responses in the gut. the range of intestinal microbes that manipulate these pathways further illustrates the importance of nf-b signals in regulating intestinal homeostasis. pathogenic bacteria and viruses exhibit an expansive repertoire of microbial mechanisms aimed at immune subversion by perturbation / activation of nf-b signalling. commensal microbes may also inhibit efficient transduction of nf-b signals in order to temper or avoid inflammatory responses. for example, enhanced nuclear export of the active nf-b subunit rela in complex with peroxisome proliferator - activated receptor- drives the anti - inflammatory activity of the human anaerobic commensal bacteroides thetaiotaomicron. taken together, these findings indicate that numerous synergistic mechanisms, both host and microbe mediated, ensure that tonic levels of intrinsic prr signalling occur constitutively in iec and that this maintains epithelial integrity and turnover, regulates the composition and penetrance of the microbiota, and prevents overt inflammation in response to commensals. intestinal antigen - presenting cells (apc) comprise heterogeneous cell populations that are present within the organized gut - associated lymphoid tissue, including peyer 's patches, isolated lymphoid follicles and draining mesenteric lymph nodes (mln) and the lamina propria. recent advances have described differences in ontogeny, phenotype and tissue location that relate to functionally distinct populations of intestinal myeloid cells, which encompass dc, monocytes and macrophages. as reviewed elsewhere within this issue, intestinal macrophages have many adaptations to prevent overt inflammatory responses to the microbiota under homeostatic conditions ; therefore, we shall mainly focus on effector functions of intestinal myeloid cells. differential expression of fractalkine receptor cx3cr1 and integrin subunit cd103 identifies two major populations of intestinal myeloid cells that have been implicated in tolerance pathways in the intestine. cd103 lamina propria dc derive from pre - classical dc lineage, whereas cd11c cx3cr1 mononuclear phagocytes (mp) may constitute a more heterogenous population of dc and macrophages that stem from ly-6c monocytes under homeostatic conditions. microbiota - dependent signals drive cd11c cx3cr1 mp accumulation in the lamina propria adjacent to the iec layer. expression of tight junction proteins by cx3cr1 mp allows projection of transepithelial dendrites in order to sample luminal contents, and this process has been suggested to contribute to resistance to enteric pathogens, such as salmonella typhimurium. however, cd11c cx3cr1 mp appear not to traffic to the draining mln or prime nave t cells, and the defective induction of systemic immune tolerance to dietary antigen (oral tolerance) observed in cx3cr1-deficient mice instead supports a role for cx3cr1 mp in enhancing tolerogenic immune responses in the gut. impaired induction of oral tolerance was associated with a failure to expand lamina propria foxp3 treg cells and with reduced production of the anti - inflammatory cytokine il-10 by intestinal myeloid cells. an important role for myeloid cell - derived il-10 in the regulation of intestinal immune responses was further supported by an independent report that an innate source of il-10 was required for maintenance of foxp3 expression and control of experimental colitis by foxp3 treg cells. thus, although foxp3 treg cells could suppress colitis induced by adoptive transfer of nave cd4 t cells into rag recipients, they were unable to prevent colitis induction in rag il-10 recipients. innate il-10 secretion by cd11b cd11c f4/80 myeloid cells was necessary to maintain foxp3 expression and suppressive functions of treg cells in the inflamed intestine. in contrast to the sub - epithelial localization of cx3cr1 mp, the anatomical location of cd11b cd103 dc is more varied, with populations dispersed both throughout the gut - associated lymphoid tissue and lamina propria. small intestinal lamia propria cd103 dc perform typical dc sentinel functions ; they can sense and take up a wide variety of foreign bacteria and innocuous antigens and migrate to the draining mln where they initiate adaptive immune responses with an intestinal tropism, such as imprinting of the gut - homing receptors ccr9 and 47 on activated t cells and induction of intestinal b - cell class switch to iga production [17, 20, 22 ]. these activities are dependent on retinoic acid (ra), a dietary metabolite derived from vitamin a, and cd103 lamina propria dc express aldh1a1 and aldh1a2 genes that encode for retinal metabolizing enzymes required for ra synthesis. dc play an important role in inducing tolerance to intestinal antigens under steady state conditions, as they migrate to the mln and promote the generation of foxp3 inducible treg cells through a transforming growth factor (tgf)- and ra - dependent mechanism. interestingly, recent evidence indicates that distinct myeloid cell compartments may co - operate to maintain intestinal tolerance as robust oral tolerance was associated with further expansion of gut - homing foxp3 treg cells in the intestinal lamina propria through interactions with cx3cr1 mp. taken together, these findings highlight that a number of heterogeneous subsets of myeloid cells can participate in the maintenance of immune tolerance in the gut (fig myeloid cells, granulocytes and innate lymphoid cells (ilc) all accumulate within the intestine during chronic intestinal inflammation and contribute to the excessive production of pro - inflammatory mediators that drive intestinal pathology [1, 2 ]. in contrast to the protective effects of tonic prr signals in iec described above, several studies have reported that persistent prr signals in leucocyte populations are crucial for the induction and perpetuation of chronic intestinal inflammation. for example, the microbiota - dependent spontaneous colitis that develops in il-10 mice was completely absent in il-10 myd88 mice, and genetic ablation of myd88 also rescued the spontaneous colitis phenotype of mice harbouring an iec - specific deletion of the ikk - regulatory subunit nemo, suggesting that functional tlr signals were required for intestinal inflammation. in addition, the innate immune - meditated typhlocolitis that develops following infection of rag mice with the gram - negative intestinal bacterium helicobacter hepaticus was completely abrogated in rag myd88 mice. furthermore, bone marrow chimeric rag mice in which myd88 signalling was selectively absent in haematopoietic cells were also completely resistant to h. hepaticus - induced intestinal pathology, indicating a specific requirement for this signalling pathway in leucocytes. similarly, selective ikk deficiency in myeloid cells (lysm ikk) ameliorated intestinal inflammation in the il-10 model of spontaneous colitis, whereas iec - specific deletion of ikk (villin ikk) did not ameliorate the disease in this setting. in other t - cell colitis models, it was shown that t - cell intrinsic myd88 signals were not essential for the induction of intestinal pathology ; however, myd88 signals in dc were required for the induction of pathogenic t - cell responses in the gut. together, these studies strongly support a crucial role for myd88 signalling in myeloid cells in driving innate and adaptive inflammatory responses in the gut. during ongoing inflammation, populations of intestinal myeloid apc develop pro - inflammatory characteristics that exacerbate immune - mediated pathology. intestinal inflammation abrogates the tolerogenic activities of cd103 mln dc, with decreased expression of aldh1a2 and tgf2 correlating with an inability to promote foxp3 treg responses. instead, under inflammatory conditions, cd103 dc produce significantly more il-6 than their steady state counterparts and promote the emergence of th1 cells. similarly, flagellin - mediated activation of cd103 lamina propria dc through tlr5 promotes the generation of th17 cells. furthermore, a cd70 cx3cr1 subset of intestinal apc can detect microbiota - derived atp through purinergic p2x and p2y receptors, resulting in the up - regulation of molecules associated with th17 lineage commitment, including il-6, il-23 and the tgf--activating integrin v8. the same study reported that administration of an atp analogue led to an increase in frequency of colonic th17 cells in germ - free animals and also exacerbated t - cell transfer colitis, suggesting a role for intestinal myeloid cells in driving colonic th17 generation under inflammatory conditions. mouse models of colitis suggest that the altered phenotypic and functional properties of myeloid cells in the inflamed intestine may partly reflect the accumulation of newly recruited cells, particularly monocyte - derived mp that respond to tlr stimulation by the elaboration of pro - inflammatory cytokines. these observations are consistent with findings that intestinal tissues from cd patients exhibit infiltration by a population of cd14 inflammatory monocytes that produce elevated levels of il-23, tnf- and il-6. a better understanding of how inflammatory myeloid cells are recruited and activated in the intestine may facilitate new therapeutic approaches in ibd. a large body of clinical and experimental evidence implicates innate leucocytes, especially myeloid cells, as critical sources of a range of inflammatory cytokines in the inflamed intestine. these may exacerbate pathological responses in several ways : through effects on tissue stromal cells and iec, through stimulation of inflammatory responses from other innate leucocytes, or through the induction of pathogenic effector t - cell responses (fig. blockade or neutralization of these factors constitutes an important new therapeutic approach, as exemplified by the successful application of anti - tnf- antibodies in ibd patients. immunohistochemical studies identified lamina propria macrophages as a key source of tnf- in the inflamed gut, although other cells including paneth cells and th1 cells may also produce tnf-. tnf- signalling can modulate a broad range of cellular responses in both haematopoietic cells and parenchymal cells, including cellular activation and proliferation, as well as potent effects on iec integrity and apoptosis. genetic ablation or antibody - mediated blockade of tnf- has been shown to reduce or ameliorate intestinal pathology in a wide range of mouse models of ibd, indicating that it is a central factor in intestinal inflammation. treatment with anti - tnf- can restore intestinal barrier function in ibd patients and this has been ascribed to reduced apoptosis of iec together with induction of apoptosis of lamina propria mononuclear cells expressing membrane - bound tnf-. il-6 is another classical pro - inflammatory cytokine that is elevated in the inflamed intestinal mucosa of ibd patients and is predominantly produced by innate leucocytes in the lamina propria. il-6 can signal both in cis-, through membrane - bound il-6r, and in trans-, through soluble il-6r (sil-6r)/il-6 complex binding to membrane - associated gp130. thus, as gp130 is widely expressed, il-6 potentially affects a broad range of cell types, which may have complex effects on intestinal homeostasis. for example, iec express the il-6r, and binding of il-6 may trigger cytoprotective nf-b activation in iec. by contrast, increased levels of il-6 in the inflamed mucosa may exacerbate inflammation in several ways, such as induction of additional inflammatory mediators from macrophages and by promoting the differentiation and survival of pathogenic th1 and th17 cells [1, 30 ]. consistent with a dominant pathogenic role of il-6 in ibd, beneficial effects were reported in a pilot trial of an anti - il-6r antibody in patients with active cd. recent findings linking genetic mutations associated with secretion of the cytokines il-1 and il-18 with ibd susceptibility have rekindled interest in their roles in intestinal homeostasis. upon activation by infection or stress, several cytosolic nlr inflammasomes, that drive auto - catalytic activation of caspase-1, a cysteine protease required for processing of pro - il-1 and pro - il-18 to their secreted active forms. polymorphisms in nlrp3, il-18 and il-18rap have all been associated with susceptibility to ibd, and both il-1 and il-18 expression are increased in inflamed ibd tissues [31, 32 ]. interestingly, myeloid cells lacking the ibd risk - associated autophagy gene atg16l1 produced increased amounts of il-1 and il-18, suggesting that a functional autophagy pathway may limit inflammasome activation. levels of il-1 are elevated in models of intestinal inflammation and blockade of il-1 signalling, using anti - il-1 or, by administration of the natural antagonist il-1ra, can ameliorate the disease [1, 31 ]. il-1 is secreted primarily from monocytic cells in the lamina propria and promotes activation of myeloid cells as well as neutrophilia and neutrophil migration into inflamed tissues. il-18 levels are increased in several models of colitis and antibody - mediated neutralization of il-18, or administration of the natural antagonist il-18bp can reduce intestinal pathology. in the chronically inflamed intestine of ibd patients, il-18 is predominately associated with mononuclear leucocytes in the lamina propria, where it may contribute to disease progression through promotion of th1 responses [32, 33 ]. however, the role of il-18 in intestinal homeostasis is complex because, as noted earlier, nlr activation has been associated with protective responses in iec. indeed, production of il-18 within the healthy gastrointestinal tract appears restricted to the intestinal epithelium, and administration of exogenous il-18 protected nlrp3-deficient mice from exacerbated pathology during dss colitis, suggesting an iec - intrinsic role for il-18 in intestinal epithelial homeostasis [5, 6 ]. similarly, genetic deficiency in either il-18 or il-18r renders mice more susceptible to dss colitis. thus, depending on the cellular location and temporal characteristics of activation, inflammasome - dependent cytokine responses may have beneficial or harmful effects on intestinal homeostasis. following epithelial damage, il-18 signalling in the intestinal epithelium plays a key role in maintaining epithelial integrity, by promoting restitution and repair pathways in iec. conversely, chronic excessive production of il-1 and il-18 by lamina propria mononuclear cells may contribute to the deleterious inflammatory responses observed in ibd patients. additional studies on inflammasome activation in the gut may offer further insights into ibd pathophysiology. genome - wide association studies associating polymorphisms in the il-23r and il-12p40 with susceptibility to cd, together with experimental studies showing that removal or blockade of il-23 could ameliorate chronic colitis, have highlighted il-23 as a central regulator of intestinal homeostasis and inflammation [1, 2 ]. production of il-23 in the intestine is induced by prr stimulation of myeloid cells, and dc appear to be a key source of il-23 both during steady state conditions and also in the inflamed intestine. in addition, a population of cd14 intestinal macrophages from the gut of cd patients have been reported to secrete large amounts of il-23. il-23 can drive intestinal pathology through excessive activation of innate and adaptive inflammatory responses [1, 2 ]. il-23 drives t - cell - dependent intestinal inflammation through direct signalling into cd4 t cells to drive the proliferation and accumulation of effector th1 and th17 cells in the gut, as well as by promoting the emergence of a population of il-17a interferon (ifn)- cd4 t cells and restraining the differentiation of foxp3 treg cells. a population of cd161 cd4 t cells able to respond directly to il-23 stimulation with production of both il-17a and ifn- has also been identified in the lamina propria of patients with ibd. in addition to cd4 t cells, several populations of innate leucocytes are able to produce il-17a, il-22 and ifn- in response to il-23. for example, t cells are enriched among the intestinal epithelial lymphocytes and can respond to il-23 and il-1 by secretion of il-22 and il-17a. however, although t cells have been shown to drive intestinal inflammation in some experimental models, their contribution to ibd remains unclear. recent studies from several groups have identified multiple populations of novel ilc that are present in the gut and have been implicated in intestinal homeostasis, inflammation and host protection. the ontogeny of different ilc populations is discussed elsewhere in this issue, but many appear to derive from a common rort precursor that is dependent upon both cytokine and commensal bacteria - derived signals for further lineage commitment. using a mouse ibd model in which infection of rag2 mice with h. hepaticus drives an il-23-dependent innate immune - mediated typhlocolitis, we identified a novel population of cd4rort thy1 sca-1 ilc that accumulated in the inflamed intestine and secreted il-17a, il-22 and ifn- in response to il-23. furthermore, neutralization of either il-17a or ifn- significantly attenuated colitis, and depletion of ilc using anti - thy1 completely abrogated intestinal inflammation. although il-17a is greatly increased in the intestinal mucosa of ibd patients, experimental models have generated conflicting evidence over whether it primarily acts as a pro- or anti - inflammatory factor in the gut [1, 2 ]. for example, il-17a appears to have a protective role in acute dss colitis whereas it appears to have a pro - inflammatory role in trinitrobenzene sulfonic acid colitis [1, 2 ]. again, these discrepancies may reflect differences in the location and temporal characteristics of il-17a secretion, as well as in differential activities of il-17a depending on the presence or absence of other cytokines in the local microenvironment. the pro - inflammatory effects of il-17a have been related to the induction of cytokines and chemokines from tissue cells, such as epithelial and endothelial cells, which in turn promotes the recruitment and activation of neutrophils. by contrast with the ambiguous activities of il-17a, the pro - inflammatory effects of ifn- in the intestine have been firmly established by the disease - attenuating effects of removal or blockade of ifn- in experimental models of chronic colitis. in the inflamed intestine, ifn--secreting cells are far more prominent than those producing il-17a, and ifn- may exert pro - inflammatory effects by directly influencing iec turnover and epithelial integrity and by activating macrophages in the lamina propria [1, 2 ]. nevertheless, the disappointing efficacy of ifn- blockade in human ibd suggests that there is a complex interplay between th1 and th17 cytokine responses in the gut and that therapies targeting both axes may be required to effectively combat chronic intestinal pathology. finally, il-23 is also a potent driver of il-22, an il-10 family member that acts to enhance innate immune defences in mucosal tissues. restricted to expression in the non - haematopoietic system, the il-22r is highly enriched on mucosal epithelial cells, especially those of the skin, lung and gut. in iec, il-22 signalling contributes to intestinal homeostasis by activation of stat3, which enhances epithelial healing and regeneration, restores goblet cells and mucus production and stimulates the production of amp. consistent with these observations, il-22 administration attenuated disease severity in the dss and t - cell receptor- colitis models. furthermore, an il-23-dependent innate source of il-22 was shown to be critical for early immunity to the attaching / effacing mouse intestinal bacterial pathogen citrobacter rodentium, and recent data suggest that this protective il-22 is derived from rort thy1 ilc. conversely, clinical evidence suggests that il-22 may be playing a pathogenic role in ibd, as patients with active cd have elevated levels of il-22 detectable in the serum, and levels of il-22 correlate with disease severity and il-23r genotype. however, whether elevated il-22 is driving disease progression or reflects a failed attempt at promoting resolution remains unclear. thus, il-23 can modulate the activation of a variety of innate and adaptive immune cells in the gut, which are mirrored by the diverse range of cytokine responses that are induced by il-23. under steady state conditions, these responses may play a critical role in the maintenance of epithelial barrier integrity and innate immune defence. however, sustained activation of il-23-driven effector pathways may underlie the chronic immune pathology found in ibd (fig. the data summarized in this review highlight that innate immune activation is a fundamental process that impacts on intestinal homeostasis on several levels (fig. it occurs not only in haematopoietic cells, but also in parenchymal cells such as iec. constitutive tonic sensing of microbial factors by prr in iec and in innate leucocytes stimulates tissue - protective innate defences that inhibit colonization and invasion by pathogens. in the steady state,. however, damage, stress or breach of the epithelium activates a plethora of innate immune mechanisms and downstream inflammatory responses, and chronic aberrant activation of these innate immune pathways may play a key role in the characteristic pathology found in ibd patients. the activities of individual factors produced by innate immune cells in the gut, such as cytokines, may be largely determined by their location, kinetics and the presence of additional factors that can modulate their activity. therefore, selective modulation of innate immune activation and of downstream mediators presents an ongoing challenge to effectively target deleterious inflammatory responses in ibd whilst sparing host - protective immunity in the intestinal tissues. | loss of intestinal immune regulation leading to aberrant immune responses to the commensal microbiota are believed to precipitate the chronic inflammation observed in the gastrointestinal tract of patients with inflammatory bowel diseases (ibd), crohn 's disease and ulcerative colitis. innate immune receptors that recognize conserved components derived from the microbiota are widely expressed by both epithelial cells and leucocytes of the gastrointestinal tract and play a key role in host protection from infectious pathogens ; yet precisely how pathogenic and commensal microbes are distinguished is not understood. furthermore, aberrant innate immune activation may also drive intestinal pathology, as patients with ibd exhibit extensive infiltration of innate immune cells to the inflamed intestine, and polymorphisms in many innate immunity genes influence susceptibility to ibd. thus, a balanced interaction between the microbiota and innate immune activation is required to maintain a healthy mutualistic relationship between the microbiota and the host, which when disturbed can result in intestinal inflammation. |
laparoscopic operations for benign adnexal pathology have been conducted for many years ; minimally invasive surgery was, in fact, initially pioneered by gynecologists. in most centers without specialized pediatric gynecologists, we have previously reported our all - in - one single - incision single - instrument (sisi) appendectomy as an essentially scarless operation with outcomes equivalent to multiport techniques at a lower cost. since 2008, we have extended the sisi approach to adnexal pathology and found it to be a useful approach for the treatment of pediatric and adolescent ovarian or paraovarian pathology, with easy conversion to standard two- or three - port laparoscopy or even open pfannenstiel, if additional visualization were needed. several authors have described the use of expensive single - site laparoscopy systems for treatment of adnexal pathology in small series of children and adolescents [28 ], and others have described similar approaches in adults [914 ], although many of these reports are for single - incision multiple - instrument methods. a few authors have also described the use of transumbilical operations for cystic ovarian lesions in small series of infants, whether laparoscopic - assisted or via minilaparotomy [1519 ]. a survey conducted among members of the international pediatric endosurgery group (ipeg) divulged that only 7% of this highly selected group 's respondents had performed single - incision pediatric endoscopic surgery (sipes) for gynecologic pathology. a quarter of respondents to this survey who did not perform sipes at all cited inadequate resources as the reason. the purpose of the current study was to describe our high - volume, extensive experience with sisi surgery applied to adnexal pathology as a low - cost, simple, efficient, and cosmetically appealing solution for pelvic surgery in children of all ages and sizes. our surgical billing database was queried for current procedural technology (cpt) codes indicating partial or total oophorectomy (58661, 58662, 58720, 58900, 58925, 58940) from january 1, 2008, to november 30, 2013. five attending surgeons performed or supervised all procedures, and all surgeons had been performing sisi appendectomy for at least 2 years. data extracted included patient age and weight at time of surgery, surgical start and end times, type of surgery performed (including surgical approach at beginning, intraoperative change of approach, and reason for conversion), final pathology and cytology results, total hospital length of stay, postoperative complications, and overall length of follow - up time. this study met criteria for exempt review, determined by the western institutional review board (case number 1 - 814812 - 1). our sisi approach utilized a 12-millimeter standard laparoscopy trocar introduced transumbilically, after placement of an indwelling bladder catheter. we then inserted an operating endoscope (karl storz, product number 26036aa, figure 1), which combines a 10-millimeter, 0-degree lens with an offset eyepiece and a 5-millimeter port through which a long instrument, such as a toothed grasper or suction, can be introduced. ovarian torsions can almost always be reduced with this instrument. for large simple cystic lesions, aspiration of the cyst fluid was performed with a spinal needle introduced through the anterior abdominal wall under direct vision while stabilizing the lesion with the grasper or by cauterizing a hole in the cyst with the long suction and then inserting it inside the cyst. if the cystic lesion was so large that it extended out of the pelvis and could be directly visualized through the umbilical incision, it was grasped with an atraumatic clamp. a purse - string suture was then placed in the cyst wall, and a suction device was inserted for decompression of fluid. once the adnexa was mobile, ovarian - sparing procedures were attempted when thought possible, with the cystic or solid lesions being dissected off the normal ovarian parenchyma, which was dropped back into the abdomen after achieving hemostasis. total oophorectomy, sparing the fallopian tube, was utilized when normal - appearing ovarian parenchyma could not be distinguished from a solid mass of undetermined character. conversion to standard laparoscopy was dependent upon the experience and comfort of the surgeon with each individual case. statistics utilized included the student t - test, chi - square test, and fisher exact test where appropriate, with p < 0.05 considered statistically significant. eight operations had two procedure codes ; five patients were excluded for nonovarian primary surgery. four patients were found to have had a repeat operation that was not elicited in our code search. the end result was 271 evaluable operations in 258 patients, 13 of whom had recurrent adnexal pathology. the most common indications for surgical intervention were pain, cyst greater than 5 centimeters in diameter, suspected torsion, and/or solid mass. demographic and clinical data by initial and final approach are delineated in table 1. over half of our initial operative attempts were via sisi (147 of 271, 54%) ; about half of these were converted to either standard laparoscopy or pfannenstiel incision, resulting in 75 patients (28% of the total cohort, 51% of those started with sisi) completing treatment with the technique. the sisi method was completed in patients ranging in age from 1 day to 19.9 years (median 15.3 years) and with weights from 4.7 to 117 kilograms (median 59.4 kilograms). reasons for change in approach are delineated in table 2 and were dominated by desire to perform contralateral oophoropexy or presentation of a solid mass. twenty - two patients underwent oophoropexy (8.1% of the total cohort) at the discretion of attending surgeon, usually in the case of residual solitary ovary such as with tumor resection or from contralateral torsion with probable gonadal compromise. with progression of time, sisi use increased and standard multiport laparoscopy use decreased ; successful sisi surgery comprised only 9.3% of all operations in 2008 compared with 34% in 2013, while multiport laparoscopy decreased from 42% to 24% over the same time period. successful sisi surgery was significantly shorter than all other approaches (46 mins versus 59 mins for standard laparoscopy, t - test, p < 0.001). length of stay (0.77 days) was shorter in the successful sisi surgery group when compared with standard laparoscopy (0.92 days), but this did not reach statistical significance. only 4.7% of the cohort was found to have a malignant or premalignant condition, and these 12 cases are broken down in detail in table 4. follow - up has ranged from 0 to 72 months (median 14.2 months). sisi converted to pfannenstiel incision had a significantly higher rate of complications (42%, p < 0.002) than either successful sisi (6.7%), sisi converted to multiport laparoscopy (15%), initial multiport laparoscopy (8.2%), or initial pfannenstiel approach (6.3%). there were no major complications of surgery, although there were five recurrences of benign disease scattered across four of the different surgical techniques, which were all treated with repeat operation. two deaths occurred in our cohort during long - term follow - up, one due to overwhelming oncologic disease (burkitt lymphoma) and the second from peritonitis 7 months postoperatively. this was associated with a peritoneal dialysis catheter and unrelated to the ovarian pathology. in regard to cytologic collection from peritoneal washings or cyst aspiration, 83 of 271 patients in the cohort (31%) had samples generated. in all but one case, the specimen yielded benign or nondiagnostic results. cytology was sent in only 6 of 12 malignant or premalignant lesions, and a single specimen in a girl with a yolk sac tumor showed atypical cells. we report here our series of single - incision, single - instrument (sisi) operations for adnexal pathology in infants, children, and adolescent girls. we feel our approach is useful as a cost - effective, simple, safe, and cosmetically appealing procedure for treating gynecologic pathology in children. we have gone on to utilize our sisi approach for other pathologies as well, including resection of meckel diverticulum and duplications, reduction of intussusception, segmental colon resection, treatment of empyema, and extraction of multiple gastric or intestinal tract magnets. we feel this technique is broadly applicable and are continuously looking for new ways to safely limit expenses and improve outcomes with its employment. one of the major decisions both prior to initiating a sisi approach and once inspecting the pelvic pathology is whether or not there is an immature or cancerous component to the pathology, for avoidance of rupture in these cases may be critical. we have found that the majority of cases can be approached with sisi, as long as the radiology is consistent with benign pathology (i.e., simple appearing cyst without solid component, hemorrhagic cyst, torsion, or solid mass consistent with teratoma based on appearance of fat or calcifications) and the hormonal markers are normal. any indication of malignancy (peritoneal studding, retroperitoneal lymphadenopathy, noncalcified or highly vascularized solid mass, or elevated hormonal markers) should discourage use of the sisi technique. in these situations, multiport or multi - instrument single - incision laparoscopy (e.g., less) can be considered, although the standard is open surgery. intraoperatively, additional care must be taken as risk of spill in ovarian malignancy must be avoided with aspiration or attempts at mobilization. intracorporeal needle decompression or periumbilical mobilization with purse - string evacuation is only undertaken in simple cystic lesions without solid component, unless the lesions are small and mobile enough to extrude easily through the umbilicus. in the present series, it is noteworthy that while malignancy was uncommon, no patient with malignancy was completed with the sisi technique. in cases of suspected malignancy or uncertain pathology, multiport laparoscopy with endobag extraction or pfannenstiel incision is utilized. this latter option affords the opportunity to extracorporeally attempt the tedious dissection often required to perform an ovarian - sparing procedure while guarding against intra - abdominal rupture in benign but very large tumors such as teratomas. occasionally, with a very large or clearly malignant tumor teratomas form an interesting group of neoplasms for which the sisi technique is particularly applicable. usually mature but occasionally massive we follow these patients for a period of at least 5 years after diagnosis, which allows small, metachronous lesions to be identified early, making ovarian preservation easier. small ovarian dermoids found during surveillance are easily treated using sisi instrumentation to exteriorize the ovary through the umbilicus for excision. based on the data gleaned from our sizable cohort, and contrary to the accepted practice in adult patients, peritoneal washing and/or cyst fluid collection for cytology may be unnecessary when benign disease is suspected. in no case did the results change either the diagnosis or the treatment, while adding a significant cost to the patient 's care. larger studies would be needed before recommending the abandonment of this diagnostic modality, but a prospective, multicenter investigation of cytology in pediatric adnexal pathology would be a fertile area for future investigation. limitations of our study include its retrospective and therefore descriptive nature. the conclusions herein are meant to encourage the application of this technique, while recognizing that surgeon judgment will always be principal when considering operative approach. our report, the largest reported experience to date, reveals that a significant subset of girls with adnexal pathology can undergo successful treatment with the single - incision single - instrument technique, allowing for scarless surgery, minimal discomfort, and same - day discharge. we advocate further implementation of this safe, low - cost, and cosmetically appealing solution to the treatment of pediatric surgical illness. | introduction. pediatric surgeons often practice pediatric gynecology. the single - incision single - instrument (sisi) technique used for appendectomy is applicable in gynecologic surgery. methods. we retrospectively analyzed the records of patients undergoing pelvic surgery from 2008 to 2013. sisi utilized a 12 mm transumbilical trocar and an operating endoscope. the adnexa can be detorsed intracorporeally or extracorporealized via the umbilicus for lesion removal. results. we performed 271 ovarian or paraovarian surgeries in 258 patients. in 147 (54%), the initial approach was sisi ; 75 cases (51%) were completed in patients aged from 1 day to 19.9 years and weighing 4.7 to 117 kg. conversion to standard laparoscopy was due to contralateral oophoropexy, solid mass, inability to mobilize the adnexa, large mass, bleeding, adhesions, or better visualization. when sisi surgery was converted to pfannenstiel, the principal reason was a solid mass. sisi surgery was significantly shorter than standard laparoscopy. there were no major complications and the overall cohort had an 11% minor complication rate. conclusion. sisi adnexal surgery is safe, quick, inexpensive, and effective in pediatric patients. sisi was successful in over half the patients in whom it was attempted and offers a scarless result. if unsuccessful, the majority of cases can be completed with standard multiport laparoscopy. |
phospholipon 90 g and phospholipon 90 h were kindly gifted by natterman gmbh germany. etoposide the hydration of the lipid film was carried out with hydroxy propyl -cyclodextrin (hpcd) solution at 60. vesicle size was reduced by subjecting the liposomes to mechanical shaking for upto 6 h on a horizontal shaker bath (expo, india) followed by intermittent sonication (30 s) carried out after every 2 h, in a bath sonicator (expo, india) at room temperature. the unentrapped drug was removed by centrifugation (20,000 g, beckman, u.s.a.). various parameters such as cholesterol concentration, lipid proportion and shaking time were optimized with respect to particle size and entrapment efficiency. the liposomal dispersion of etp was freeze dried using trehalose as cryoprotectant (labconco, england). the freeze dried etp - liposomes were stored in a refrigerator in sealed vials until use. thermal behaviour of etp liposomes before and after freeze drying was studied using dsc (dt40, shimadzu japan). the particle size distribution of the liposomes was assessed on a malvern mastersizer (malvern instruments, usa) after rehydration with saline. transmission electron microscopy (tem) images were visualized using the electron microscope (jeol jem-1010, japan) and photographed. surface topography of the freeze dried liposomes was determined using environmental scanning electron microscope (quanta-200, fei). to visualize the reformation of liposomes in presence of saline, the dry powder was wetted with a drop of saline, and observed over a period of 5 min under esem and photographed. the chromatographic system consisted of jasco pu 980 intelligent pump, jasco uv 975, a uv - vis detector version 1.50 build 15 (jasco - borwintm, japan) and rheodyne injector valve bracket fitted with a 20 l sample loop. hplc separations were performed on a stainless - steel technochrom kromacil ; c-18 analytical column (2504.6 mm) packed with 5 m diameter particles. data was processed using chromatography software, hercule 2000 chromatography interface star 800 interface module interface version 2.0 (jasco - borwintm, japan). the composition of the mobile phase was methanol : water : glacial acetic acid 0.1%, (60:40:1). the injection volume was 20 l and the retention time was found to be 4.3 min. for ee of etp liposomes, 0.25 ml of liposomal preparation was diluted to 4 ml with distilled water and centrifuged at 20000 g at 80.5 for 30 min using allegra tm 64 r centrifuge (beckman, u.s.a.). the supernatant was appropriately diluted in distilled water and the amount of drug present in the supernatant was determined by reverse phase hplc. entrapment efficiency was calculated as the difference between the initial amount of drug added and that present in the supernatant as free drug, and was expressed as g of drug entrapped per mm of total phospholipids (g / mm of lipid). in vitro drug release profile of freeze dried etp liposomes was assessed in simulated lung fluid (ph 7.4) at 37 by static method. after periodic intervals of time, the liposomal aliquots were centrifuged and supernatant was evaluated for the drug content by hplc. the respirable fine particle fraction (fpf) of the etp liposomes was determined using a twin - stage impinger, apparatus a (tsi), official in indian pharmacopoeia. the liposomes reconstituted in saline was nebulized using the nebulizer (omron cx3) attached to the tsi by means of rubber collars. the fractions collected in each stage were analyzed for etp content by hplc using the chromatographic conditions as described above. fine particle fraction (fpf) i.e., respirable fraction is expressed as the ratio of the amount of drug recovered from the lower stage of the impinger to the sum of drug recovered from the capsule shells, the inhaler device and the upper and lower stages of the twin stage impinger. stability evaluation of aqueous liposome suspension and freeze dried liposomes was carried out at 2 - 8 and ambient condition (284) for a period of six months. samples withdrawn at specified time intervals (1, 3 and 6 month) were visually observed for appearance, ease of reconstitution, and sedimentation. also the liposomes were evaluated for etp content, particle size and fpf. the hydration of the lipid film was carried out with hydroxy propyl -cyclodextrin (hpcd) solution at 60. vesicle size was reduced by subjecting the liposomes to mechanical shaking for upto 6 h on a horizontal shaker bath (expo, india) followed by intermittent sonication (30 s) carried out after every 2 h, in a bath sonicator (expo, india) at room temperature. the unentrapped drug was removed by centrifugation (20,000 g, beckman, u.s.a.). various parameters such as cholesterol concentration, lipid proportion and shaking time were optimized with respect to particle size and entrapment efficiency. the liposomal dispersion of etp was freeze dried using trehalose as cryoprotectant (labconco, england). the freeze dried etp - liposomes were stored in a refrigerator in sealed vials until use. thermal behaviour of etp liposomes before and after freeze drying was studied using dsc (dt40, shimadzu japan). the particle size distribution of the liposomes was assessed on a malvern mastersizer (malvern instruments, usa) after rehydration with saline. transmission electron microscopy (tem) images were visualized using the electron microscope (jeol jem-1010, japan) and photographed. surface topography of the freeze dried liposomes was determined using environmental scanning electron microscope (quanta-200, fei). to visualize the reformation of liposomes in presence of saline, the dry powder was wetted with a drop of saline, and observed over a period of 5 min under esem and photographed. the chromatographic system consisted of jasco pu 980 intelligent pump, jasco uv 975, a uv - vis detector version 1.50 build 15 (jasco - borwintm, japan) and rheodyne injector valve bracket fitted with a 20 l sample loop. hplc separations were performed on a stainless - steel technochrom kromacil ; c-18 analytical column (2504.6 mm) packed with 5 m diameter particles. data was processed using chromatography software, hercule 2000 chromatography interface star 800 interface module interface version 2.0 (jasco - borwintm, japan). the composition of the mobile phase was methanol : water : glacial acetic acid 0.1%, (60:40:1). the injection volume was 20 l and the retention time was found to be 4.3 min. for ee of etp liposomes, 0.25 ml of liposomal preparation was diluted to 4 ml with distilled water and centrifuged at 20000 g at 80.5 for 30 min using allegra tm 64 r centrifuge (beckman, u.s.a.). the supernatant was appropriately diluted in distilled water and the amount of drug present in the supernatant was determined by reverse phase hplc. entrapment efficiency was calculated as the difference between the initial amount of drug added and that present in the supernatant as free drug, and was expressed as g of drug entrapped per mm of total phospholipids (g / mm of lipid). in vitro drug release profile of freeze dried etp liposomes was assessed in simulated lung fluid (ph 7.4) at 37 by static method. after periodic intervals of time, the liposomal aliquots were centrifuged and supernatant was evaluated for the drug content by hplc. the respirable fine particle fraction (fpf) of the etp liposomes was determined using a twin - stage impinger, apparatus a (tsi), official in indian pharmacopoeia. the liposomes reconstituted in saline was nebulized using the nebulizer (omron cx3) attached to the tsi by means of rubber collars. the fractions collected in each stage were analyzed for etp content by hplc using the chromatographic conditions as described above. fine particle fraction (fpf) i.e., respirable fraction is expressed as the ratio of the amount of drug recovered from the lower stage of the impinger to the sum of drug recovered from the capsule shells, the inhaler device and the upper and lower stages of the twin stage impinger. stability evaluation of aqueous liposome suspension and freeze dried liposomes was carried out at 2 - 8 and ambient condition (284) for a period of six months. samples withdrawn at specified time intervals (1, 3 and 6 month) were visually observed for appearance, ease of reconstitution, and sedimentation. also the liposomes were evaluated for etp content, particle size and fpf. the advantage provided by liposomes as drug carrier is based on their ability to alter favuorably the pharmacokinetic profile of the encapsulated species and thus provide selective and prolonged pharmacological effects at the site of administration. etp, being practically insoluble in water, may precipitate out on hydration of liposomes. many studies report the use of hpcd drug complexes to improve the entrapment and impart stability to liposomes. to determine the effect of hydration medium on entrapment efficiency of etp, the solution of etp hpcd complex is able to travel inside the liposomes during hydration and lead to increase in entrapment efficiency. entrapment efficiency also depends on the formulation variables i. e., lipid composition used. to study this aspect, several batches of etp liposomes were prepared with varying lipid compositions. lipid composition of various batches, their ee and particle size observed are listed in table 1. decreased ee, after increasing cholesterol may be attributed to the lipophilic nature of cholesterol and hence possibly competing with etp in the lipid bilayer and reducing the entrapment of etp. effect of freeze drying on drug content and particle size increasing the amount of phospholipon 90 g increased the ee of etp. this could be attributed to the fact that unsaturated phospholipids are relatively more flexible in nature, and thereby provide less hindrance for etp to be retained in lipid bilayer. increasing the saturated lipid, phospholipon 90 h resulted in reduced ee ; this could be due to the saturated lipid behaving in the same way as cholesterol. this could be attributed to better solubilization of etp in hpcd and subsequent increased ee. further investigations were carried out on batch el 7 as it had shown better drug content as well as particle size. various approaches have been developed and evaluated to impart long term stability to liposomes and one of them is lyophilization. to maintain the same particle size distribution and to avoid leakage of the encapsulated drug from liposomes after the freeze - drying / rehydration cycle, suitable cryoprotectants like disaccharides need to be added, which prevent fusion, aggregation and leakage of encapsulated compounds. in the present study, several disaccharides like lactose, mannitol, maltodextrin, and trehalose were evaluated for their cryoprotective effects at varying ratios. lipid : trehalose at ratio of 1:10 was found to be most suitable, as less drug leakage and minimal change of particle size was observed after freeze drying. 1showed one sharp endotherm at 288.9 with onset temperature 269.5 and recovery temperature 297.3. freeze dried liposomes gave thermal events in the range of 117.5 - 144.1, and a big endothermic peak at 279.4 immediately followed by exotherm with peak temperature at 296.5. this event indicated the interaction of trehalose with phospholipids of liposomes during freeze drying. dsc thermograms for (a) etoposide, (b) cholesterol, (c) pg90h, (d) pg90 g, (e) trehalose, (f) freeze dried liposomes, (g) freeze dried liposomes without trehalose, (h) blank liposomes without trehalose, which shows there is interaction of trehalose with phospholipids of liposomes during freeze drying. the sharp endotherm of etp at 288.9 was masked when freeze dried etp - loaded liposomes were analyzed. the broad endotherm started at 245.6 with peak of endotherm at 279.4 and followed immediately by exotherm at 297.3. so to detect the presence of etp in liposomes, a dsc scan was carried out on freeze dried blank and etp loaded liposomes without trehalose. blank liposomes did not show any thermal event for the entire temperature range studied, while a small broad endotherm was observed at 283 in etp loaded liposomes indicating presence of the drug. the shift in the endotherm from 288.9 may be due to the presence of lipids. the liposomes appeared as bright spheres surrounded by dark thick layer showing large internal aqueous core by one or two lamelle. the esem analysis showed the freeze dried trehalose particles as irregular porous particles as shown in fig. esem photomicrograph of freeze dried trehalose particles and etp liposomes esem photomicrograph shows that freeze dried trehalose particles (a) were of irregular porous particles and freeze dried liposomes (b) appeared as aggregated particles with lipids on the surface. esem was used to study liposome formation from freeze dried liposomes and to provide an alternative method to observe liposome formation after rehydration. dynamic formation of liposomes was monitored by placing drop of saline on the tab and analyzing over a period of 5 min. the esem photographs of hydrated freeze dried liposomes at 0 min, 2 min and after complete hydration (5 min) are shown in figs. 4a4c respectively. at 0 min, after adding few drops of saline on tab with freeze dried liposomes, trehalose started dissolving in saline and liposome formation was initiated due to budding off mechanism. following sufficient hydration of the lipids on trehalose particles, spherical structures were clearly seen, after 2 min liposomes were completely formed, and after 5 min entire area was filled with formed liposomes. esem photomicrograph of freeze dried etp liposome hydrated with saline shows initiation of formation of liposome at 0 min (a), further at 2 min there was sufficient hydration of lipids and spherical structures indicates complete formation of liposomes (b), and finally at 5 min entire area was filled with formed liposomes (c). the particle size increased in all the liposomes irrespective of the formulation variables indicating aggregation of liposomes after freeze drying. however, no significant difference was observed in drug content after freeze drying of etp liposomes indicating minimum leakage. table 1 shows the result of drug content and particle size before and after freeze drying. the drug release was sustained for more than 70 h for all batches but there was significant difference in release rate of the drug between the batches as shown in fig. the formulation variables like cholesterol content, and lipid content (90 g and 90 h), played a major role in the drug release. 5 shows the time required for 80% drug release (t80%) with respect to lipid ratio [lipid (90g:90h):cholesterol ]. thus it can be concluded that, the permeability of the bilayer is strongly influenced by its constituents. it can be seen that high amount of cholesterol significantly reduced the t80%.the fpf of both liposomal dispersions as well as freeze dried liposomes ranged around 50%, revealing good respirable properties. effect of composition of liposomes on t80% it shows the time required for 80% drug release (t80%) with respect to lipid composition, [lipid (90g:90h):cholesterol ] stability of freeze dried liposomes and aqueous dispersion of etp liposomes was assessed at 2 - 8 and at ambient conditions (284). stored samples were evaluated periodically for change in colour, appearance, particle size and drug leakage. the change in particle size as well as drug leakage from the freeze dried liposomes and aqueous suspension of liposomes in stability studies are as given in table 2. drug leakage and particle size of stability samples of etp liposomes sedimentation of lipid phase was observed in aqueous liposomes which upon gentle shaking easily get redispersed, and no colour change was evident upon storage. microscopic observation of aqueous liposomes showed absence of drug precipitation ; however the particle size was markedly increased. at 2 - 8, aqueous liposomes showed lesser aggregates compared to the ones stored at ambient conditions in which aggregates of liposomal vesicles were observed. particle size distribution of aqueous liposomes stored at different conditions showed shifting towards increasing the particle size and the range of particle size several reports on light scattering studies of the coalescence kinetics of liposomes have revealed that the average cluster size grows with time following a characteristic behaviour. liposomes were easily formed when stored samples of freeze dried liposomes were mixed with saline on gentle shaking, and no color change was seen upon storage. no significant difference was observed in the particle size of freeze dried liposomes stored for a period of six month at different conditions. the drug leakage was significantly less in freeze dried liposomes compared to aqueous liposomes, and was observed to be in accordance with the increase in particle size. it was observed that increase in particle size was directly associated with the drug leakage. the effect on fpf of the freshly prepared liposomes fpf was determined using tsi. for freeze dried liposomes, increase in storage time did not significantly change the fpf in six months storage at both the storage conditions. on the other hand, aqueous dispersion showed decrease in fpf at the different storage conditions. drug delivery to or via the respiratory tree has been a long standing pharmaceutical objective. for locally acting agents it is desirable to confine the action of the drug to the lungs in order to eliminate unintended side effects, which might result following absorption and distribution to other extra vascular sites. oral inhalation is often the preferred route in order that such effects can be minimized. here, etp - loaded liposomes were developed optimized and evaluated for its suitability for pulmonary sustained drug delivery for treatment of cancer. further in vivo safety and deposition studies in suitable animal models will give better insight for clinical applications of the inhalable etp liposomes. | etoposide is a semisynthetic compound, widely used in treatment of non small cell lung cancer. however, frequent dosing and adverse effects remain a major concern in the use of etoposide. liposomal systems for pulmonary drug delivery have been particularly attractive because of their compatibility with lung surfactant components. in the present investigation, pulmonary liposomal delivery system of etoposide was prepared by film hydration method. various parameters were optimized with respect to entrapment efficiency as well as particle size of etoposide liposomes. for better shelf life of etoposide liposomes, freeze drying using trehalose as cryoprotectant was carried out. the liposomes were characterized for entrapment efficiency, particle size, surface topography, and in vitro drug release was carried out in simulated lung fluid at 37 at ph 7.4. the respirable or fine particle fraction was determined by using twin stage impinger. the stability study of freeze dried as well as aqueous liposomal systems was carried out at 2 - 8 and at ambient temperature (284). the freeze dried liposomes showed better fine particle fraction and drug content over the period of six months at ambient as well as at 2 - 8 storage condition compared to aqueous dispersion of liposomes. |
the present case involved a 62-year - old male with a large left - sided inguinoscrotal hernia. a ct scan and a clinical examination led to a diagnosis of a giant left - sided amyand 's hernia. the hernia was repaired using the ultrapro hernia system (uhs), and the patient exhibited an uneventful postoperative course. a 62-year - old male visited our hospital due to a left inguinoscrotal hernia, which had been gradually increasing in size for a few years. in addition to increasing in size, the hernia had also become incarcerated within the past 10 months, and while it was reducible it was causing the patient considerable discomfort, especially during walking and standing. a large left - sided inguinoscrotal hernia, which extended to the midpoint of the inner thigh while the patient was standing, was detected during a clinical examination (fig.1). the scrotal skin did not display any signs of cellulitis or ulceration, nor did the skin over the hernia exhibit tenderness. a right - sided inguinal hernia, which did not extend to the scrotum, was also detected. dislocation of the ileum, appendix, cecum, and ascending colon into the left - sided inguinoscrotal hernial sac was depicted by computed tomography (fig.2). a computed tomography scan depicting dislocation of the ileum, appendix, cecum, and ascending colon (arrows) into a left - sided inguinoscrotal hernial sac, and a right - sided inguinal hernia (arrowhead). under general endotracheal anesthesia, a medial incision was made in the lower abdomen, and the ileum, appendix, cecum, and ascending colon were reduced into the peritoneal cavity. then, bilateral standard oblique inguinal incisions were made, before the hernial sac was excised. next, a large ultrapro hernia system (uhsl) was used to reinforce the bilateral inguinal regions (fig.3). the uhs underlays (arrows) were installed into the bilateral preperitoneal spaces and (b) uhs. monitoring of the patient 's airway pressure determined that compartment - enlarging procedures were not necessary. specifically, he was extubated during the immediate postoperative period and did not require any mechanical ventilatory support. amyand 's hernia, which is a rare condition, involves the entrapment of the appendix within an inguinal hernia. the incidence of a normal appendix becoming trapped within an inguinal hernial sac is about 1%. this is probably because right - sided inguinal hernias are more common than left - sided hernias, and the appendix is normally found on the right side. left - sided amyand 's hernias are rare, and it has been suggested that they could be associated with a mobile cecum, situs inversus, or intestinal malrotation. the present case involved a mobile cecum, but not situs inversus or malrotation of the gut. regarding appendectomy, the indications for the procedure in cases of amyand 's hernia depend on the mode of presentation. in cases involving patients with normal appendices, appendectomy is not necessary. on the other hand the presence of pus and perforation of the appendix are absolute contraindications for mesh - based hernia repair procedures. appendectomy was not performed in the present case because the patient 's appendix was not inflamed, and the procedure could have resulted in a prosthetic infection. hernias that extend below the midpoint of the inner thigh while the patient is standing are known as giant inguinoscrotal hernias. such hernias are rare and are usually only seen in cases in which the patient has ignored the problem for years. the forced reduction of hernial contents into the peritoneal cavity could affect the patient 's intra - abdominal and/or intra - thoracic pressure levels (i.e., induce abdominal compartment syndrome), which might increase the risk of severe respiratory or cardiac failure and result in a worsening of morbidity and mortality rates. in addition, closing the abdominal wall under tension has been linked with a greater risk of wound breakdown ; i.e., 30% of patients who undergo such procedures suffer from hernia recurrence and/or wound dehiscence. concerning loss of domain, various methods for combatting this issue based on the debulking of the abdominal contents and the progressive distention of the abdominal wall have been described. as for abdominal wall distention, older techniques such as phrenicectomy, iatrogenic incisional hernia, and musculoskeletal flaps are no longer used. goni moreno was the first to describe the use of preoperative pneumoperitoneum preparation (ppp) during the treatment of large ventral hernias, and the technique has subsequently been successfully employed to treat inguinal hernias. however, it has been reported to cause thin hernial sacs (instead of the abdominal cavity) to expand. however, a number of techniques for separating the components of the abdominal wall have been reported and have produced good results. regarding the debulking of the abdominal contents, this method, which is usually applied to the small intestine, greater omentum, or colon, results in the acquisition of a sufficiently spacious abdominal cavity. however, previous studies have found that this approach is associated with a greater risk of complications, e.g., prosthetic infection or anastomotic leakage. in our case, monitoring of the patient 's airway pressure determined that compartment - enlarging procedures were not required. repairing giant hernias using conventional methods thus, we employed the uhs, which strengthens the posterior wall of the inguinal canal in two ways. initially, the floor of the inguinal canal is supported from the posterior side by placing an underlay under the transversalis fascia, as is performed in laparoscopic repair, and then the anterior side of the canal is supported by installing an overlay, as is performed in lichtenstein repair. instead of using the conventional method, giant hernias should be repaired with synthetic mesh due to the high recurrence rate associated with the former approach. thus, appendectomy should not be performed in cases in which the appendix is not inflamed. | key clinical messagethe present case involved a 62-year - old male with a large left - sided inguinoscrotal hernia. a ct scan and a clinical examination led to a diagnosis of a giant left - sided amyand 's hernia. the hernia was repaired using the ultrapro hernia system (uhs), and the patient exhibited an uneventful postoperative course. |
ischaemic heart disease (ihd) is the single leading cause of death worldwide, accounting for 11.2% of all deaths globally in 2011, the last year for which a reliable estimate is available. our group has previously studied the global epidemiology of ihd from 1995 to 2009. in this paper we provide an update, reporting on the burden of ihd worldwide from 2001 to 2012 using mortality data collected by the world health organisation (who). our update also provides a geographical analysis of ihd mortality using a cartogram, a world map in which the area of each country is algorithmically transformed so that it is proportional to a measured variable for that country, in this case, number of deaths. the value of cartograms over and above typical map display is that it illustrates the spatial representation of a variable of interest whilst retaining the semblance of a world map. analysing the emerging global pattern of ihd mortality at regular time intervals is necessary to inform and update public health strategy. collection of raw mortality data categorised by cause of death is integral to this, but limited raw data seem to be available even from countries whose resource position might be expected to permit an exemplary role in the promptness and completeness of data collection and disclosure. public health bodies such as the who and the institute for health metrics and evaluation (ihme) therefore use sophisticated methods to provide estimates. the disadvantage of estimation is the inevitable introduction of some degree of error. in this paper this comprises all deaths registered by national civil registration systems submitted to the who, with underlying cause of death coded by the relevant national authority using the international statistical classification of diseases and related health problems (icd) 9th and 10th revisions. gross national income per capita was extracted from the national accounts main aggregates database from the united nations statistics division. countries were categorised into high (greater than us $ 12,275), upper middle ($ 3976 to $ 12,275 inclusive), lower middle ($ 1006 to $ 3975 inclusive) and low (less than $ 1006) income countries based on world bank income grouping. ihd deaths for countries with available age - specific data were standardised to the who world standard population to allow direct comparison of ihd mortality rates between countries. the small numbers of deaths in the unspecified age group were discounted in this analysis. a cartogram was designed to illustrate the worldwide distribution of ihd deaths using 2010 estimates of ihd deaths from the ihme. the area for each country is algorithmically transformed so that it is proportional to the number of deaths from ihd in each country. after transforming the area to represent number of deaths (the first variable of interest), we use gradations of colour to represent standardised mortality ratio (the second variable of interest) with darker shades of red representing countries with higher standardised mortality ratios. the cartogram was generated using scapetoad - v11 and r (version 3.0, r foundation) with the following packages : shapefiles, rgdal, rcolorbrewer and classint. this comprises all deaths registered by national civil registration systems submitted to the who, with underlying cause of death coded by the relevant national authority using the international statistical classification of diseases and related health problems (icd) 9th and 10th revisions. gross national income per capita was extracted from the national accounts main aggregates database from the united nations statistics division. countries were categorised into high (greater than us $ 12,275), upper middle ($ 3976 to $ 12,275 inclusive), lower middle ($ 1006 to $ 3975 inclusive) and low (less than $ 1006) income countries based on world bank income grouping. ihd deaths for countries with available age - specific data were standardised to the who world standard population to allow direct comparison of ihd mortality rates between countries. the small numbers of deaths in the unspecified age group were discounted in this analysis. a cartogram was designed to illustrate the worldwide distribution of ihd deaths using 2010 estimates of ihd deaths from the ihme. the area for each country is algorithmically transformed so that it is proportional to the number of deaths from ihd in each country. after transforming the area to represent number of deaths (the first variable of interest), we use gradations of colour to represent standardised mortality ratio (the second variable of interest) with darker shades of red representing countries with higher standardised mortality ratios. the cartogram was generated using scapetoad - v11 and r (version 3.0, r foundation) with the following packages : shapefiles, rgdal, rcolorbrewer and classint. table 1 shows the burden of ihd deaths in 2010 in countries for which data were available. of these 71 countries, russia, the united states of america and ukraine account for the largest numbers of deaths. startlingly, ukraine had almost as many deaths as the united states of america yet the united states of america 's population is over 6 times larger than that of ukraine. the global distribution of deaths from ihd has been estimated for the majority of countries in 2010. 1 in the form of a map where country area has been transformed to provide a visual representation of the numbers of deaths : larger country areas indicate larger numbers of deaths. the five countries with the greatest numbers of estimated deaths are india, china, russia, the united states of america and ukraine in descending order. the five with the highest rates are turkmenistan, ukraine, belarus, uzbekistan and kazakhstan in descending order. 2a d shows the large increase in mortality in each sex in the 4 countries selected (ukraine, russia, the united kingdom and japan) because their mortality rates are some of the highest and lowest in the world and recent data was available. in men there was a 2.3- to 2.9-fold increase in ihd mortality per decade and a 3.2- to 4.5-fold increase in women. fig. 3a and b shows the trends in crude death rates and age standardised mortality respectively in 20 countries between 2001 and 2012 for which extensive longitudinal data were available, predominantly european countries. in these countries, in contrast, there has been a general decline in directly standardised mortality rates in these countries between 2001 and 2012 (fig. these data include both high income countries as well as countries in eastern europe where, despite high standardised mortality rates, an improvement in reducing these rates is noticeable in recent years. at least one year of ihd mortality data was available for 137 countries between 2001 and 2012. data were not available for any country for 2013. the who states that this is because countries usually submit data to who within 1218 months after the closure of their records for the calendar year. over half of the world 's countries (113/216) have provided ihd mortality data for 2008 or later. table 1 shows the burden of ihd deaths in 2010 in countries for which data were available. of these 71 countries, russia, the united states of america and ukraine account for the largest numbers of deaths. startlingly, ukraine had almost as many deaths as the united states of america yet the united states of america 's population is over 6 times larger than that of ukraine. the global distribution of deaths from ihd has been estimated for the majority of countries in 2010. 1 in the form of a map where country area has been transformed to provide a visual representation of the numbers of deaths : larger country areas indicate larger numbers of deaths. the five countries with the greatest numbers of estimated deaths are india, china, russia, the united states of america and ukraine in descending order. the five with the highest rates are turkmenistan, ukraine, belarus, uzbekistan and kazakhstan in descending order. 2a d shows the large increase in mortality in each sex in the 4 countries selected (ukraine, russia, the united kingdom and japan) because their mortality rates are some of the highest and lowest in the world and recent data was available. in men there was a 2.3- to 2.9-fold increase in ihd mortality per decade and a 3.2- to 4.5-fold increase in women. fig. 3a and b shows the trends in crude death rates and age standardised mortality respectively in 20 countries between 2001 and 2012 for which extensive longitudinal data were available, predominantly european countries. in these countries, in contrast, there has been a general decline in directly standardised mortality rates in these countries between 2001 and 2012 (fig. these data include both high income countries as well as countries in eastern europe where, despite high standardised mortality rates, an improvement in reducing these rates is noticeable in recent years. at least one year of ihd mortality data was available for 137 countries between 2001 and 2012. data were not available for any country for 2013. the who states that this is because countries usually submit data to who within 1218 months after the closure of their records for the calendar year. over half of the world 's countries (113/216) have provided ihd mortality data for 2008 or later. ihd mortality data show that the largest numbers of ihd deaths occurred in russia, the united states of america and ukraine (table 1). however, estimates indicate that india and china, countries for which no recent mortality data is available, have even larger numbers of deaths (fig. 1). the marked age dependence of ihd mortality rate is concordant between countries even when the countries have very different mortality rates. provision of ihd mortality data from most countries is limited for reasons which are not clear. displaying a modified map with country areas representing the numbers of deaths is a convenient way to convey to the reader the global geographical distribution of ihd mortality events. simultaneously the colour of each country can be used to represent the standardised mortality rate in that country. this allows the reader to both assess the relative cardiovascular risk of different countries and the magnitude of its contribution to the global burden of ihd deaths. the cartogram shows that india and china contribute a large proportion of the planet 's ihd deaths out of proportion to their true land area. part of this is because of higher population density in these countries. in the case of india there is a contribution of higher standardised mortality rate as can be seen from its colour being darker than, for example, western european countries. however, the standardised mortality rate in china is comparable to that of western europe as can be seen from their colours. the cartogram shows that many countries of the former soviet union and central asia have a high standardised mortality rate as evidenced by the dark colouring. only just over half the countries have provided any data after 2007. of the remainder a small group of 24 countries the remaining 79 have never provided ihd mortality data in the icd9 or icd10 formats to the who. these countries include india and china which are estimated to be experiencing large numbers of ihd deaths making a major contribution to the global burden of ihd mortality. whilst resources may be limited in some countries like india, they may be less so in countries like china and should not be so in the united arab emirates whose gni per capita is in excess of us $ 40,000. where data are missing, methods can be used to estimate mortality but there is a risk of introducing error. provision of this information to the who would assist with evaluation of the global burden of disease and in national and international health resource planning. the task is not insuperable, as evidenced by delivery of data from countries such as serbia and the republic of moldova with creditable timeliness. displaying a modified map with country areas representing the numbers of deaths is a convenient way to convey to the reader the global geographical distribution of ihd mortality events. simultaneously the colour of each country can be used to represent the standardised mortality rate in that country. this allows the reader to both assess the relative cardiovascular risk of different countries and the magnitude of its contribution to the global burden of ihd deaths. the cartogram shows that india and china contribute a large proportion of the planet 's ihd deaths out of proportion to their true land area. part of this is because of higher population density in these countries. in the case of india there is a contribution of higher standardised mortality rate as can be seen from its colour being darker than, for example, western european countries. however, the standardised mortality rate in china is comparable to that of western europe as can be seen from their colours. the cartogram shows that many countries of the former soviet union and central asia have a high standardised mortality rate as evidenced by the dark colouring. only just over half the countries have provided any data after 2007. of the remainder a small group of 24 countries the remaining 79 have never provided ihd mortality data in the icd9 or icd10 formats to the who. these countries include india and china which are estimated to be experiencing large numbers of ihd deaths making a major contribution to the global burden of ihd mortality. whilst resources may be limited in some countries like india, they may be less so in countries like china and should not be so in the united arab emirates whose gni per capita is in excess of us $ 40,000. where data are missing, methods can be used to estimate mortality but there is a risk of introducing error. provision of this information to the who would assist with evaluation of the global burden of disease and in national and international health resource planning. the task is not insuperable, as evidenced by delivery of data from countries such as serbia and the republic of moldova with creditable timeliness. in many countries which have provided substantial longitudinal data there has been a continuing trend of reduction in age - specific death rates. data do not become available for international comparison quickly, with 103 of the world 's 216 countries not having provided any data after 2007. greater focus on providing reliable data would greatly assist international efforts on strategic healthcare planning. visual display of ihd mortality on a cartogram can assist recognition of the separate but related issues of age - standardised mortality and total ihd mortality burden in individual countries. however, there are many countries with much higher age - standardised mortality rates which may in the future contribute progressively greater numbers to global ihd deaths. | backgroundischaemic heart disease (ihd) is the leading cause of death worldwide and its prevention is a public health priority.methodwe analysed worldwide ihd mortality data from the world health organisation as of february 2014 by country, age and income. age - standardised mortality rates by country were calculated. we constructed a cartogram which is an algorithmically transformed world map that conveys numbers of deaths in the form of spatial area.resultsof the countries that provided mortality data, russia, the united states of america and ukraine contributed the largest numbers of deaths. india and china were estimated to have even larger numbers of deaths. death rates from ihd increase rapidly with age. crude mortality rates appear to be stable whilst age - standardised mortality rates are falling. over half of the world 's countries (113/216) have provided ihd mortality data for 2008 or later. of these, 13 countries provided data in 2012. no countries have yet provided 2013 data. of the 103 remaining countries, 24 provided data in 2007 or earlier, and 79 have never provided data in the icd9 or icd10 format.conclusionsin the countries for which there are good longitudinal data, predominantly european countries, recent years have shown a continuing decline in age - standardised ihd mortality. however, the progressive aging of populations has kept crude ihd mortality high. it is not known whether the pattern is consistent globally because many countries have not provided regular annual data including wealthy countries such as the united arab emirates and large countries such as india and china. |
according to world health statistics 2015, which was published by the world health organization (who), the use of cesarean section in clinical delivery was very high in all countries for the period 20072014. in china, the reported cesarean rate was 27%, which is significantly higher than the goal of less than 15% proposed by who during the 1980s. an even higher percentage of patients receiving in vitro fertilization - embryo transfer (ivf - et) accept cesarean section due to the higher cost of embryo implantation and the concerns for the babies. with abolishing of the single - child policy in china, the number of patients with a history of cesarean section choosing ivf - et as an alternative strategy for their second progeny will increase, as expected. on the one hand, it has been reported in the literature that a cesarean section scar can reduce the chance of embryo implantation and lead to spontaneous abortion. det increases the embryo implantation and pregnancy rate in an ivf transfer cycle. on the other hand, det will significantly increase the incidence of iatrogenic multiple pregnancies (imps) as well. most uterine rupture cases occur in women with a uterine scar, and multiple pregnancies are a risk factor for uterine rupture for patients who have had a previous cesarean section [79 ]. when a pregnancy implants on a cesarean fibrous tissue scar, a cesarean scar pregnancy (csp) occurs. multi - embryo transfer is also a risk factor for csp. as a rare and dangerous type of ectopic pregnancy in ivf - et, the severe complications of csp include placenta previa / accreta, uterine rupture, and life - threatening hemorrhage [1215 ]. what role should previous cesarean section play in affecting clinical pregnancy outcomes and avoiding the complications of ivf ? we conducted a retrospective study to investigate if previous cesarean section affects embryo implantation rate and pregnancy outcomes in patients with previous cesarean section compared to patients with a history of vaginal delivery. moreover, in this article, we focus on the number of transferred embryos and assess the clinical efficacy and safety of eset in patients who have a previous cesarean scar. institutional ethics board approval was obtained from the reproductive medical ethics committee of nanjing drum tower hospital (protocol number : 2014002). all participating patients were formally informed of the potential academic use of their clinical data in the future when admitted in the hospital, and the written informed consents were obtained from all participants as well. a total of 231 patients with history of delivery who received ivf - et technology in the reproductive medical center of our hospital from january 2012 to september 2014 were enrolled in this study. patients who did not receive embryo transfer during the oocyte retrieval cycle were excluded. according to the previous ways of delivery, patients were divided into two groups : previous cesarean section group (n=130) and vaginal delivery group (n=101). after the patients were informed of all benefits and risks of single - embryo or double - embryo transfer, they were permitted to choose one - embryo transfer (eset) or double - embryo transfer (det) depending on their own desire. therefore, patients in the cesarean section scar group were further divided into a, eset group (n=56) and a det group (n=74). one patient experienced two previous cesarean deliveries ; however, the others experienced just one. ultrasound measurement of scar thickness on the uterus of those who previously gave birth by caesarean section was done before ivf. the mid - luteal lupron (luteal phase down - regulation) protocol was applied for all participants. for the patients with ovulation, administration of gonadotrophin - releasing hormone agonist (gnrh - a) was initiated in mid - luteal phase. oral contraceptives were first given to the patients without ovulation for 1517 consecutive days starting from the fourth day of menses. then, gnrh - a was administered to them for 1415 consecutive days as well. ovarian stimulation was carried out with either recombinant follicle - stimulating hormone (fsh) or human menopausal gonadotropin (hmg). when follicles reached pre - ovulatory size (1822 mm), 5,000 iu of human chorionic gonadotropin (hcg) was administered. fertilization and embryo culture manipulation were conducted according to the general protocol of our center. briefly, short - term fertilization or intracytoplasmic sperm injection (icsi) was performed 3 or 4 hours later after oocyte retrieval. formation of pro - nucleus was monitored 1719 hours after insemination. at 6668 hours after insemination, embryos without multi - nucleus displaying more than 6 blastomeres and fewer than 20% fragments were selected for b - ultrasound - guided transfer. urine pregnancy tests or blood hcg tests were initially conducted 1416 days after embryo transfer. transvaginal ultrasound was performed 3035 days after embryo transfer to confirm the progeny by observation of a gestational sac or fetal heartbeat. basic information about the patients, such as age, years after cesarean section, body mass index (bmi), gonadotropin (gn) dose, causes of infertility, fertilization methods, and endometrial thickness for embryo transfer, was recorded. for the comparison of fertilization and embryo development between groups of patients, the fertilization rate, cleavage rate, number of cells in the transferred embryo, and embryo cryopreservation cycle rate were recorded and compared. moreover, pregnancy rate, embryo implantation rate, rate of multiple pregnancies, early and mid - term abortion rate, ectopic pregnancy rate, take - home baby rate, gestational age of delivery, birth weight, and the rate of occurrence of birth defects were compared between groups as well for understanding the pregnancy, delivery outcomes, and neonatal conditions comparison of mean values between two groups was assessed by the student s t test. institutional ethics board approval was obtained from the reproductive medical ethics committee of nanjing drum tower hospital (protocol number : 2014002). all participating patients were formally informed of the potential academic use of their clinical data in the future when admitted in the hospital, and the written informed consents were obtained from all participants as well. a total of 231 patients with history of delivery who received ivf - et technology in the reproductive medical center of our hospital from january 2012 to september 2014 were enrolled in this study. patients who did not receive embryo transfer during the oocyte retrieval cycle were excluded. according to the previous ways of delivery, patients were divided into two groups : previous cesarean section group (n=130) and vaginal delivery group (n=101). after the patients were informed of all benefits and risks of single - embryo or double - embryo transfer, they were permitted to choose one - embryo transfer (eset) or double - embryo transfer (det) depending on their own desire. therefore, patients in the cesarean section scar group were further divided into a, eset group (n=56) and a det group (n=74). one patient experienced two previous cesarean deliveries ; however, the others experienced just one. ultrasound measurement of scar thickness on the uterus of those who previously gave birth by caesarean section was done before ivf. the mid - luteal lupron (luteal phase down - regulation) protocol was applied for all participants. for the patients with ovulation, administration of gonadotrophin - releasing hormone agonist (gnrh - a) was initiated in mid - luteal phase. oral contraceptives were first given to the patients without ovulation for 1517 consecutive days starting from the fourth day of menses. then, gnrh - a was administered to them for 1415 consecutive days as well. ovarian stimulation was carried out with either recombinant follicle - stimulating hormone (fsh) or human menopausal gonadotropin (hmg). when follicles reached pre - ovulatory size (1822 mm), 5,000 iu of human chorionic gonadotropin (hcg) was administered. fertilization and embryo culture manipulation were conducted according to the general protocol of our center. briefly, short - term fertilization or intracytoplasmic sperm injection (icsi) was performed 3 or 4 hours later after oocyte retrieval. formation of pro - nucleus was monitored 1719 hours after insemination. at 6668 hours after insemination, embryos without multi - nucleus displaying more than 6 blastomeres and fewer than 20% fragments were selected for b - ultrasound - guided transfer. urine pregnancy tests or blood hcg tests were initially conducted 1416 days after embryo transfer. transvaginal ultrasound was performed 3035 days after embryo transfer to confirm the progeny by observation of a gestational sac or fetal heartbeat. basic information about the patients, such as age, years after cesarean section, body mass index (bmi), gonadotropin (gn) dose, causes of infertility, fertilization methods, and endometrial thickness for embryo transfer, was recorded. for the comparison of fertilization and embryo development between groups of patients, the fertilization rate, cleavage rate, number of cells in the transferred embryo, and embryo cryopreservation cycle rate were recorded and compared. moreover, pregnancy rate, embryo implantation rate, rate of multiple pregnancies, early and mid - term abortion rate, ectopic pregnancy rate, take - home baby rate, gestational age of delivery, birth weight, and the rate of occurrence of birth defects were compared between groups as well for understanding the pregnancy, delivery outcomes, and neonatal conditions. comparison of mean values between two groups was assessed by the student s t test. generally, age was considered as the key factor affecting the clinical outcome of ivf. patients in different age stages have different pregnancy rates. in our study, the mean age of the group with cesarean section scar (33.614.21 years) was lower than that of the vaginal delivery group (34.854.03 years). to illustrate the influence of age, we divided the patients into groups according to age (table 1). patients were further divided into sub - groups based on their age (37 years), and no significant difference was observed between the two groups. as demonstrated in table 1, other factors such as bmi, dosage of gn, causes of infertility, fertilization methods, and endometrial thickness were also similar between the two groups. statistically significant differences were not observed for embryo development - related factors such as the number of retrieved oocytes (9.954.60 vs. 9.394.20), fertilization rate (87.79% vs. 89.35%), cleavage rate (97.80% vs. 97.99%), cell number of transferred embryo (8.150.97 vs. 7.970.90), and embryo cryopreservation cycle rate (74.62% vs. 72.28%). the presence of a previous cesarean section scar did not affect fertilization and embryo development after ivf. pregnancy and delivery outcomes of the participants receiving ivf - et are shown for the previous cesarean section group and the previous vaginal delivery group (figure 1). despite the fact that the percentage of patients receiving single - embryo transfer in the previous cesarean section group was significantly higher than that in the previous vaginal delivery group (43.08% vs. 6.93% ; p=0.000), the clinical pregnancy rates (58.46% vs. 54.46%) and take - home baby rates (45.38% vs. 40.59%) between the two groups were similar. the implantation rate in the previous cesarean section group was higher than that in the previous vaginal delivery group (49.02% vs. 38.46% ; p=0.034). the occurrence of abnormal pregnancy such as multiple pregnancies, abortion, and ectopic pregnancy also was similar between the two groups (32.89% vs. 38.18%, 21.05% vs. 23.64%, and 1.32% vs. 0) (table 2). there were no statistically significant differences between the previous cesarean section group (22.02%,13/59) and the previous vaginal delivery group (12.20%, 5/41) regarding preterm birth related to delivery history. in singleton deliveries among the two groups, however, a significantly lower gestational age of delivery (35.242.46 vs. 37.001.24 ; p=0.022) and higher preterm birth rates (58.82% vs. 21.43% ; p=0.036) were observed in patients in the previous cesarean section group who experienced multiple delivery (table 3). we further divided patients who had previous cesarean section into groups receiving eset or det. for general information such as age, years after cesarean section, bmi, dosage of gn, causes of infertility, fertilization methods, and endometrial thickness although the fertilization rate in the eset group was lower than that in det group (84.14% vs. 90.37% ; p=0.024), the fertilization rates in both groups were higher than the 65% industry standard (table 4). the implantation outcomes (50.00% vs. 48.65%) for the eset and det groups were similar ; the clinical pregnancy rate (50.00% vs. 64.86%), abortion rate (25.00% vs. 18.75%), and take - home baby rate (37.50% vs. 51.35%) in these two groups also did not display statistically significant differences (table 5). however, 50% of ivf pregnancies were multiples in the det group and carried higher risk to the neonates compared with singleton pregnancy (table 5). at delivery, multiple det babies had significantly lower gestational age and birth weight than singleton eset babies. there was one case of csp and three cases of birth defects (one case of congenital pyloric obstruction and two cases of congenital heart disease) in the det group (table 6). generally, age was considered as the key factor affecting the clinical outcome of ivf. patients in different age stages have different pregnancy rates. in our study, the mean age of the group with cesarean section scar (33.614.21 years) was lower than that of the vaginal delivery group (34.854.03 years). to illustrate the influence of age, we divided the patients into groups according to age (table 1). patients were further divided into sub - groups based on their age (37 years), and no significant difference was observed between the two groups. as demonstrated in table 1, other factors such as bmi, dosage of gn, causes of infertility, fertilization methods, and endometrial thickness were also similar between the two groups. statistically significant differences were not observed for embryo development - related factors such as the number of retrieved oocytes (9.954.60 vs. 9.394.20), fertilization rate (87.79% vs. 89.35%), cleavage rate (97.80% vs. 97.99%), cell number of transferred embryo (8.150.97 vs. 7.970.90), and embryo cryopreservation cycle rate (74.62% vs. 72.28%). the presence of a previous cesarean section scar did not affect fertilization and embryo development after ivf. pregnancy and delivery outcomes of the participants receiving ivf - et are shown for the previous cesarean section group and the previous vaginal delivery group (figure 1). despite the fact that the percentage of patients receiving single - embryo transfer in the previous cesarean section group was significantly higher than that in the previous vaginal delivery group (43.08% vs. 6.93% ; p=0.000), the clinical pregnancy rates (58.46% vs. 54.46%) and take - home baby rates (45.38% vs. 40.59%) between the two groups were similar. the implantation rate in the previous cesarean section group was higher than that in the previous vaginal delivery group (49.02% vs. 38.46% ; p=0.034). the occurrence of abnormal pregnancy such as multiple pregnancies, abortion, and ectopic pregnancy also was similar between the two groups (32.89% vs. 38.18%, 21.05% vs. 23.64%, and 1.32% vs. 0) (table 2). there were no statistically significant differences between the previous cesarean section group (22.02%,13/59) and the previous vaginal delivery group (12.20%, 5/41) regarding preterm birth related to delivery history. in singleton deliveries among the two groups, however, a significantly lower gestational age of delivery (35.242.46 vs. 37.001.24 ; p=0.022) and higher preterm birth rates (58.82% vs. 21.43% ; p=0.036) were observed in patients in the previous cesarean section group who experienced multiple delivery (table 3). we further divided patients who had previous cesarean section into groups receiving eset or det. for general information such as age, years after cesarean section, bmi, dosage of gn, causes of infertility, fertilization methods, and endometrial thickness, no significant difference was observed between the two groups (table 4). although the fertilization rate in the eset group was lower than that in det group (84.14% vs. 90.37% ; p=0.024), the fertilization rates in both groups were higher than the 65% industry standard (table 4). the implantation outcomes (50.00% vs. 48.65%) for the eset and det groups were similar ; the clinical pregnancy rate (50.00% vs. 64.86%), abortion rate (25.00% vs. 18.75%), and take - home baby rate (37.50% vs. 51.35%) in these two groups also did not display statistically significant differences (table 5). however, 50% of ivf pregnancies were multiples in the det group and carried higher risk to the neonates compared with singleton pregnancy (table 5). at delivery, multiple det babies had significantly lower gestational age and birth weight than singleton eset babies. there was one case of csp and three cases of birth defects (one case of congenital pyloric obstruction and two cases of congenital heart disease) in the det group (table 6). cesarean section is applied in high - risk pregnancies by using a surgical method. with advances in cesarean section technology and improvements of anesthesia as well as perioperative monitoring, the safety of cesarean section is guaranteed. however, abuse of cesarean section without medical needs resulted in rapid rising of the cesarean section rate throughout the twentieth century with a substantial increase in the last 30 years all over the world. the caesarean section rates of 1820% in developed countries and 27% in china far exceed the 15% that is recommended by the who. with the single - child policy recently abolished, there will be an increased need for patients who have previously given birth by caesarean section to choose ivf technology as an alternative strategy for their second pregnancy. cesarean section does not affect function of the ovary or quality of the ovum unless the hemorrhage caused by previous surgery affects the blood supplies for the uterus or the ovary. in our study, patients with a uterine scar had a 58.46% clinical pregnancy rate, which was similar to that in vaginal delivery group ; this suggests that the existence of a uterine scar does not affect embryo implantation. however, the location of embryo implantation is the major factor affecting the pregnancy condition in patients with a cesarean scar. if the location of embryo implantation is outside the scar region, prior cesarean section has little impact on the outcome in the early stage of pregnancy. however, it is more difficult to conduct surgical abortion if the embryo is implanted on the cesarean scar. moreover, due to the weak myometrial fibers at the scar and the increased volume of gestational sac wrapping in scar tissue, there will be increased risk for massive hemorrhage or even uterine rupture for csp. furthermore, the risk of spontaneous uterine rupture is also increased in patients experiencing multiple cesarean sections. according to the regulation in china, the number of transferred embryos is limited to fewer than two during the application of assisted reproductive technology unless the recipient s age is more than 35 years or there was a previous failure of ivf - ef. with double - embryo ivf - et, the risk of iatrogenic multiple births is significantly increased. in this study, no significant difference was observed for embryo implantation rates, pregnancy rates, abortion rates, and take - home baby rates between eset or det in patients receiving previous cesarean section. however, 50% of ivf pregnancies were multiples in the det group, which may lead to a higher incidence of complications. twin pregnancy in previous cesarean scar patients was associated with increased risk of placenta previa and uterine scar rupture, preterm birth, and reduced birth weights of twin infants. in a previous report to assess the benefits of single - embryo transfer, a significantly increased incidence of preterm births (including the rate of very preterm births) and low neonatal birth weights, respiratory complications, sepsis, and jaundice in neonates were observed for twin pregnancies. therefore, there are more benefits if patients with a previous cesarean scar prefer eset. in our study, only 43.08% patients with previous cesarean section and 6.93% patients with previous vaginal delivery accepted eset. patients are more willing to choose det to achieve a better pregnancy rate, and the urging for progeny causes patients to ignore the risk related to twin pregnancy. moreover, concerns about the low pregnancy rate with eset and the cost for another embryo transfer cycle also affect patients decisions regarding the number of transferred embryos. the primary goal of assisted reproductive technology is to have a healthy baby. although prior cesarean delivery is not shown to be associated with an increased risk of stillbirth in a subsequent pregnancy, increased uterine volume caused by multiple pregnancies may increase intrauterine pressure and lead to ruptures at late stage of pregnancy or delivery, especially for the cases with prior cesarean deliveries. in our study, pregnancy outcomes were similar for eset and det patients with a previous cesarean section scar. reducing the number of embryos transferred to patients is the most effective method to prevent the multiple pregnancies. therefore, there is no need to transfer more embryos to improve the implantation outcome, while iatrogenic multiple pregnancies result both maternal and fetal risk. physicians should guide patients properly for accepting high - quality single - embryo ivf - et to ensure a safer labor and delivery, as well as better neonatal outcome. due to the similar pregnancy efficiency and clinical outcome of eset and det, our data provide valuable information for guiding future patients who previously gave birth by cesarean section to accept eset in order to ensure the safety of delivery. since the patients enrolled in our study were limited, a large - scale investigation is need to validate our conclusion. | backgroundwhat role should previous cesarean section play in affecting clinical pregnancy outcomes and avoiding the complications of in vitro fertilization ? in this article, we focus on elective single - embryo transfer (eset) versus double - embryo transfer (det) and assess the clinical efficacy and safety of eset in patients who have a previous cesarean scar.material/methodsthe pregnancy, delivery, and neonatal outcomes of 130 patients who had a previous cesarean scar and received in vitro fertilization - embryo transfer (ivf - et) were retrospectively analyzed. the number of transferred embryos was chosen depending on patients desire after acknowledging all benefits and risks, including eset (eset group, n=56) and det (det group, n=74). a total of 101 patients with previous vaginal delivery receiving ivf - et in the same period were included as a control group.resultsthe pregnancy rates, multiple birth rates, abortion rates, ectopic pregnancy rates, gestational age at delivery, preterm birth rates, neonatal birth weight, and take - home baby rates were similar between the previous cesarean section group and the previous vaginal delivery group. a previous cesarean section scar did not affect embryo implantation and pregnancy outcomes in ivf. in the eset and det groups of previous cesarean section patients, the embryo implantation rates, pregnancy rates, abortion rates, and take - home baby rates were similar. however, the rate of multiple pregnancies reached 50% in the det group, which led to more preterm births and lower birth weight.conclusionselective single - embryo transfer is a well - accepted strategy to avoid multiple pregnancies and improve the obstetric and neonatal outcomes of singleton pregnancy in ivf patients with a previous cesarean section. |
hand eczema is one of the commonest eczemas encountered in clinical practicehand eczema is the commonest eczema encountered in patch test clinics outside india but in india footwear dermatitis and airborne contact dermatitis predominatesmale predominance is seen in hand eczema and chromate, fragrance mix and nickel are commonest allergens seen in these cases. hand eczema is one of the commonest eczemas encountered in clinical practice hand eczema is the commonest eczema encountered in patch test clinics outside india but in india footwear dermatitis and airborne contact dermatitis predominates male predominance is seen in hand eczema and chromate, fragrance mix and nickel are commonest allergens seen in these cases. hand eczema is usually a chronic and recurring disorder with a prevalence of about 8% in adult population. classification of hand eczema is done either on the basis of morphology or the etiological factors associated with it. morphologically, hand eczema is divided into many types, the commonest of which are fingertip eczema, hyperkeratotic eczema, apron eczema, ring eczema and pompholyx. on the etiological front hand eczemas a number of endogenous and exogenous factors play a role in the development of hand eczema. atopy is the most important of the endogenous factors and among exogenous factors, contact sensitization to different allergens, either occupational or nonoccupational is the most important. while contact sensitization is the primary cause of hand eczema in 2060% cases, it also plays a crucial role in aggravation of other types of hand eczema. that is the reason why patch testing is considered mandatory in all those patients in whom hand eczema persists for more than 1 month. results obtained from a patch test have to be interpreted in a proper manner in patients with hand eczema. the most important part of this interpretation is the assessment of the relevance of patch test result with clinical condition. only after assessing the relevance of the patch test result can a proper advice be given and thus the benefit of the test can be passed on to the patient. we are reporting hereby the results of patch testing in hand eczema patients over a period of 7 years in ethnic kashmiri population. a total of 800 patients were seen at the contact dermatitis clinic of our institute from may 2006 to may 2013 out of which 278 patients had a primary diagnosis of hand eczema. the demographic profile and clinical details were filled in a preformed format at the first visit of each of these patients. these demographic details included the age, sex, occupation, rural / urban residence, hobbies and other daily activities of each patient. clinical evaluation of the patients included the morphological type of eczema, the sites involved, the duration of eczema, the seasonal and photo - exposure related variation and any coexistent skin disorders including atopic diathesis. all the patients were patch - tested using the indian standard series as recommended by the contact and occupational dermatosis forum of india and marketed by systopic labs india ltd. a standard protocol was employed in each patient wherein the test antigens were applied on the upper back in a prefixed pattern using aluminium patch - test chambers mounted on a micropore tape. the test patches were removed after 2 days and the sites were marked with a permanent marker pen. readings were taken 1530 min after removing the test patches on day 2 and then again on day 4 as well. the reactions were interpreted according to the international contact dermatitis research group criteria and graded accordingly. the patients were also told to report if there were any further reactions at the patch - test area on day 7. after grading the test reactions, the relevance of the patch test report was analyzed by a thorough evaluation of the patient 's clinical and exposure history and the details were added to the patient 's data. the chronological association of the eczema and exposure to the probable allergen on the basis of patch test report was assessed and the relevance was thus reported as almost all age groups were represented in the study group with our youngest patient being just 12-year - old while the oldest patient having an age of 56 years. majority of the patients, however, belonged to 3 and 4 decade of their life. female sex was preponderant and in the 278 patients tested in the present study, 171 were females representing approximately 62% of the whole group [table 1 ]. the commonest occupation group represented was housewives followed by manual workers, students, masons and office workers [table 1 ]. masons constituted about 17% of the male population in this study (18 out of 107 males). the commonest morphological types of hand eczema seen were the finger - tip eczema, vesicular hand eczema and hyperkeratotic hand eczema in 45, 38 and 34 patients respectively [figure 1 ]. other morphological patterns of hand eczema seen were chronic acral dermatitis, ring eczema and pompholyx in 28, 25 and 17 patients respectively. in the remaining 91 patients (32.7%), the hand eczema could not be classified into any particular morphological type [table 1 ]. hand eczema in a mason with chromate sensitivity atopic diathesis was not clinically evident in majority of the study population and only 11 patients had clinical features suggestive of atopy. a positive patch - test report was obtained in 135 patients out of the total of 278 patients tested. out of these, 97 patients showed a positive reaction to a single patch - test antigen while 38 others showed positive patch test reactions to multiple (2 or more) antigens. nickel and potassium dichromate were found to be the two commonest allergens in the study population with positive reactions in 45 and 27 patients respectively. thus, nickel and potassium dichromate together accounted for about 38% of the total positive test reactions. other common allergens found in our population included benzocaine, neomycin, fragrance mix, mercaptobenzothiazole and paraben mix in 21, 17, 17, 11 and 10 patients respectively. contact allergen positivity in patients with hand eczema females were seen to show higher patch - test positivity then males. out of a total of 171 females, a positive patch - test was seen in 94 (54.9%) patients while only 41 out of the107 males (38.3%) showed a positive patch test reaction. the commonest allergen found in female patients was nickel (in 35 patients) while in males, highest number of positive reactions were seen with potassium dichromate, nickel and fragrance mix in 14, 10 and 8 patients respectively. the positive test reactions were analyzed for their clinical relevance and majority of these reactions were found to be clinically relevant. in fact, a total of 122 reactions out of 189 (64.5%) had either a present or past relevance to the clinical picture. the highest clinical relevance was seen in patients with metal sensitivity. in patients with chromate sensitivity, occupational exposure to relevant substances was seen in 14 patients (all masons) while 9 others had a nonoccupational exposure. thus the overall relevance of this subgroup was about 80% (23 out of 27 patients). in patients with metal allergy on patch testing, clinical relevance was evident in 77.7% patients out of whom 35.5% had relevance to the current dermatitis while 42.2% others had a past relevance in the form of a past exposure to metallic substances especially in females in the form of ear piercing. a peculiar finding that we observed in our patients was the simultaneous involvement of hands and lips in many patients with the primary diagnosis of hand eczema. these patients were seen to be predominantly sensitive to nickel and the involvement of lips was probably because of the use of metallic spoons for taking rice based diet which is the staple diet for ethnic kashmiri population. however, dimethylglyoxamine test was not done to prove this association. kashmir valley is a province of jammu and kashmir state with a temperate climate and it has a population of about 7 million with a unique ethnic character. a good proportion of the adult population in the valley is involved with agriculture, horticulture and handicraft sectors. this custom is completed in early childhood in most of the females and by the age of 10 years almost all females are through with this procedure. additionally, the female population is also almost universally involved with cooking meals at home and in the villages these females are also involved with gardening and farming practices. our institute was the first to start a patch testing facility in the valley about 8 years back and we have been able to patch test about 800 individuals belonging to the ethnic kashmiri population till date. we found hand eczema to be quite common in this population and in fact, it formed the commonest diagnosis in patients who underwent patch testing at our institute. this finding is in conformity with many similar studies from different parts of the world. on the other hand, in indian context, footwear dermatitis and air - borne contact dermatitis are the two commonest eczemas seen in patch - tested patients. the high incidence of hand eczema in our patch - tested population, in fact, formed the basis of conducting this study as we tried to figure out the reasons behind this high incidence and the allergens responsible for it in this population. we patch - tested all population and occupational groups and the study population included students, office - goers, handicraft workers, housewives, farmers and other occupational groups as well. about 48.5% of hand eczema patients that we patch tested showed one or more positive reactions in this study. this figure is comparable to those quoted by most of the studies especially on this subject. this is probably because of a much higher incidence of nickel sensitivity in our female population. in fact, nickel proved to be the predominant allergen causing hand eczema in females irrespective of their occupational status. all this can be easily explained on the basis of the universal practice of ear - piercing in our female population. the custom of wearing jewellery especially on religious festivals and marriage ceremonies adds to the chances of developing a nickel - sensitivity in this population subgroup. additionally, the earthen - ware that was used commonly for cooking purposes in the past has been replaced totally by steel utensils over the last 2 - 3 decades. in males the patch test results were mixed as there was no single predominant allergen and potassium dichromate, para - phenylenediamine and fragrance mix were the commonest allergens in this sub - group. additionally, males showed a significantly less incidence of positive reactions to the patch - test allergens than females, a phenomenon that has been reported in many studies till date. this sex - disparity has been explained on the basis of a relatively much higher rate of nickel sensitivity in females and this was evident in our series as well. among topical antimicrobials neomycin was the commonest allergen with 17 patients (6.1%) showing a positive reaction to this compound. in all these patients, there was a history of using topical antimicrobial combinations and thus the patch test result had a strong clinical relevance in all of them. the importance of gentamicin and neomycin emerging as common sensitizers has been stressed upon in many studies from india and abroad. this allergen, in addition to being a constituent of many cosmetic products, cross reacts with chloroxylenol, an active ingredient of dettol, the use of which has increased dramatically in recent years. an interesting fact that we observed in our patients was that there was a tendency to switch over to dettol soap or at least add dettol solution to bath water as the first measure to tackle any dermatitis. hand eczema is more common in females than males in ethnic kashmiri population and females also show a higher rate of patch test positivity in this population. nickel and potassium dichromate constitute the two commonest allergens responsible for hand eczema, nickel being especially common in females. hand eczema is the commonest eczema seen in a patch test clinic in kashmir valleyfemales predominate and even show more patch test positivity in kashmiri patients with hand eczemanickel is the commonest allergen followed by potassium dichromate in these cases. hand eczema is the commonest eczema seen in a patch test clinic in kashmir valley females predominate and even show more patch test positivity in kashmiri patients with hand eczema nickel is the commonest allergen followed by potassium dichromate in these cases. hand eczema is the commonest eczema seen in a patch test clinic in kashmir valleyfemales predominate and even show more patch test positivity in kashmiri patients with hand eczemanickel is the commonest allergen followed by potassium dichromate in these cases. hand eczema is the commonest eczema seen in a patch test clinic in kashmir valley females predominate and even show more patch test positivity in kashmiri patients with hand eczema nickel is the commonest allergen followed by potassium dichromate in these cases. hand eczema is the commonest eczema seen in a patch test clinic in kashmir valleyfemales predominate and even show more patch test positivity in kashmiri patients with hand eczemanickel is the commonest allergen followed by potassium dichromate in these cases. hand eczema is the commonest eczema seen in a patch test clinic in kashmir valley females predominate and even show more patch test positivity in kashmiri patients with hand eczema nickel is the commonest allergen followed by potassium dichromate in these cases. | background : hand eczema is one of the commonest eczemas encountered in dermatology practice. contact allergens responsible for causing hand eczema vary from one geographical area of the world to another. aim : the study tries to identify the commonest allergens causing hand eczema in ethnic kashmiri population.material and methods : a total of 800 patients were patch tested at a sole patch testing facility for the local population over a 7-year period out of which 278 were diagnosed with hand eczema. the morphological type of hand eczema was noted and the patch testing results were analyzed. past or present relevance of the patch - test result to the clinical diagnosis was also analyzed.results:hand eczema was the most common type of eczema patch tested over the 7-year period, constituting 34.75% of the patch - tested population. a positive patch - test result was obtained in 135 patients (48.5%) out of which it was found to be clinically relevant in about 65% cases. nickel and potassium dichromate were found to be the two commonest allergens causing hand eczema in our population with positive patch test reactions in 45 and 27 patients respectively. females showed a much higher incidence of hand eczema as well as higher patch test positivity than males (54.1% vs. 38.1%).conclusions : hand eczema with a positive patch - test report was found to be much more common in females than males and nickel and potassium dichromate were seen to be the commonest allergens causing hand eczema in ethnic kashmiri population. |
although surgical resection is the best curative treatment option,1,2 there are several obstacles that limit surgical resection such as tumor number, location, hepatic reserve, and patient performance status.3 therefore, alternative techniques such as percutaneous ethanol injection (pei), microwave therapy, radiofrequency ablation (rfa), and transcatheter arterial chemoembolization (tace) have been practiced for the treatment of hcc.4 - 6 rfa is an effective loco - regional treatment for hepatocellular carcinoma that was first introduced in 1996.7 as a minimally invasive procedure, rfa is considered as a relatively simple and safe modality for treatment of hcc. post - rfa complications such as liver abscess, tumor seeding, pleural effusion, pneumothorax, gastrointestinal perforation, intraperitoneal bleeding, and skin burn have been reported from 5.2% to 9.5% in literature.8 - 10 hemobilia is defined as hemorrhage from the biliary tree. the causes of hemobilia are known as trauma, infection, tumor, inflammation, and gallstones. hemobilia has been reported as a rare biliary complication after percutaneous hepatic intervention.11 hemocholecyst secondary to hemobilia is also a rare sequelae of hemobilia. as we experienced hemobilia accompanied with hemocholecyst after rfa for treatment of hcc, a 69-year - old man visited the emergency room due to right upper quadrant abdominal pain for two days. he had a medical history of hypertension, cerebral infarction, and liver cirrhosis secondary to chronic hepatitis b virus infection. two weeks ago, the patient was treated with rfa for 14 mm sized hcc in segment 8 (fig. 1a, b). physical examination and laboratory findings were within normal range when rfa was performed. percutaneous rfa was performed under local anesthesia using cool - tip electrode with a 3-cm exposed tip (act2030, valleylab, boulder, colorado, u.s.a) according to manufacture 's manual. laboratory finding showed increased serum levels of alanine transaminase (242 iu / l from 24 iu / l), total bilirubin (3.6 mg / dl from 0.4 mg / dl), and decreased hemoglobin level (12.1 g / dl from 15.1 g / dl). acute infection was suggested by leukocyte count (20,180/mm), c - reactive protein (14.2 mg / dl). abdominal computed tomogram (ct) showed gallbladder (gb) filled with high density material, considered as hematoma (fig. endoscopic retrograde cholangiopancreatography (ercp) showed an irregular shaped filling defect in gb with normal cholangiogram (fig. abdominal pain gradually decreased with antibiotic therapy after endoscopic sphincterectomy to decompress the bile duct. the follow up ct scan three days after ercp showed a decreased but remnant mass - like lesion within gb (fig. the pathologic examination showed hemorrhage and diffuse infiltration of inflammatory cells at gb wall without primary gb pathology (fig. hemobilia involving the gb can present with signs and symptoms of upper gastrointestinal bleeding and eventually lead to gb rupture and hemoperitoneum, when cystic duct is obstructed by blood clots.12,13 the mechanism of hemobilia after rfa is mainly due to direct traumatic injury to biliary and vascular structures during electrode advancement and thermal injury may also contribute to bile duct and vascular damage.14 hemobilia related to rfa has been reported ranging from 0.02% to 0.25%.12 - 14 the term " hemocholecyst ", introduced in 1961, refers to describe hemorrhage of gb. originally, this term applies only to atraumatic bleeding confined to the gb.15,16 however, it might be very difficult to differentiate hemocholecyst from hemobilia in clinical practice. hemocholecyst can cause hemobilia, or vice versa, hemobilia may result in hemocholecyst.17,18 causes of primary hemocholecyst are limited including gb cancer, cholelithiasis, vascular disorders, and the presence of heterotopic gastrointestinal mucosa within the gb. secondary hemocholecyst can be resulted from iatrogenic trauma, such as liver biopsy and rfa.12,13,16 the diagnosis of hemocholecyst is difficult. the sonographic appearance of blood clots within the gb appears as clumps of echogenic material. ct scan can show high density mass - like lesion in distended gb and high density mass.19 ercp can identify evidence of bleeding at duodenal ampullar in only 30% of patients. in the present case, we identified high attenuating mass like lesion in gb through sonography and abdominal ct scan, which showed hematoma density about 60 hounsfield number in ct scan. ercp failed to show evidence of bleeding in biliary system in present case, which is mainly due to time delay of 2 weeks between ercp and rfa. direct gb injury from rfa might be an another etiology of hemocholecyst, but, considering the fact that the ablation site is not close from the gb, we presumed that hemocholecyst is associated with hemobilia after rfa. although definite hemobilia could not be identified, no histologic evidence of primary gb disease causing hemorrhage made it diagnosed as hemocholecyst associated with hemobilia it is difficult to provide an algorithm for management of hemocholecyst associated with hemobilia. once the clot in the gb begins to organize, it is very unlikely to dissolve. therefore in most cases, open or laparoscopic cholecystectomy should be performed.20 laparoscopic cholecystectomy was performed in present case due to persistent pain and the patient successfully recovered. we assume the thermal injury during rfa caused hemobilia subacutely which eventually resulted in hemocholecyst. eventually the patient was successfully managed by laparoscopic cholecystectomy. in conclusion, although hemocholecyst associated with hemobilia is a very rare complication of rfa, we should suspect possibility of this complication in patients presenting with continued abdominal pain and decrease in hemoglobin level without obvious cause. early recognition and management is crucial not to miss the chance for the opportune treatment. | radiofrequency ablation (rfa) is performed as an alternative to surgical resection for primary or secondary liver malignancies. although rfa can be performed safely in most patients, early and late complications related to mechanical or thermal damage occur in 8 - 9.5% cases. hemocholecyst, which refers to hemorrhage of the gallbladder, has been reported with primary gallbladder disease or as a secondary event associated with hemobilia. hemobilia, defined as hemorrhage in the biliary tract and most commonly associated with accidental or iatrogenic trauma, is a rare complication of rfa. here we report a case of hemocholecyst associated with hemobilia after rfa for hepatocellular carcinoma that was successfully managed by laparoscopic cholecystectomy. |
primary cardiac malignancy is very unusual with an estimated incidence ranging from 0.001% to 0.03%, nine percent of which, are ascribed to cardiac leiomyosarcoma (cl), making it extremely rare. diagnoses are most often made after a mass in the chambers of a heart is found or abnormal cardiac hemodynamic caused by an occupying tumor is noticed. while the exact oncogenesis is not known, cl is highly progressive and locally invasive. it can be either a primary tumor in heart or a metastatic tumor arising from other primary sites. a 67-year - old woman was admitted with recurrent chest tightness for four years, progressive dyspnea for six months, and nausea for two months. the past history included hypertension for over 30 years, and diabetes and hyperlipidemia for two years. a left mastectomy for breast cancer was performed 19 years ago, followed by local radiation therapy of three months. her physical examination revealed the blood pressure of 105/80 mmhg and the heart rate of 118 beat / min in sinus rhythm. a grade iv pan - systolic murmur could be heard at the lower left sternal edge. echocardiography (figure 1) showed the hypokinesis in the middle to apex sections of the left ventricle and the paradoxical septal motion in the apex while the continuous interruption in the septum between the apex and middle sections with left to right shunting was observed, and left ventricular ejection fraction (lvef) was 34%. the echocardiography performed three years ago demonstrated mild hypertrophy of the ventricular septum, normal myocardium motion and heart chambers, and lvef from 58% to 65% while the recent echocardiographic results indicated the hypokinetic development of her left ventricular walls and lvef from 43% to 48%. a coronary artery angiogram showed atherosclerosis in the mid - left anterior descending artery (lad), and the luminal stenosis of 60% compared with 50% three years ago. there were multiple coronary fistulae in the lad draining into the left ventricle and fistulae in the end of left circumflex (lcx) and the right coronary artery (rca), compared with small left coronary fistulae in the lad and none in the lcx and rca three years ago. the single photon emission computed tomography (spect) carried out two years ago illustrated an irregular radionuclide distribution in the anterior, inferior, posterior and septum segments of the myocardium with no evidence of ischemia after exercise. the left ventricle was enlarged with diffuse hypokinesis of the ventricular wall, particularly at the apex. there was a shunt of 10 mm in diameter in the septum near the apex as blood flowed through from the left to right ventricle. cardiac resynchronization therapy was performed to improve her progressive congestive heart failure, but the beneficial effect was not sustained. an intra - aortic balloon pump (iabp) was inserted, which improved her conditions significantly. the iabp was inserted again and the patient underwent surgical repair of her ventricular septal defect. at the operation, a left apical aneurysm was present, but no mass was found in the chambers. the myocardium motion was not recovered and the patient died from refractory cardiac failure one week post operatively. the histopathology report of the myocardial specimen confirmed the presence of malignant spindle cell neoplasm with prominent pleomorphism (figures 2). there were signs of mitotic activities with atypical features with coarse grain chromatin, thin cytoplasm and diffuse foliated necrosis. immunohistochemical stains were positive for vimentin, smooth muscle actin, s-100 and caldesmon, and negative for pan - cytokeratin, myoglobin, cd34 and cd 99. the patient presented the progressive development of chest tightness and heart failure, and did not positively respond to the treatment in accordance with myocardial ischemia. spect demonstrated the irregular radionuclide distribution in the myocardium segments, which was not bonded to the myocardial ischemia. the above observations illustrated that the abnormality of the myocardium existed and may be related to the invasion of the cl. the infiltration of malignant cells into the myocardium could cause the injury of the myocardium, resulting in the abnormal motions of the ventricular walls and the ventricular septal defect. the coronary fistulae resulted in the ischemia at the end of the myocardium, which enhanced the abnormal motions, and further led to the formation of the ventricular aneurysm and ventricular septal perforation. (a) : hematoxylin - eosin (he) stains of a myocardial specimen revealed a prominently pleomorphic mass containing signs of mitotic activities with atypical figures and diffuse necrosis (200) ; (b) : the he stains showed clostridial - shaped tumor cells containing coarse grain chromatin and thin cytoplasm arranged in dense lines. pathological splitting can be identified in these cells due to the presence of foliated necrosis (400). the uniqueness of this case is that various examinations and even the surgical investigation failed to recognize the presence of cardiac mass or neoplasm. the final diagnosis of the cl was made from the myocardial biopsy and the cl originated most likely from the heart. the results of the positron emission tomography (pet) and tumor marker examinations were normal, which led to no evidence of the cl from other tumor metastasis. to date, there has not been any report of a cancer caused by local breast cancer radiotherapy. the radiation destruction or radiotherapy has not been known to cause etiology of the cl. thus, it is impossible that the cl was caused by the local breast cancer radiotherapy. to our knowledge, this is the first reported case of cl associated with multiple coronary fistulae and ventricular perforation, but without a mass. in conclusion, this case is the primary cl characterized by the progressive congestive heart failure and coronary fistulae and ventricular septal perforation without a mass, which has not been previously reported. the lesson learned was that the possible existence of a myocardial disease or even cl, should be suspected if the presentation of usual coronary heart disease with congestive heart failure but with poor response to the corresponding conventional therapy has existed. | coronary fistulae and ventricular septal perforation are very rare clinically, and even less caused by cardiac leiomyosarcoma. a case is reported that a 67-year - old female had cardiac leiomyosarcoma with progressive heart failure and coronary fistulae and ventricular septal perforation. this case was special since all ante - mortem examinations and cardiac surgery failed to detect the presence of any abnormal cardiac mass. therefore, the malignant cardiac tumors could appear in an invasive form without mass and be one of the causes of the coronary fistulae and ventricular septal perforation. |
migraine and tension - type headache prevalence has been determined to be 16 (21.8% for women and 10.9% for men) and 31.7%, respectively, in turkey based on a structured cross - sectional study for headache carried out in 13 centers with 2,007 participants. however, structured studies in certain age and target groups are rather limited in turkey [28 ]. migraine prevalence in university students has been reported in a few studies [5, 7, 8 ]. those studies may be limited when the negative impact of migraine on the quality of life for each age is considered. it is unequivocal that migraine data vary according to the populations and detailed evaluations are much more informative. nonetheless, it is undeniable that a reliable and validated screening test can analyze this widespread disorder in populations quickly. these tests are advantageous and practical for clinicians to provide a preliminary data in large populations., new york, usa), which is a quick and appropriate test, has been used as a screening test in primary health services. sensitivity, specificity and positive predictive value of this test in primary care have been defined as 81, 75 and 93%, respectively. the turkish version of the i d migraine screening test has already been validated [11, 12 ]. karli. reported 91.8% sensitivity, 63.4% specificity, 71.9% positive predictive value and 88.4% negative predictive value for i d migraine in their study. it has rarely been used in large series and primarily for validation purposes [9, 13, 14 ]. for example, kim. used i d migraine for diagnostic purposes in patients with temporomandibular joint disease, whereas karli. validated the test in outpatient clinics. in this prospective study, we aimed to determine the prevalence of migraine and non - migraine headaches by using a questionnaire including i d migraine for university students from various academic departments. the study was approved by the hospital ethics committee and consent was received from each subject. a total of 1,310 students were selected randomly among the 20032004 curriculum list (16,252 students in total) recieved from the university administration. the study was designed cross - sectionally and migraine prevalence was accepted as 20% in university students (worst acceptable 1723%) and sample size was calculated as 1,100 students with 80% power and 99% reliability range. of the 1,310 students, 26 refused to participate in the study and 28 students were chosen randomly from faculties of medicine, science and social sciences of different terms to ensure standardization. second term includes 3rd year medicine and dentistry students and 2nd year of other faculties. third term comprises 4th year medicine and dentistry students and 3rd year students of other faculties, whereas fourth term includes all seniors of all faculties. the study was performed based on the questionnarie merely, without a face - to - face interview with the students. due to this the questionnarie was first applied to 40 students who were not enrolled in the study and after this, it was revised into its final form. in the first part, questions related to social and demographic aspects of the students such as familial, economic, housing, school life, success levels, loss of term, etc., were found. the second part comprised questions related to headache. subjects having headache were those who answered yes to the questions : did you ever have headache in your lifetime ? and did you ever have headache in the last 3 months ? the final three - item screening questions of the i d migraine test were as follows (questions 41, 42 and 43 in the questionnaire) : during the last 3 months, (1) did you feel nauseated or sick in your stomach with your headaches ? (2) did light bother you when you had a headache (a lot more than when you do not have headaches) ? (3) did your headache limit your ability to work, study or do what you needed to do for at least 1 day ? the cut - off point for a test - diagnosis of migraine headache was at least two positive responses. a total of 1,310 students were selected randomly among the 20032004 curriculum list (16,252 students in total) recieved from the university administration. the study was designed cross - sectionally and migraine prevalence was accepted as 20% in university students (worst acceptable 1723%) and sample size was calculated as 1,100 students with 80% power and 99% reliability range. of the 1,310 students, 26 refused to participate in the study and 28 were excluded due to lack of questionnaire or inappropriate internal consistency test. students were chosen randomly from faculties of medicine, science and social sciences of different terms to ensure standardization. second term includes 3rd year medicine and dentistry students and 2nd year of other faculties. third term comprises 4th year medicine and dentistry students and 3rd year students of other faculties, whereas fourth term includes all seniors of all faculties. the study was performed based on the questionnarie merely, without a face - to - face interview with the students. due to this the questionnarie was first applied to 40 students who were not enrolled in the study and after this, it was revised into its final form. questions related to social and demographic aspects of the students such as familial, economic, housing, school life, success levels, loss of term, etc., were found. the second part comprised questions related to headache. subjects having headache were those who answered yes to the questions : did you ever have headache in your lifetime ? and did you ever have headache in the last 3 months ? the final three - item screening questions of the i d migraine test were as follows (questions 41, 42 and 43 in the questionnaire) : during the last 3 months, (1) did you feel nauseated or sick in your stomach with your headaches ? (2) did light bother you when you had a headache (a lot more than when you do not have headaches) ? (3) did your headache limit your ability to work, study or do what you needed to do for at least 1 day ? the cut - off point for a test - diagnosis of migraine headache was at least two positive responses. of the 1,310 subjects, 1,256 students (529 female and 727 male) were enrolled in the study. their mean age was 21.9 2.1 years (1731 years). different types of headache were identified in 1,129 (89.9%) subjects, whereas 127 were headache - free. migraine - type headache was detected in 265 subjects (21.9%) based on the i d migraine evaluation. table 1 displays social and demographic characteristics of the students. table 1social and demographic characteristics of the studentsmigraine % (n) non - migraine % (n)headache free % (n)psex females14535762 23.5 (60)24.2 (198)22.4 (17)0.989 3501,000 $ 55.3 (141)53.2 (436)53.9 (41) 1,000 $ < 21.2 (54)22.6 (185)23.7 (18)social activities never or rarely2.6 (53)2.3 (210)26.6 (21)0.935 occasionally43.8 (95)48.7 (442)46.8 (37) frequently or always27.2 (59)28.8 (264)26.6 (21) social and demographic characteristics of the students of these, 145 (11.5%) were female and 120 (9.6%) were male (female / male ratio : 1.2/1). therefore, migraine was diagnosed in 145 of 529 female subjects (28.1%) and in 120 of 727 male subjects (17.3%). non - migraine type headache was identified in 864 subjects, 357 female and 507 male (table 1). a total of 334 students (26.6%) were in the first term, 298 (23.3%) in the second term, 312 (24.8%) in the third term and 312 (24.8%) in the fourth term. migraine frequency was found to be 26.6% in the first term, being relatively higher compared to other terms, though not statistically significant. no significant difference was found in non - migraine - type headaches among different terms. nevertheless, the frequency of non - migraine headaches (71.5%), and the overall headache frequency (93.5%) in the 4th term were higher than in the other terms. gender only had a significant relation in all groups (subjects with migraine, subjects with non - migraine headache and headache free) (p < 0.005). in 21.9% of 1,256 university students, migraine diagnosis was established based on the data of the questionnaire using i d migraine. female / male ratio was 1.2/1.0 (145/120) in subjects with migraine in our study. non - migraine - type headache was found in 71.5% of the subjects considering the questionnaire results, whereas 6.5% of the subjects defined no headache. there are few studies on migraine in university students in turkey [5, 7, 8 ] female / male ratio was 3.4/1.0 (99/29) and 1.6/1.0 (224/138) in subjects with migraine in the first two studies, respectively. in a recent study from our department, aimed to determine all types of headache in senior medical students, migraine was found in 24.4% (n 31/127). it might be speculated that headache in cases who were invited but did not attend the study had a less negative impact on their life. our results using i d migraine were not much different from the other studies using distinct methodologies in the literature. these ratios are higher than the ratios in the above - mentioned study. in a multicenter study for headache prevalence in turkey, migraine prevalence was found to be 20.9% in the mediterranean region and 24.0% in the southeastern region, whereas the prevalence in agean, mid - anatolian, marmara, and karadeniz regions was 20.6, 11.7, 11.4 and 14.7%, respectively. in a recent multi - center study (mira), including ours, migraine prevalence was found to be 24.9% in outpatient clinics irrespective of subgroup analysis. migraine prevalence was found to range between 2.4 and 40.2% in various studies based on the questionnaire in the literature. the prevalence in the studies by mitsikostas, muniz, deleu, demirkirkan, kurt, bigal 19 and amayo 20 was 2.4, 7.58, 12.2, 12.4, 24.9, 25 and 38%, respectively, whereas sanvito 21 found 40.2% only in medical students of similar age. migraine prevalence found by using i d migraine in our study (21.9%) is quite consistent with the results derived from the multicenter study in turkey. these variations may be due to the study designs as well as the variations in the study groups. however, our own results seem to be consistent with our country s data. on the other hand, in 71.5% of 1,256 subjects however, the high frequency rate of migraine and non - migraine - type headache in a student population is quite interesting. as has been known, tension - type headache is the most common in the age group we have investigated. in the multicenter study for headache prevalence in turkey, frequency of tension - type headache in the mediterranean and southeastern regions were 59 and 36.6%, respectively, being consistent with the rates in our study. regarding the latter, it is not surprising that migraine prevalence in our study is higher than in other turkish university studies since most of the students in cukurova university are from the mediterranean and southeastern regions [5, 8 ]. considering the stress factors for migraine in the population mentioned above in detail, gender had a statistically significant relation with migraine. on the other hand, it was quite interesting that factors such as parental health and their marital status, school success, income and social activites had no significant relation. a limitation of our study is that sensitivity and specificity ratios and predictive values could not be assessed due to its design. as a conclusion ; it is straightforward that a face - to - face verbal interview is the gold standard for migraine diagnosis and specifically designed further studies are necessary. nevertheless, i d migraine, as a valid, reliable and quick screening tool in primary care migraine recognition, may encourage the clinician due to the similarities between the results of face - to - face interview and screening questionnaires in migraine studies. it would be practical and beneficial to the clinician to perform the i d migraine in larger populations for preselection of the patients for further face - to - face interview. | in this prospective study, we aimed to determine the prevalence of migraine and non - migraine headaches using a questionnaire, including i d migraine, for university students. the study was designed cross - sectionally and a questionnaire consisting of 43 questions was applied to 1,256 students. the questionnaire consisted of questions related to demographic, social, curriculum, housing and headache characteristics of the subjects. three - item screening questions of the i d migraine test were included at the end of the questionnaire aimed at migraine diagnosis. the mean age of 1,256 students (529 females and 727 males) enrolled in this study was 21.9 2.1 years (1731 years). migraine - type headache was detected in 265 subjects (21.9%) based on the i d migraine evaluation. of these, 145 (54.7%) were female and 120 (45.3%) were male (female / male ratio : 1.2/1). non - migraine - type headache was identified in 864 subjects, with 357 females and 507 males. as a conclusion, i d migraine screening test might be practical and beneficial when a face - to - face interview is not possible or to pre - determine the subjects for a face - to - face interview for migraine diagnosis in larger populations. |
precancerous lesions are related to the development of tumors in several organs, such as intestinal - type gastric cancer that develops through the progression of various sequential lesions that frequently start with a helicobacter pylori infection. this infection causes superficial gastritis that can progress to chronic atrophic gastritis, intestinal metaplasia, dysplasia, and, finally, carcinoma. intestinal metaplasia, characterized by the differentiation of gastric stem cells into intestinal - phenotype cells, is associated with more than 80% of intestinal - type adenocarcinoma. besides this metaplasia - dysplasia - carcinoma pathway, gastric carcinogenesis can originate from gastric ulcer, a lesion in the mucosa that develops in low acid concentration sites and severe inflammation [5, 6 ]. eighty - five percent of gastric ulcer cases occur in the presence of h. pylori infection [5, 7 ]. the progression of the lesions cascade depends on many genetic factors in both the host and the bacterium, besides environmental factors. this results in a great amount of inflammatory mediators and the reactive oxygen and nitrogen species that induce genetic and epigenetic changes in protooncogene and tumor suppressor genes, influencing the emergence of cancer. such bacteria populations are heterogeneous and contain virulence factors such as caga (cytotoxin - associated gene a antigen), which produces a protein that acts in many cellular events such as cytoskeleton rearrangement, cellular polarity breaking, and mitogenic and proapoptotic responses. this virulence genotype, however, is not found in all the strains ; its occurrence has a relation with major gastric mucosa inflammation and high risk of gastric cancer development. outstanding among the inflammatory mediators that activate the immune response cascade are the anti - inflammatory proteins annexin - a1 (anxa1) and galectin-1 (gal-1), involved in various inflammatory processes [13, 14 ]. anxa1 belongs to a protein superfamily characterized by binding to cellular membranes in a calcium - dependent manner, acting on several cellular and molecular processes. throughout the inflammatory process, this protein externalizes in the plasmatic membrane, blocking the interaction between leukocytes and endothelium in order to stop the inflammatory cells transmigration to the damaged site. its anti - inflammatory action is also related to the induction of neutrophil apoptosis and inhibition of phospholipase a2 (pla2). furthermore, it also has a connection to cellular differentiation mechanisms, growth, signal transduction, and cytoskeleton formation. thus, changes in their expression and subcellular location can contribute to inflammatory diseases and cancer [1922 ]. it has many cellular functions, such as apoptosis, cellular signaling, adhesion, migration, and proliferation, and its action depends on its cellular concentration and location. its anti - inflammatory function relates to the induction of apoptosis of t - activated cells [24, 27 ] and the decreased production of proinflammatory cytokines [28, 29 ]. furthermore, galectin-1 promotes differentiation of treg cells controlling their immunosuppressive mechanisms [30, 31 ] and suppresses dendritic cells maturation favoring a tolerogenic microenvironment. in the tumor microenvironment, the presence of gal-1 contributes to immunosuppression, angiogenesis promotion, metastasis, and cell transformation. several types of cancers show changed expression of this anti - inflammatory mediator [36, 37 ], associated with the increase of cellular proliferation and metastasis. studies on annexin - a1 and galectin-1 in gastric cancer and precancerous lesions are few and reveal contradictory results. some of them found anxa1 and gal-1 downregulation [3941 ], while others reported increase of these proteins in the lesions and tumor tissue [4246 ]. considering that gastric cancer has a high incidence and poor prognosis when diagnosed at a late stage, the study of precancerous lesions can provide important information about the initial phases of gastric carcinogenesis, thereby contributing to prevention strategies which may lead to a decrease in its incidence through the identification of potential biomarkers. recently, we showed a similarly increased expression of annexin - a1 and galectin-1 in chronic gastritis and in gastric cancer, evidencing deregulation in the expression of these proteins during the initial step of gastric carcinogenesis. based on this finding, we decided to investigate the expression and location pattern of both proteins in other precancerous gastric lesions that participate in the progression cascade to gastric cancer. in this study, we evaluated the quantitative mrna expression of genes anxa1 and lgals1 and the expression of both proteins in intestinal metaplasia and gastric ulcer lesions. in addition, we also investigated the occurrence of association between the expression levels and infection by h. pylori and its caga virulence genotype, besides other risk factors associated with gastric carcinogenesis. this research was approved by the local research ethics committee (cep - ibilce / unesp, number 059/11), and written informed consent was obtained from all participants, who also filled out a questionnaire from which we obtained family and occupational data, besides information on smoking and drinking habits. dna and cdna samples of gastric biopsies (antrum and corpus) of the lesion area and adjacent normal mucosa stored in our laboratory from a previous study were used. all subjects were recruited from the service of endoscopy, hospital de base, so jos do rio preto, sp, brazil. a total of 75 samples were evaluated, 36 of which of intestinal metaplasia of individuals without gastric cancer (im19 women, 17 men ; mean age : 60.53 12.21 years), 29 of gastric ulcer (gu8 women, 21 men ; mean age : 54.62 12.42 years), and 10 from individuals with histologically normal gastric mucosa without dyspepsia, used as controls (c3 women, 7 men ; mean age : 34.20 9.73 years). subjects with prepyloric or nonsteroid anti - inflammatory drug - induced ulcers were excluded from this study. dna samples of normal mucosa adjacent to gastric lesions were subjected to multiplex pcr containing primers for bacterial genes urea and tsaa and for the human constitutive gene cyp1a1, to verify dna integrity and efficiency of the pcr reaction (table 1). we used 1x buffer, 0.15 m of each deoxyribonucleotide, 2 mm mgcl2, 0.6 m of each primer, 100 ng genomic dna, and 1.8 u platinum taq dna polymerase (invitrogen) in a final volume of 25 l. the reaction was processed in an automatic thermocycler, with denaturation performed at 94c for 3 minutes, followed by 35 cycles at 94c for 45 seconds, 60c for 30 seconds, and 72c for 1 minute, and a final extension of 10 minutes at 72c. the reaction products were subjected to electrophoresis on 3.0% agarose gel stained with ethidium bromide. the h. pylori - positive samples were subjected to a second pcr assay, to investigate the caga virulence genotype. the reaction solution contained 1x buffer, 0.1 m of each deoxyribonucleotide, 2 mm mgcl2, 0.6 m of each primer (table 1), 300 ng genomic dna, and 1 u taq dna polymerase (invitrogen). the reaction conditions were 94c for 5 minutes for denaturation, 40 cycles of 1 minute at 94c, 1 minute at 56c and 1 minute at 72c, and a final extension of 7 minutes at 72c. relative quantification of mrna was performed in an abi prism 7300 sequence detection system (applied biosystems, california, usa), using the sybrgreen pcr core reagent (applied biosystems, california, usa) protocol and specific primers for the genes anxa1, lgals1, and actb, as described in previous studies. gene actb was used as endogenous control (reference gene) because it had shown the lowest variation amplification in gastric tissue. beyond the i m and gu groups, 10 samples of normal gastric mucosa were mixed and submitted to quantitative pcr and used as a calibrator for analysis (standard sample). relative quantification (rq) of genes anxa1 and lgals1 was performed according to the model proposed by pfaffl, compared with pool of samples from normal gastric mucosa and normalized to the actb reference, according to the formula r = (e target)/(e endogenous). rq was expressed as log2 mean sd, and cases with a value of rq > 2 were considered upregulated. immunohistochemical analysis was performed on 25 samples of i m, 10 of gu, and 6 of normal mucosa. after deparaffinization, histological sections were submitted to antigen retrieval in citrate buffer and blocking endogenous peroxide activity. the sections were incubated overnight at 4c with the primary rabbit polyclonal anti - anxa1 and anti - gal-1 antibodies (zymed laboratories, cambridge, uk) diluted to 1 : 1000 and 1 : 500, respectively, in 1% bovine serum albumin (bsa in phosphate - buffered saline (pbs)). after incubation with a secondary biotinylated antibody (dako, cambridge, uk), staining was detected using a peroxidase - conjugated streptavidin complex, revealed with 3,3-diaminobenzidine substrate (dako, cambridge, uk), and counterstained with hematoxylin. all experiments were carried out with a negative control consisting of a tissue section incubated with pbs and 1% bsa instead of the primary antibody, with all other steps kept equal, confirming that the staining is specific. immunostaining was evaluated in stroma, epithelial nuclei, and cytoplasm by densitometric analysis using an arbitrary scale from 0 to 255, performed with axiovision software under a zeiss - axioskop ii light microscope. sixty points, equally distributed in each one of the regions, were scored, and the resulting values were expressed as mean se. fisher 's exact test was used to determine if there were any significant differences among the groups regarding the presence of bacteria and the virulence of genotype caga. to determine whether an association exists between groups and relative gene expression, we used the unpaired t - test with welch 's correction. the same test was used to investigate the existence of an association of risk factors (age, gender, smoking, and drinking) and h. pylori infection with the values of relative gene expression. the nonparametric mann - whitney test was used to determine whether there was a correlation between the virulence of genotype caga and the relative gene expression. for protein expression, the mean values obtained by densitometry for each region were compared in groups c, i m, and gu, using the anova followed by the bonferroni test. to determine the association between h. pylori infection and the values of protein expression we used the nonparametric mann - whitney test. forty - six percent (29/63) of all gastric lesion samples were found to be positive for h. pylori ; 38.9% (14/36) of them belonged to the i m group and 55.5% (15/27) to the gu group. among them, 75.9% (22/29) were also positive for the caga genotype, 85.7% (12/14) belonging to the i m group and 66.7% (10/15) to the gu group. no significant difference regarding the bacterium infection (p = 0.21) or the presence of the caga genotype (p = 0.39) was found between the i m and gu groups. all 10 samples of normal mucosa were confirmed by molecular testing as h. pylori negative. the relative gene expression of anxa1 was upregulated in 100% of both the i m (mean rq = 6.22 1.43) and the gu cases (mean rq = 6.69 1.56). however, for lgals1, both groups showed basal gene expression, with overexpression in only 36.1% (13/36) of the i m cases (mean rq = 0.35 1.37) and 44.8% (13/29) of the gu cases (mean rq = 0.69 1.65) (figure 1 and table 2). there was no significant difference between the i m and gu groups regarding the mean gene expression values for either the anxa1 or the lgals1 gene (p = 0.21 and p = 0.38, resp.). we also evaluated a possible association among the relative expression levels of anxa1 and lgals1 mrna and the risk factors age, gender, smoking, drinking, h. pylori infection, and caga genotype (table 2). we found a significant increase only in the mean level of lgals1 mrna of the women (0.80 1.30) compared to the men (0.15 1.29) in the i m group (p = 0.03). in normal mucosa, the anxa1 and gal-1 proteins showed low expression in the stroma, while in the epithelium there was no immunostaining for gal-1, and anxa1 presented low immunoreactivity (figures 2(a) and 3(a)). in the i m and gu groups, both proteins exhibited high immunostaining in the epithelial nuclei and cytoplasm as well as in the stroma. for gal-1, these groups showed high staining throughout the extension of the epithelial cytoplasm (figures 3(b) and 3(c)). however, regarding the anxa1 protein, the gu samples presented higher immunostaining in the basal portion of the epithelial cytoplasm (figure 2(c)), while the i m group showed immunoreactivity in the whole cytoplasm (figure 2(b)). the specificity of the reactions was confirmed by negative controls (figures 2(d) and 3(d)). the mean optical densitometry values for anxa1 ranged from 147.5 to 218.2 in the three regions analyzed (epithelial nucleus, cytoplasm, and stroma) and, for gal-1, from 131.6 to 215.8. there was a significant difference in the expression of both proteins in the epithelial cytoplasm and nuclei and in the stroma of the i m and gu groups compared to the control group (p < 0.05). moreover, we found a significant difference (p < 0.05) between the i m and gu groups regarding the nuclear expression of the anxa1 protein in the gastric epithelium (figures 2(e) and 3(e)). this is the first study that revealed increased annexin - a1 expression in human intestinal metaplasia and gastric ulcer and increased galectin-1 expression in gastric ulcer, two precancerous gastric lesions, indicating that these anti - inflammatory mediators can exert effects on the initial steps of stomach carcinogenesis. our results also demonstrated the location of these proteins in the affected tissue and showed that gene expression alterations occur regardless of h. pylori infection and caga virulence genotype. the relative gene expression of anxa1 was 6.2- and 6.7-fold increased, respectively, in intestinal metaplasia and gastric ulcer and these results were confirmed by axna1 protein expression analysis. however, the protein expression was significantly higher in both intestinal metaplasia and gastric ulcer compared to normal mucosa. in the present study, the qpcr and immunohistochemistry techniques performed are reliable due to the absence of quantification and immunostaining, respectively, in the negative controls. several studies have shown minimal or limited correlation between mrna and protein levels, particularly when using average expression values [50, 51 ]. this is justified by the existence of posttranscriptional processes such as translation, posttranslational mechanisms, and degradation, which influence the protein abundance in a specific tissue [5052 ]. the increase of annexin - a1 expression in the lesions analyzed indicates a possible involvement in the progression of gastric carcinogenesis from early lesions, as observed in our previous study in chronic gastritis and now in intestinal metaplasia and gastric ulcer, to gastric cancer. the role of this protein in carcinogenesis is still uncertain, and its effect may seem tissue specific and dependent on its subcellular location. particularly in gastric cancer, the few studies show conflicting results, some of them revealing overexpression of anxa1 [44, 46, 53 ] and others downregulation [39, 41 ]. regarding precursor lesions, martin. observed increased expression of this protein in healing areas of gastric ulcer induced in rats, but we did not find any study in intestinal metaplasia. interestingly, other studies in cancers that originate from multistep processes also show alterations in the anxa1 expression, both in precursor lesions and carcinoma, indicating a greater proximity between precancerous and cancerous stages [19, 21 ]. for example, in oral squamous cell carcinoma, this protein presented decreased expression in the plasmatic membrane of tumor cells and premalignant lesions compared to normal oral mucosa. similarly, alves. also observed reduced expression of anxa1 in premalignant lesions diagnosed as oral leukoplakia and in laryngeal squamous cell carcinoma. the molecular pathway of gene anxa1 in the modulation of carcinogenesis is related to its action as a substrate to the epidermal growth factor receptor (egfr) and protein kinase c (pkc), which implies its involvement in signal transduction pathways to cancer [54, 55 ]. the anxa1 expression influences the mitogen - activated protein kinases (mapks) pathway that is associated with the regulation of biological functions such as cell proliferation, differentiation, and apoptosis. however, its way of acting on these pathway members remains uncertain. increased anxa1 expression was found to be associated with constitutive expression of extracellular signal - regulated kinases- (erk-)1 and 2 in macrophages and vascular smooth muscle cells, contributing to the reduction in the cell proliferation rate through downregulation of cyclin d1. yet, in prostate cancer, the involvement of anxa1 in this pathway was not by an antiproliferative but rather a proapoptotic action through p38 and jnk (c - jun n - terminal kinase) activation. thus, the action of anxa1 on proliferation seems to depend on the tissue type. an antiproliferative activity was found in lung adenocarcinoma, macrophages, and smooth muscle tissue. on the other hand, proliferation stimulation was observed in hepatocytes, in which anxa1 was related to egf, and in breast cancer ; in the latter, it was associated with formyl peptide receptor (fpr2) binding and increased levels of cyclin d1. in gastric cancer patients, the high expression of this protein was related to the promotion of invasiveness and shorter survival, and this relationship occurred through the fpr / erk / itgb1bp1 pathway. the gal-1 protein shows increased expression in various types of cancer and is associated in most of the cases with aggressiveness and metastatic potential [36, 37, 6163 ]. in gastric cancer, however, bektas. did not find expression of this protein in most of the cases studied, whereas jorge. chen. observed increased expression of the lgals1 mrna in the stomach cell line tmc-1, suggesting that this protein is important in the metastasis process. another in vitro study showed that the tumor suppressor gene rassf1a positively regulates the lgals1 mrna level, leading to the suppression of the nf - kb signaling pathway, which indicates that gal-1 expression may be related to cell cycle arrest. regarding gastric lesions, we previously observed elevated protein expression in chronic gastritis, but bektas. did not detect any increased expression in tumor - associated metaplasia and dysplasia, and we found no reports about this finding in gastric ulcer. however, in the present study, gal-1 presented increased expression in both intestinal metaplasia not associated with cancer and gastric ulcer. in contrast, in colorectal adenoma, a precancerous lesion of the colon presented downregulation of gal-1 compared to normal mucosa, showing a change in gene expression in early stages of colorectal carcinogenesis. high expression of this protein in the tumor microenvironment contributes to the development and progression of the tumor by promoting environmental immunosuppression, angiogenesis, and metastasis. gal-1 induces fas - mediated apoptosis in immature thymocytes and activated t lymphocytes, resulting in the activation of caspase 8 and increased mitochondrial membrane potential ; in lymphoblastoid jurkat cells, the apoptosis triggered by gal-1 occurs via jnk / c - jun / ap-1 (activating protein-1 transcription factor). thus, gal-1 contributes to conferring immune privilege to tumors through apoptosis of t cells. gal-1 released in the tumor microenvironment also acts on activated endothelial cells, promoting h - ras signaling via the raf / mapk / mek / erk pathway, which results in the proliferation and migration of endothelial cells and consequent generation of new vessels. taken together, these data, combined with the results of the present study, indicate that galectin-1 can also influence the gastric carcinogenesis process, contributing to the progression of the lesions cascade from the initial stages. besides the change in expression levels, the location of these anti - inflammatory proteins may play an important role in the development of different pathological conditions. we analyzed the location of both proteins in the premalignant lesions, which presented increased expression in relation to normal mucosa in the stroma and in the epithelial cytoplasm and nucleus. exposure to hydrogen peroxide, heat, arsenic, and egf promotes its translocation to the nucleus. in gastric cancer, nuclear expression of this protein was found to be associated with advanced disease and poor prognosis, and in oral carcinoma it is a predictor of lower survival. however, we can not affirm how significant the nuclear expression of this protein was in the precancerous lesions studied here. differently, gal-1 has both cytoplasmic expressions as nuclear, while stromal expression is associated with differentiation stage and metastasis in gastric cancer. our results showed significantly higher expression of this protein in the stroma of intestinal metaplasia and gastric ulcer samples compared to normal mucosa, which may contribute, as shown by hittelet. in colon cancer, to the progression from a precancerous to a cancerous stage. when we analyzed the association of anxa1 and lgals1 mrna expression levels with the risk factors in the two lesions studied, we only found an association between the relative expression of lgals1 and gender in the i m group, with women showing higher expression than men (rq = 0.80 versus 0.15). we did not find any association between the expression levels of both genes and h. pylori infection, nor with the caga genotype. nevertheless, considering the important role of bacterial infection in the development and progression of gastric cancer, investigations in larger populations are needed to confirm these results. regarding the role of the higher expression of galectin-1 in women, von wolff. reported elevated expression of this protein in human endometrium during the menstrual cycles and in maternal deciduas, suggesting a role of this protein in maintaining pregnancy. other studies suggest that sex steroids may regulate lgals1 expression in female reproductive tissues of humans and rats [70, 71 ]. had observed increased expression of this gene in the uterus of ovariectomized rats six hours after treatment with 17-estradiol and 12 hours after inoculation of progesterone, the effects being blocked by antagonists of these hormones. it has therefore been suggested that estrogen and progesterone receptors are involved in galectin-1 expression, so the higher lgals1 mrna expression in women can be explained by hormonal factors. in conclusion, our results evidence that both the anxa1 and gal-1 anti - inflammatory proteins are overexpressed in precancerous gastric lesions such as intestinal metaplasia and gastric ulcer. these results, together with the data of our previous study in chronic gastritis and gastric cancer, allow suggesting their involvement in gastric carcinogenesis from the early stages through the tumor progression cascade, possibly due to the inflammatory process of the gastric mucosa. however, the deregulated expression occurs regardless of the h. pylori infection and caga virulence genotype. further investigations are necessary to clarify the mechanisms of action of these proteins in the progression from precancerous lesions to cancer. | objective. annexin - a1 (anxa1/anxa1) and galectin-1 (lgals1/gal-1) are mediators that play an important role in the inflammatory response and are also associated with carcinogenesis. we investigated mrna and protein expression in precancerous gastric lesions that participate in the progression cascade to gastric cancer, such as intestinal metaplasia (i m) and gastric ulcer (gu). methods. quantitative real - time pcr (qpcr) and immunohistochemical techniques were used to analyze the relative quantification levels (rq) of anxa1 and lgals1 mrna and protein expression, respectively. results. increased relative expression levels of anxa1 were found in 100% of cases, both in i m (mean rq = 6.22 0.06) and in gu (mean rq = 6.69 0.10). however, the lgals1 presented basal expression in both groups (i m : mean rq = 0.35 0.07 ; gu : mean rq = 0.69 0.09). immunohistochemistry revealed significant positive staining for both the anxa1 and gal-1 proteins in the epithelial nucleus and cytoplasm as well as in the stroma of the i m and gu groups (p < 0.05) but absence or low immunorectivity in normal mucosa. conclusion. our results bring an important contribution by evidencing that both the anxa1 and gal-1 anti - inflammatory proteins are deregulated in precancerous gastric lesions, suggesting their involvement in the early stages of gastric carcinogenesis, possibly due to an inflammatory process in the gastric mucosa. |
a 56-year - old previously healthy woman presented with a 3-month history of weight loss (25 lb), dyspnea at rest, weakness, intermittent palpitations, nervousness, and heat intolerance. she denied any hair or skin changes, anorexia, diarrhea, cough, or hemoptysis. her examination revealed a blood pressure of 100/60 mmhg and decreased oxygen saturation (89% on room air). jugular venous distention, a pansystolic murmur at the left sternal border, hepatomegaly, abdominal distention, and bilateral leg edema were also noted. laboratory evaluation revealed a white blood count of 13,800/l with normal differential, hemoglobin (hgb) 9 g / dl, hematocrit 30%, platelet count 157,000/l, normal electrolytes, bilirubin 1.9 mg / dl, alkaline phosphatase 187 u / l, aspartic transaminase 170 u / l, alanine transaminase 177 u / l, tsh 0.0 iu / ml, and free t4 9.8 ng / dl. sedimentation rate, rheumatoid factor, antinuclear antibodies, and rapid protein reagents were all normal. however, the uptake was performed after the contrast computed tomography of the chest, which might have interfered with the iodine uptake. (the free iodine load of contrast media interferes with iodine uptake in the thyroid compromising diagnostic thyroid uptake for about 2 months after administration of contrast media.)1 the heart rate was controlled with intravenous diltiazem, and the patient was anticoagulated. the patient was not given any specific treatment for hyperthyroidism and was discharged on metoprolol and warfarin to be followed up in the outpatient clinic. upon because of atrial fibrillation and signs and symptoms suggestive of cardiac dysfunction, an echocardiogram was performed. this revealed right atrial and ventricular dilatation, right ventricular systolic dysfunction, severe pulmonic and tricuspid regurgitation, and an elevated pulmonary artery systolic pressure of 75 mmhg. other causes of pulmonary hypertension, such as alcohol abuse, hiv infection, and vitamin deficiency were ruled out. the patient was treated with methimazole and in a few weeks became euthyroid in sinus rhythm. at that time liver function tests normalized and a repeat echocardiogram revealed a normal size and function of the right ventricle and only mild tricuspid regurgitation. high output biventricular heart failure with normal or decreased systemic and pulmonary vascular resistance is the expected cardiovascular complication of hyperthyroidism.24 isolated right heart failure, variable degrees of tricuspid regurgitation, pulmonary hypertension, or different combinations of the three in patients with thyrotoxicosis have been infrequently reported.522 possible mechanisms of these cardiac abnormalities include increased blood volume12,17,23,24 or increased right ventricular load because of increased cardiac output and venous return.24 the thin - walled structure of the right ventricle, as opposed to the left ventricle, may render the right ventricle more susceptible to volume overload. high cardiac output - induced or autoimmune - induced pulmonary vascular endothelial injury may lead to pulmonary hypertension.12,25,26 increased pulmonary vascular resistance could also result from increased metabolism of certain pulmonary vasodilating substances.26 these cardiac abnormalities in the reported cases were characterized by the reversibility of the pathology and the benign clinical course once thyrotoxicosis was treated and patients became euthyroid.522 however, one report described a case of pulmonary hypertension and thyrotoxicosis that was complicated with further arrhythmias and asystole because of worsened thyrotoxicosis by amiodarone given for atrial fibrillation.5 atrial fibrillation was documented in only 2 of 14 reported cases of thyrotoxicosis and isolated right heart failure. both of these cases had pulmonary hypertension and tricuspid regurgitation that resolved or improved after treatment of hyperthyroidism (table 1).522table 1characteristics of the current case and other reported cases (data taken from various studies522)type of cardiac abnormalitycurrent caseother reported casesatrial fibrillationyesdocumented in 2 of 14 casespulmonary hypertension (method of evaluation)yes (by echocardiography only)13 of 14 cases (8 cases by echocardiography only and 5 by echocardiography and right heart catheterization)systolic pulmonary artery pressure elevation75 mmhg3371 mmhgtricuspid regurgitationyes (severe)14 of 14 cases8 cases of severe trright ventricular dysfunction / dilatationyes14 of 14 cases characteristics of the current case and other reported cases (data taken from various studies522) in the present case, thyrotoxicosis was associated with pulmonary hypertension, right ventricular failure, and severe tricuspid regurgitation, which resolved when the patient became euthyroid. secondary causes of pulmonary hypertension, such as left ventricular failure, pulmonary embolus, chronic obstructive pulmonary disease, obstructive sleep apnea, alcohol abuse, hiv, vitamin deficiency, and vasculitides were ruled out. our case is consistent with other reported cases, though it was at the extreme end of the spectrum regarding the severity of pulmonary hypertension (table 1). this may illustrate that even severe pulmonary hypertension can be reversed when the underlying cause is corrected. there has been at least one report of a thyrotoxic patient presenting with isolated right ventricular failure with normal pulmonary vascular resistance measured by right heart catheterization.17 right ventricular failure and tricuspid regurgitation might be directly related to thyrotoxicosis independently from pulmonary hypertension. most of these cases of thyrotoxicosis and right ventricular failure and pulmonary hypertension were diagnosed as graves disease, indicating a possible autoimmune mechanism for this association.27 a prospective echocardiographic study of 39 hyperthyroid patients and 39 matched controls revealed that the mean pulmonary artery pressure in the hyperthyroid patients was significantly greater than in controls (38 vs 27 mmhg). moderate to severe tricuspid regurgitation was significantly more common in the hyperthyroid group (7 vs 1). these abnormalities resolved in most patients after 14 months of follow - up. this study and others suggest that isolated right - sided heart failure and pulmonary hypertension are more common than once thought. if an echocardiogram is done routinely on patients with hyperthyroidism, more of these cardiovascular abnormalities may be identified.28 in the setting of hyperthyroidism, pulmonary hypertension and right ventricular dysfunction may be underdiagnosed. because the cardiovascular findings resolve when the patient becomes euthyroid, patients who had these findings might not be evaluated for them.522 it is important to consider hyperthyroidism in patients presenting with pulmonary hypertension or unexplained right heart failure. furthermore, thyroid function evaluation should be included in the work up of primary pulmonary hypertension. because amiodarone may trigger hyperthyroidism in patients with multinodular goiter or worsen coexisting thyrotoxicosis owing to its iodine content, it should not be used for rhythm or rate control of atrial fibrillation without ruling out hyperthyroidism or susceptibility to such condition.5 it remains unclear why only a subgroup of patients with thyrotoxicosis is prone to have isolated right heart failure with or without pulmonary hypertension. | hyperthyroidism may present with signs and symptoms related to dysfunction of a variety of organs. cardiovascular pathology in hyperthyroidism is common. a few case reports describe isolated right heart failure, tricuspid regurgitation, and pulmonary hypertension as the prominent cardiovascular manifestations of hyperthyroidism. although most textbooks do not mention hyperthyroidism as a cause of pulmonary hypertension and isolated right heart failure, the literature suggests that some hyperthyroid patients may develop reversible pulmonary hypertension and isolated right heart failure. we report a case of hyperthyroidism presenting with signs and symptoms of isolated right heart failure, tricuspid regurgitation, and pulmonary hypertension, which resolved with treatment of hyperthyroidism. |
gold copper (au cu) phases were employed already by pre - columbian civilizations, essentially in decorative arts, whereas nowadays, they emerge in nanotechnology as an important catalyst. the knowledge of the phase diagram is critical to understanding the performance of a material. however, experimental determination of nanophase diagrams is rare because calorimetry remains quite challenging at the nanoscale ; theoretical investigations, therefore, are welcomed. using nanothermodynamics, this paper presents the phase diagrams of various polyhedral nanoparticles (tetrahedron, cube, octahedron, decahedron, dodecahedron, rhombic dodecahedron, truncated octahedron, cuboctahedron, and icosahedron) at sizes 4 and 10 nm. one finds, for all the shapes investigated, that the congruent melting point of these nanoparticles is shifted with respect to both size and composition (copper enrichment). segregation reveals a gold enrichment at the surface, leading to a kind of core shell structure, reminiscent of the historical artifacts. finally, the most stable structures were determined to be the dodecahedron, truncated octahedron, and icosahedron with a cu - rich core / au - rich surface. the results of the thermodynamic approach are compared and supported by molecular - dynamics simulations and by electron - microscopy (edx) observations. |
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midgut malrotation is an infrequent congenital anomaly, with an estimated incidence of 0.2% in pediatric population. when symptomatic, it is usually diagnosed within the first months after birth and more than 90% of symptomatic patients present during the first year of life. therefore, adult midgut malrotation is a very rare condition, affecting less than 0.16% of adult population (1). nevertheless, acute or chronic complications may occur in adults, such as midgut volvulus. midgut volvulus as the most frequent complication is not an easy diagnosis because of its low incidence and unspecific presentation symptoms, such as abdominal pain, vomiting and food intolerance (2). diagnostic imaging techniques play an important role in the diagnosis, especially computed tomography (ct) (which reveals the abnormal position of midgut) as well as barium studies of gastrointestinal tract. we described the diagnosis of a midgut volvulus presenting in adult, with the typical barber pole sign in ct angiography that is better shown in three - dimensional volume rendering reconstructions. a 41-year - old man, with no significant medical history, was referred from a gastroenterologist consult for long - term (more than 10 years) intermittent abdominal pain, bloating and vomiting, mostly 2 - 3 hours after eating. the ultrasound imaging revealed severe gastric dilatation, with food and gas retention that prevented proper assessment of retroperitoneal structures (figure 1). barium gastrointestinal transit confirmed gastric dilatation extended to the proximal duodenum without bulbar deformity (figure 2), together with a depressed duodenojejunal flexure and deviation of the midgut usual trajectory. the fourth part of duodenum was downward and the jejunal loops were located in the right upper quadrant. a linear mark corresponding to the ladd s band was seen (figure 3) at the duodenojejunal transition and the jejunal loops were not dilated from this point. with suspicious to midgut malrotation, worsening of symptoms led to abdominal ct study request. abdominopelvic ct study was performed with a multi - detector row scanner (brightspeed 16 ; ge medical systems, milwaukee, usa). angiographic images were obtained after intravenous injection of iodinated contrast, at injection rate of 3 ml / s, using bolus test tracking located at descending aorta. these showed gastric and small intestine dilatation with food retention, abnormal rotation of the small intestine and vessels around the root of the mesentery (whirl sign), beak - like appearance and cecum located in the right upper quadrant, confirming the diagnosis of midgut malrotation and volvulus. collateral intestinal perfusions supplied by an inferior mesenteric artery via a hypertrophic riolan arc and by the jejunal branches of the celiac artery were also seen. an enlarged superior mesenteric vein (smv) in abnormal location posterior and left of the superior mesenteric artery (sma) was found (figure 4). angiographic images depicted clockwise twisting of sma, with corkscrew appearance, showing the barber pole sign in the volume rendering three - dimensional reconstructions (figure 5). with the diagnosis of intermittent partial bowel obstruction due to midgut malrotation with volvulus, the patient was admitted for scheduled surgical treatment, including duodenal release with supraantral duodenal and gastric partitioning performing omega - foot bypass loop, colonpexia and appendectomy. the patient had an uneventful postoperative recovery and was discharged after one week of postsurgical stay without any incidence. midgut malrotation is a rare diagnosis, with a reported incidence lower than 0.2%, and is usually asymptomatic. when symptoms occur, normally present during the first year after birth, although a few of them may remain quiescent during childhood and appear in adult life, presenting with long - term unspecific abdominal complains such as pain, bloating and vomiting due to intermittent volvulation, a dynamic lesion with frequent spontaneous remissions and relapses. both its low incidence and unspecific symptoms make this pathologic entity rarely considered on clinical diagnosis. this explains often diagnostic delay, accounting for many years of abdominal complains before diagnosis (4). midgut malrotation is an embryological anomaly. from the fifth to the tenth week of fetal development, the embryonic gut undergoes rapid elongation exceeding the capacity of the abdominal cavity causing umbilical herniation. during this period, midgut goes through counter - clockwise rotation in a 270 degree, gradually returning to the abdominal cavity. failure in this process can cause different degrees of anomalies in the location of small bowel, caecum, colon and appendix. peritoneal fibrous bands fixing the duodenum, known as the ladds bands, may persist and eventually compress the duodenum causing extrinsic obstruction. malrotation results in malposition of the bowel and malfixation of the mesentery, resulting in a narrow pedicle at the mesenteric attachment, which facilitates volvulation. in symptomatic patients, imaging techniques may reveal a non - suspected diagnosis, shortening the time gap between debut of symptoms and surgical treatment if required. plain abdominal radiography is often the first imaging study performed in these patients, but conventional radiography has low utility. nevertheless, there are a few findings such as abnormal gas - bowel distribution, right sided jejunal loops and absent colon in the right lower quadrant, which may raise the suspicion of abdominal anomaly and lead to more specific imaging studies. it usually shows duodenum and duodenojejunal flexure in the right half of the abdomen, as well as abnormal location of jejunum loops. however, these studies have lower sensitivity for detecting associated complications and are not very specific ; a normal barium study can not rule out gut malrotation (5). ultrasound study, especially indicated in pediatric patients, may reveal abnormal location and caliber of bowel loops ; in some cases a dilated duodenum with distal tapering is appreciable. using color doppler atypical situation of sma may be shown as well as twisted mesentery around the sma ; these features are known as the whirlpool sign, however in adult patients, technical difficulties limit its use (6). nowadays, ct has been considered the most useful imaging technique for demonstrating adult midgut malrotation with or without volvulus ; it can depict smv in anomalous location posterior and left of the sma (7) ; this is a frequent finding but is not entirely diagnostic. patients with malrotation may have a normal sma - smv orientation, and patients with abnormal sma - smv relationship may not have malrotation corresponding to normal variant (8). in addition, abnormal disposition of midgut and duodenum not crossing the spine can be shown. in case of malrotation with volvulus, besides the above findings, ct can show small bowel and mesentery twisted around the narrow sma pedicle showing the whirl - pool sign, first described by fisher (9). also a range of abnormalities like dilatation of small intestine, circumferential bowel thickening, halo appearance, beak - like appearance or closed loops can be seen (10). in specific circumstances, mesenteric angiography has nowadays limited use in diagnostic management of suspected malrotation, because of invasive nature and high cost, but in the past, it was used to demonstrate abnormal mesenteric vessels, with typical corkscrew appearance of whirling sma and its branches, showing the barber pole sign (3). in addition to this finding, tapering or abrupt termination of mesenteric vessels, prolonged contrast transit time, absent venous opacification, or dilated smv can be detected. angiographic ct volume rendering reconstructions can depict similar findings helping in surgical approach (12), although in articles previously cited, similar cases were reported, value of volume rendering reconstructions replacing diagnostic angiography is the outstanding point in this case report. when symptomatic, midgut malrotation requires surgical intervention whether open or laparoscopic, habitually following the ladd s procedure or any of its variations. meanwhile, management of asymptomatic patients is controversial (13) ; there are studies that reveal low risk of volvulus at follow - up (14) as well as studies that recommend surgery to prevent catastrophic complications (15). opinion of patient, clinical condition and potential risks of the procedure weighted against benefits should be considered in each case. | adult midgut volvulus is a challenging diagnosis because of its low incidence and nonspecific symptoms. diagnostic delay and long - term complaints are frequent in this clinical scenario. we reported a patient referred to our diagnostic imaging unit with intermittent abdominal pain, bloating and episodic vomiting for several years. he underwent barium gastrointestinal transit and abdominal ultrasound, which revealed severe gastric dilatation, food retention and slow transit until a depressed duodenojejunal flexure, with malrotation of the midgut and jejunal loops being located in the right upper quadrant. computed tomography angiography was performed, showing rotation of the small intestine around the mesentery root, suggestive of midgut malrotation. in addition, an abnormal twisted disposition of superior mesenteric artery with corkscrew appearance was seen, shaping the pole - barber sign which was evident in volume rendering three - dimensional reconstructions. the patient underwent scheduled surgical treatment without any complication and had good outcome after hospital discharge and follow - up. computed tomography plays an important role in evaluation of adult midgut volvulus. in addition, angiographic reconstructions can help us to assess the anatomic disposition of mesenteric vascular supply. both of these assessments are useful in preoperative management. |
the spectrum of cardiovascular diseases represents a significant cause of morbidity and mortality worldwide, including lebanon. the healthcare system in lebanon is mainly private, in a country with a multiethnic population of nearly 4.5 million. the lebanese order of physicians (lop) counts 602 cardiologists (as of august 1, 2014), and only 400 cardiologists are registered with the lebanese society of cardiology (lsc). the lsc is affiliated to the european society of cardiology (esc), which allows both societies to share common issues and exchange different and complementary strategies. the main mission of the lsc is to decrease the burden of cardiovascular diseases in lebanon, through education and prevention, while improving the healthcare system and making it more accessible. the current healthcare system in lebanon shows major discrepancies regarding the availability and equity of care ; this fact is related to political, social, financial and medical factors. faced with this hard reality, the ministry of health (moh), the syndicate of hospitals (soh), the lsc and lop must come together in an attempt to improve the healthcare system, and to fight against medical sectarianism and inequity. the current lsc board (20132015) has considered and implemented many plans to improve cardiology in lebanon, as a science and as a practice. in order to implement such plans, the lsc needs close cooperation with other medical authorities, especially the lop, soh and moh ; more importantly, the individual commitment of every cardiologist is of the utmost importance. the lsc has established many plans and perspectives within its aim to improve prevention and management of cardiovascular diseases, taking into account financial, social and medical considerations. these plans and perspectives conform to the mission of the esc, which include the following : reduce the burden of cardiovascular diseases, identify and alleviate health care inequalities in different regions, promote humanitarianism globally, and especially in vulnerable population groups.1 the lsc emphasizes humanitarian values and the patient s right to medical care, regardless of his or her background. moreover, the lsc is focused on prevention of cardiovascular diseases and public awareness, and programs for that purpose are implemented, in conjunction with the industry. the role of medical directors in this process is essential ; they can serve as local health governors, especially in a primarily private healthcare system.2 the lsc has set a road map that consists mainly of the following plans and perspectives : invest more into scientific research and medical congresses. implement international guidelines. supervise and advise national media regarding any health - related information. moreover, the lsc adopted the assessment of cardiologists scientific activities according to international standards : nature and volume of publications, active contributions to scientific organizations, and leadership in organizing scientific conferences.3 in june 2014, nearly one year after initiation of the road map, we conducted a self - evaluation using some indicators and markers, and the verdict was grave. despite the progress, the road is long, advancement is made little by little, against multiple obstacles and challenges. one of the major obstacles facing the lsc is the overwhelming dominance of the private sector ; uniform processes or policies can not easily be imposed on a national level. instead, every institution may set up its own local regulations and medical directors are local health governors. in light of this, the lsc is exerting influence on medical facilities by using its scientific authority. in a healthcare system where private medicine is prevalent, individuals are focused on their own progress, and the concept of teamwork is nearly absent. because of this, sensitizing cardiologists to the process of cme is of utmost importance, and acquiring minimal cme credits should be a pre - requisite for maintaining a medical practice license.4 the continuous political and social unrest in lebanon has impacted medical practice including cardiovascular practice. almost all media is private and has a political or religious background, and many the lsc encourages such messages when they aim to improve public awareness and when they are pre - controlled to ensure quality and to avoid advertising objectives. moreover, relatively little is invested in research and scientific activities, in part because of the difficult socio - economic situation in a private medical system where physicians time and energy is mainly consumed by the tasks of daily medical practice. for instance, a registry regarding atrial fibrillation or pacemakers is still missing in lebanon. furthermore, the organization of some scientific events by private individuals or societies without the support of the lsc has raised many objections and suspicions among cardiologists ; such events are parallel to those of the lsc, with sometimes significant industry involvement, creating thereby a conflict of interest. the pharmaceutical and medical device industries must be committed more to the national scientific societies, herein to the lsc, to encourage more investment in research and prevention for better management of cardiovascular diseases. the current lsc (20132015) has utilized high - level strategy to face the real needs of the medical community in a rapidly evolving world where healthcare service is becoming more and more expensive. in order to create a framework for better practice, to fight against medical sectarianism and to decrease the burden of cardiovascular disease it must be more involved in real medical and social life, and this requires a full implementation of plans and perspectives with the aid of the lop, moh and soh. otherwise, private and individual interests may prevail over the public interest, with grim prospects for the future. our aim of a high professional level of cardiology in lebanon may quite simply become a slogan, and the concept of good practice of cardiology in lebanon may become difficult to implement or even may simply vanish. in this paper, we emphasize the constructive role of the lsc in order to improve the quality of care and to decrease the burden of cardiovascular diseases in lebanon. the progression in implementing the function of the lsc is facing many difficulties and in this regard, we addressed general concepts according to the policy and regulations of the lsc. in this view, we did not imply any personal judgment or reflection on any individual or collective entity. in this paper, we emphasize the constructive role of the lsc in order to improve the quality of care and to decrease the burden of cardiovascular diseases in lebanon. the progression in implementing the function of the lsc is facing many difficulties and in this regard, we addressed general concepts according to the policy and regulations of the lsc. in this view, we did not imply any personal judgment or reflection on any individual or collective entity. | scientific societies in medicine theoretically aim to improve a medical field as a science ; however, this role is expanding nowadays to seeking also the improved practice of a certain medical field. in this regard, the current lebanese society of cardiology (20132015) has laid many plans and considered many perspectives. these concern mainly, but not exclusively, the increase of public awareness regarding prevention, investment in research, implementation of guidelines, support of continuous medical education, organization of cardiology symposia and congresses, and achievement of national registries regarding main cardiac conditions, as well as the society s main objective of decreasing the burden of cardiovascular diseases in lebanon. nonetheless, the implementation of such plans and perspectives is facing contrary winds related to a multifaceted phenomenon : the dominance of private medicine with a subsequent lack of teamwork, the dominance of private media, the social and political unrest in lebanon, significant discrepancies in the scientific background of cardiologists, and the absence of a standardized national cardiology licensing exam. importantly, the implementation of such plans and perspectives requires individual commitment, along with the cooperation of the order of physicians, the syndicate of hospitals in lebanon (representing private hospitals) and the ministry of health. moreover, industry must be more committed to medical scientific societies ; the support of cardiology events organized without the auspices of the lebanese society of cardiology is not encouraged because of the presence of significant conflict of interest. |
systemic lupus erythematosus (sle) is a heterogeneous, multi - system autoimmune disease that presents with a wide range of clinical and laboratory abnormalities. challenges in the clinical management of sle include the identification of new and relevant therapeutic targets and of specific biomarkers that can be used to optimize diagnosis, assessment of disease activity (severity of disease) and prediction of flares (periodic worsening of symptoms). dna microarray technology allows a hypothesis - free method to comprehensively identify the genes and biological pathways that are associated with clinically defined conditions. for sle, accordingly, several publications have reported microarray studies of peripheral blood cells to identify gene expression signatures in sle. these studies mainly confirm and extend the central role of the type i interferons in sle. in the may issue of bmc medicine, arasappan and colleagues describe a new meta - analysis method that allows analysis across different dna - microarray datasets to identify genes and processes relevant to sle. because of the complexity of microarray technology and frequently sub - optimally powered studies, verification of results is an essential step in microarray analysis. therefore, combining analyses from different studies is important for increasing power, reliability and validation. however, several important challenges need to be considered when integrating microarray datasets for meta - analysis. first, sample collection, annotation, processing and preparation need to be performed according to quality controlled and compatible, preferably standardized, procedures. there is considerable inter - individual variability in the transcriptomes of sle patients, which is inherent to the heterogeneity of the disease and affects analyses. third, appropriate and properly used data analysis practices are required. in order to establish quality criteria and allow comparisons across independent datasets, recent reports from the microarray quality control (maqc) consortium confirm that microarray technology is robust and should be able to reliably reveal differentially expressed genes across samples using different datasets. several approaches have been used for meta - analysis of microarray data to enable comparative analyses across multiple datasets, to minimize noise and to generate multivariate metrics for clinical use. initial studies compared statistical measures of differentially expressed genes for each dataset to classify samples. others revealed that the concordance between datasets improved markedly when the quantity of differential expression was used for gene selection, rather than the statistical significance. alternatively, the inter - gene correlations between datasets and a ' meta - review ' method for ranking genes using the genuine published evidence for each study have been applied. all these approaches are gene - based, that is, they aimed to identify commonly expressed genes between studies. arasappan and colleagues present a new strategy for microarray meta - analysis that is based on the identification of pathways that are coordinately expressed in multiple disease datasets. they used as input microarray datasets of peripheral blood mononuclear cell samples obtained from sle patients, and healthy controls derived from four different studies, of which two involved only children and the other two only adults. transcriptional profiles were generated and low stringency and fold change cut - offs were applied to select differentially expressed genes. for each dataset, ingenuity pathway analysis was used to identify biological pathways that were differentially expressed between sle patients and controls. validation with the leave - one - out permutations method revealed three main biological pathways that were consistently enriched in sle patients. subsequently, a meta - signature consisting of 37 genes involved in diverse processes was generated. each gene that was selected met the original criteria, was involved in at least one relevant pathway and had a fold change of over 2 in at least one of the datasets. this signature differentiated well between children with sle and healthy controls in a fifth independent dataset. comparison of sle with healthy controls could generate insights into the underlying immune dysfunction and thus help identify therapeutic targets for sle. signatures that were found to be consistently enriched between the different datasets included interferon signaling, corroborating and extending earlier findings, and interleukin-10 signaling, which may reflect a dysregulated inflammatory process linked to humoral immune activation and janus kinase (jak)/signal transducer and activator of transcription (stat) signaling. finally, a glucocorticoid receptor signaling signature was also implicated, although corticosteroid therapy, frequently used for sle, may be a confounding factor. specific signatures such as the (low density) neutrophil, immunoglobulin and lymphopenic signatures that were previously reported to be part of one of the datasets were not listed. this finding supports observations from studies directly and indirectly comparing cohorts of children and adults with sle, which revealed that no known unique physiological or genetic pathways were identified that can explain the variability in the disease phenotypes. overall, the pathway - based approach of arasappan and colleagues seems to offer a simple and valuable way to increase the power of microarray data meta - analysis. using different pathway - level stringencies and approaches, such as gene set enrichment analysis, panther and metacore for confirmation of pathway signatures, may increase the robustness of the results. the value of the different meta - analysis methods will become apparent in comparative studies. such benchmarking, together with incorporation of properly annotated demographic and clinical data, would allow optimization of meta - analysis approaches. the future challenge is to use meta - analysis strategies to identify gene signatures and biomarkers that can improve measures for diagnosis and disease activity in sle. in an attempt to use a blood leukocyte gene expression profile to improve the diagnosis of sle, chaussabel and colleagues went one step further and compared a dataset from sle with datasets from several other diseases, including those that show an interferon signature. to compare datasets across multiple diseases, they used a custom meta - analysis strategy for diagnostic biomarker selection using statistical significance (p 3) sledai score, confirming results from other independent studies. one such study was performed by chaussabel and colleagues, who used a ' modular analysis framework ' based on the identification of coordinately expressed genes in disease datasets that form transcriptional modules. their approach predicted severity of disease more accurately than the sledai in some cases, demonstrating that the blood leukocyte gene expression profile might be useful for discovering diagnostic and prognostic biomarkers and monitoring disease progression. unique to transcriptome analyses is the identification of gene signatures that represent biological networks, such as the interferon system, that are relevant in disease pathogenesis and thus provide a starting point for a systems biology approach. successive research activities on these networks, together with approaches using complementary platforms such as (epi)genetics, multiplex fluorescence - activated cell sorting and advanced metabolomics / proteomics, will provide a complete insight into the mechanism and other network components of processes and pathways relevant to disease. for example, interferon - based genetic studies led to the identification of polymorphisms that are strongly associated with sle in genes encoding interferon regulatory factor 5 (irf5), stat4, interleukin-1 receptor - associated kinase 1 (irak1), autophagy protein 5 (atg5) and three prime repair exonuclease 1 (trex1), genes that are all connected with deregulated interferon activity. proteomics on downstream components revealed that a composite chemokine score for the interferon - regulated chemokines cxcl10 (ip-10), ccl2 (mcp-1) and ccl19 (mip-3b) in patients with a sledai of 4 or less were predictive of a lupus flare over the ensuing year. thus, besides identifying clinically relevant transcriptome markers, dna - microarray technology provides a basis for an evidence - based systems biology approach to delineate pathogenic processes and reveal other relevant markers. meta - analysis methods will be instrumental in helping to select those exploratory markers for further biomarker validation, which will pave the way for clinical development and benefit patients. | systemic lupus erythematosus is a systemic, heterogeneous autoimmune disease. understanding of its molecular complexity is incomplete and there is a need to identify new therapeutic targets and to optimize criteria for its diagnosis, assessment and prognosis. recently, arasappan and colleagues have described a new meta - analysis method that enables data analysis across different dna - microarray datasets to identify genes and processes relevant to systemic lupus erythematosus. their study provides a simple and valuable meta - analysis method for the selection of biomarkers and pathways in disease.see related research by arasappan. : http://www.biomedcentral.com/1741-7015/9/65 |
metabolic syndrome (mets) is defined as a cluster of risk factors that identifies persons with increased risk of cardiovascular disease (cvd). the prediction of cvd onset does not have to be better than the framingham score and systematic coronary risk evaluation (score), based on the main factors of cardiovascular risk (age, sex, systolic blood pressure, smoking, total, and hdl and ldl cholesterol). the epidemiological proportions of mets prevalence support its importance in the past 5 decades in countries where the population was found to have increased food consumption and insufficient physical activity. recent studies indicate that mets is inferior in establishing rules for the prediction of either type 2 diabetes mellitus (dm2) or coronary heart disease (chd). mets, as a predictor of cvd, has also been studied due to the existence of different definitions. the most frequently mentioned definitions are the third report of the national cholesterol education program (ncep) expert panel on detection, evaluation and treatment of high blood cholesterol in adults, adult treatment panel iii, the new world health organization (who) definition, the modified international diabetes federation (idf) definition [810 ], and that of the american college of endocrinology (aace). the who and the european group for the study of insulin resistance guidelines (egir) have been primarily proposed for the needs of research, and the ncep and idf definitions were designed for clinical use. this indicates that the role of mets as a cvd predictor is still uncertain and insufficiently researched. differences in the definition of mets create confusion in the timely detection of persons with increased cardiovascular risk in general practitioners practices. although there are studies about mets in the republic of croatia [1319 ], there have been few population studies that analyzed the predictive relevance of the association of each definition with cvd been sparse. we aimed to determine if, defined on the basis of the who, ncep, or idf criteria, mets was associated with an increased risk of developing cvd in the population under a family physicians care, living in various life conditions (region, rural vs. urban), and depending on age and sex. this study was conducted within the randomized clinical research of the cardiovascular risk and intervention study in croatia - family medicine (crisic - fm) in the republic of croatia and was registered as a clinical trial (international standard randomized controlled trial number register exclusion criteria were the inability to communicate due to conditions such as dysphasia, aphasia, serious dementia or psychiatric decompensation, and an expected survival of less than 6 months. the first phase was to establish a quadruple stratified representative sample of family medicine physicians according to regions (coastal and continental), population size (up to 3999 inhabitants ; 4000 to 9999 ; 10 000 to 29 999 ; 30 000 to 89 999 ; and 90 000 and over), rural area (4000 inhabitants), and the number of the insured individuals contracted between family medicine practitioners and the croatian health insurance (hzzo) in 2007 (up to 1399 ; 1400 to 1799 ; and 1800). for each initially contacted physician, a reserve sample of 4 more gps was made, according to the 4-fold stratum. if a gp declined to participate, the nearest gp from a reserve sample was invited. all gps were verbally informed in detail about the study and then signed a consent form to participate in the research. the sample size needed to reach 95% confidence interval and the desired power of statistical tests. of the 82 gps invited to participate in this study, 64 of them accepted (78%), of which 5 declined participation at first follow - up, so the total number of gps in the final sample was 59. in the second stage, each gp chose a systematic, disproportionate sample of the first 55 patients who visited the practice for any reason from the day the study began, and who met the inclusion criteria and confirmed their consent by signing a written informed consent. all the participating gps included the same number of patients (n=55), regardless of the total number of insured persons they have contracted with cihi, and the total number of patients from the target population they examined. this was corrected by post - hoc weight factors prior to statistical analysis (figure 1). a standardized, validated crisic - fm questionnaire with 140 questions, designed for the study, participants height and weight were measured twice (standardized, identical measuring scales) as well as their waist circumference (wc) and hip circumference (with plasticized inelastic tape measure), and their waist - hip ratio (whr) was then calculated. mean arterial pressure (mercury sphygmomanometer) and pulse frequency were assessed. a blood sample for the analysis of total cholesterol concentration, hdl and ldl cholesterol, triglycerides, fasting glucose (fg), and uric acid was taken from each participant. the modified who, idf, and ncep definitions (table 1) were used for the mets analysis. coronary disease was defined by previous myocardial infarction, angina pectoris, and/or revascularisation of coronary arteries, and cerebrovascular disease was defined by previous cerebral insult and/or transitory ischaemic attack. peripheral artery disease (pad) was defined by anamnestic data of intermittent claudication (fatigue, cramping and pain during walking) and 4000 inhabitants), and the number of the insured individuals contracted between family medicine practitioners and the croatian health insurance (hzzo) in 2007 (up to 1399 ; 1400 to 1799 ; and 1800). for each initially contacted physician, a reserve sample of 4 more gps was made, according to the 4-fold stratum. if a gp declined to participate, the nearest gp from a reserve sample all gps were verbally informed in detail about the study and then signed a consent form to participate in the research. the sample size needed to reach 95% confidence interval and the desired power of statistical tests. of the 82 gps invited to participate in this study, 64 of them accepted (78%), of which 5 declined participation at first follow - up, so the total number of gps in the final sample was 59. in the second stage, each gp chose a systematic, disproportionate sample of the first 55 patients who visited the practice for any reason from the day the study began, and who met the inclusion criteria and confirmed their consent by signing a written informed consent. all the participating gps included the same number of patients (n=55), regardless of the total number of insured persons they have contracted with cihi, and the total number of patients from the target population they examined. this was corrected by post - hoc weight factors prior to statistical analysis (figure 1). a standardized, validated crisic - fm questionnaire with 140 questions, designed for the study, was administered. participants height and weight were measured twice (standardized, identical measuring scales) as well as their waist circumference (wc) and hip circumference (with plasticized inelastic tape measure), and their waist - hip ratio (whr) was then calculated. mean arterial pressure (mercury sphygmomanometer) and pulse frequency were assessed. a blood sample for the analysis of total cholesterol concentration, hdl and ldl cholesterol, triglycerides, fasting glucose (fg), and uric acid was taken from each participant. the modified who, idf, and ncep definitions (table 1) were used for the mets analysis. coronary disease was defined by previous myocardial infarction, angina pectoris, and/or revascularisation of coronary arteries, and cerebrovascular disease was defined by previous cerebral insult and/or transitory ischaemic attack. peripheral artery disease (pad) was defined by anamnestic data of intermittent claudication (fatigue, cramping and pain during walking) and 65 years of age) because the association of mets with all - cause mortality has already been proven in these women, but, in men, such an association has not been proved. still, in continental europe, (e.g., germany), research showed a higher prevalence in men, mostly between 60 and 79 years of age. our study showed a higher prevalence of mets in the continental region (due to diet), and in urban vs. rural residents (more physical activity). similar results were shown by a study in portugal, where a higher prevalence was found in older women in the continental region. the prevalence of mets, according to the ncep definition, in the sample of 9 dalmatian islands in 2006, was 34%, even reaching 47.2% on the island of vis. the results of our study of the prevalence of mets in the coastal region, with 26.6% (ncep) and 26.1% (idf), are very similar to the results shown by the study of mets on the island of hvar in 20078, where it was 25% and 38.5%, based on ncep and idf definitions, respectively. data on the prevalence of mets among the mainland croatian populations is limited. a study conducted in a region of continental croatia (baranja) showed that the prevalence of mets, assessed by the ncep criteria, was 40% (35% in males and 42% in females). the prevalence of mets was much higher than in our study, but it is necessary to emphasize that our study was conducted in the whole territory of the republic of croatia. the main study limitation is the inability to meet the exact who criteria, due to difficulties in establishing microalbuminuria diagnosis in family medicine. furthermore, gp s engagement in the intervention group could not be completely controlled, and therefore possibly was not equal among all physicians randomized in that group. the sample s subjects were patients registered by gps, which does not entirely correspond to the general population sample. in addition, we did not examine microalbuminuria, which is part of the who definition, due to its unavailability at the primary health care level in croatia. the sample s subjects were patients registered by gps, which does not entirely correspond to the general population sample. in addition, we did not examine microalbuminuria, which is part of the who definition, due to its unavailability at the primary health care level in croatia. although all 3 definitions of mets were associated with a higher risk for chd, association was the greatest and most consistent when using the ncep definition. according to that definition, the prevalence of mets is higher in the continental region (possibly due to different diet type) and in urban residents (probably due to less physical activity and more sedentary lifestyle), in older people, and in males. | backgroundthis study compared the association between the 3 definitions of metabolic syndrome (mets) suggested by the world health organization (who), national cholesterol education programme (ncep atp iii), and international diabetes federation (idf), and the risk of cardiovascular diseases (cvd) and shows the prevalence and characteristics of persons with mets in continental vs. coastal regions and rural vs. urban residence in croatia.material/methodsa prospective multicenter study was conducted on 3245 participants 40 years, who visited general practices from may to july 2008 for any reason. this was a cross - sectional study of the cardiovascular risk and intervention study in croatia - family medicine project (isrctn31857696).resultsall analyzed mets definitions showed an association with cvd, but the strongest was shown by ncep atp iii ; coronary disease or 2.48 (95% ci 1.803.82), cerebrovascular disease or 2.14 (1.193.86), and peripheral artery disease or 1.55 (1.042.32), especially for age and male sex. according to the ncep atp iii (idf), the prevalence was 38.7% (45.9%) [15.9% (18.6%) in men, and 22.7% (27.3%) in women, and 28.4% (33.9%) in the continental region, 10.2% (10.9%) in the coastal region, 26.2% (31.5%) in urban areas, and 12.4% (14.4%) in rural areas. older age, male sex, and residence in the continental area were positively associated with mets diagnosis according to ncep atp iii, and current smoking and mediterranean diet adherence have protective effects.conclusionsthe ncep atp iii definition seems to provide the strongest association with cvd and should therefore be preferred for use in this population. |
str (or microsatellite) loci consist of simple tandem repeated sequences of 16 bp in length. as with the larger variable number tandem repeat (vntr or minisatellite) loci, strs may exhibit a high degree of length polymorphism owing to variation in the number of repeat units displayed (1). however, unlike vntrs, which occur predominantly in telomeric regions, strs appear to be abundant throughout the human genome and occur, on average, every 610 kb (1). because of their abundance, polymorphic nature, and amenability to amplification by pcr, strs are ideal markers for genomic mapping and genetic linkage analysis (2). in addition to their suitability for mapping and linkage analysis, strs provide a source of highly informative loci for use in the identification of individuals. dna profiling based on pcr amplification of strs has the advantage of being more sensitive than conventional techniques. furthermore, because of their small allele sizes (generally < 300 bp), str systems are more likely to be successful on old or poorly stored specimens that contain only degraded dna (35). also, the ability to resolve pcr products differing in size by just 1 base on polyacrylamide gels allows precise allele designation, thus eliminating the need for the continuous allele distribution models currently employed with vntr systems (6, 7). high mutational rates of microsatellites lead to extensive polymorphism and increase the probability that isolated populations diverge rapidly at these loci. such characteristics make them particularly useful in the study of population genetics of species showing limited variability at other markers (8). it has been almost a decade since the 13 genetic markers that form the core of the fbi laboratory s combined dna index system (codis) were selected in november 1997 (911). are csf1po, fga, th01, tpox, vwa, d3s1358, d5s818, d7s820, d8s1179, d13s317, d16s539, d18s51, and d21s11. these loci have become the common currency of data exchange for human identity testing both in forensic casework and paternity testing largely because of their ease of use in the form of commercial str kits (12). finding suitable and specific markers for ethnic groups or populations would be useful in different studies such as forensic, population diversity, and paternity. blood samples (n = 308 for tpox and n= 392 for vwa) were collected from unrelated, randomly selected individuals throughout khuzestan province. the total genomic dna was extracted from the leukocytes using diatom dna prep extraction kit (gen fanavaran, iran) according to the instructions provided by the manufacturer. the dna samples were used to genotype vwa and tpox str loci using polymerase chain reaction (pcr). the pcr reactions were carried out in a 25 l total volume containing 0.2 mm of dntp and 2 units taq - dna polymerase (cinagen, iran) in a pcr buffer [50 mm tris - hcl ph 8.8, 15 mm (nh4)2so4, 1.5 mm mgcl2, 0.01% gelatin ] (13). the amplification cycles were as : 1 cycle at 94c for 4 min ; 30 cycles (94c 30 sec, 60c for 45 sec, 72c for 1 min), followed by 1 cycle 72c for 5 min. the motives and respective locations of these loci are as follows : tpox : (aatg)n, intron 10 of the thyroid peroxidase gene (2p23 - 2pter) ; (may 2004, ncbi build 35) (genbank accession m68651) vwa : (tcta)n, intron 40 of the von willebrand factor gene (12p12-pter) ; (may 2004, ncbi build 35) (genbank accession m25858) the pcr products (2 l) were separated on a vertical 15 cm 25 cm 0.75 mm polyacrylamide gel electrophoresis (page). visualization was undertaken by the silver staining method (14) using the uv tec gel documentation apparatus (uv tec, uk). allele size was determined by comparison with the 20 bp dna ladder (fermentase, ca). the 20 bp marker contains dna fragments in the size range from 20 to 300 base pair (figure 1, figure 2). one - dscan 1.3 software (scanalytics, billerica, ma) was used to analyze the gel run. statistical analysis was undertaken using the following statistical packages : arlequin software version 3.1 was used to assess hardy - weinberg equilibrium (hwe) and to determine population parameter () and gene diversity indices (gd) (15) ; the frequency of each allele for each locus tested was calculated by power marker version 3.25 (16). polymorphic information content (pic), observed heterozygosity (ho), expected heterozygosity (he) were performed with power marker version 3.25. blood samples (n = 308 for tpox and n= 392 for vwa) were collected from unrelated, randomly selected individuals throughout khuzestan province. the total genomic dna was extracted from the leukocytes using diatom dna prep extraction kit (gen fanavaran, iran) according to the instructions provided by the manufacturer. the dna samples were used to genotype vwa and tpox str loci using polymerase chain reaction (pcr). the pcr reactions were carried out in a 25 l total volume containing 0.2 mm of dntp and 2 units taq - dna polymerase (cinagen, iran) in a pcr buffer [50 mm tris - hcl ph 8.8, 15 mm (nh4)2so4, 1.5 mm mgcl2, 0.01% gelatin ] (13). the amplification cycles were as : 1 cycle at 94c for 4 min ; 30 cycles (94c 30 sec, 60c for 45 sec, 72c for 1 min), followed by 1 cycle 72c for 5 min. the motives and respective locations of these loci are as follows : tpox : (aatg)n, intron 10 of the thyroid peroxidase gene (2p23 - 2pter) ; (may 2004, ncbi build 35) (genbank accession m68651) vwa : (tcta)n, intron 40 of the von willebrand factor gene (12p12-pter) ; (may 2004, ncbi build 35) (genbank accession m25858) the pcr products (2 l) were separated on a vertical 15 cm 25 cm 0.75 mm polyacrylamide gel electrophoresis (page). visualization was undertaken by the silver staining method (14) using the uv tec gel documentation apparatus (uv tec, uk). allele size was determined by comparison with the 20 bp dna ladder (fermentase, ca). the 20 bp marker contains dna fragments in the size range from 20 to 300 base pair (figure 1, figure 2). one - dscan 1.3 software (scanalytics, billerica, ma) was used to analyze the gel run. statistical analysis was undertaken using the following statistical packages : arlequin software version 3.1 was used to assess hardy - weinberg equilibrium (hwe) and to determine population parameter () and gene diversity indices (gd) (15) ; the frequency of each allele for each locus tested was calculated by power marker version 3.25 (16). polymorphic information content (pic), observed heterozygosity (ho), expected heterozygosity (he) were performed with power marker version 3.25. genotyping and allele frequency of tpox and vwa loci were investigated. in table 1, the characteristics of the str loci examined are illustrated. the observed allele frequencies of tpox and vwa loci in arab and non - arab populations are shown in table 2. moreover, the expected (he) and observed (ho) heterozygosity, pic, gd,, and p - values were calculated and presented in table 3. the results obtained revealed that neither of the markers studied were in agreement with the hardy - weinberg expectations in this population. the differences between observed and expected heterozygosis were significant, which demonstrates an excess of homozygotes in our sample. this disequilibrium can be due to sub - population and too many consanguineous marriages (17, 18). for vwa, the 17 and 18 alleles in arab population and the 19 and 17 alleles in non - arab population had the highest frequency (table 2). for tpox, the 9 allele in non - arab population and the 8 and 9 alleles in arab population had the highest frequency (table 2). both str loci showed a high degree of pic values (0.83 for vwa and 0.87 for tpox). the high level of heterozygosity (0.78) for vwa marker indicated that this locus potentially can be a very promising marker and could be used in individual and ethnic group identification and also for identity testing in khuzestan province. moreover, at the tpox locus the fairly even allele distribution within the examined population makes it particularly informative for forensic and paternity testing purposes. polymorphic information content (pic) analysis for both strs (vwa and tpox) showed relatively similar values for forensic and individual identification purposes (see table 3). these loci were very informative based on observation of a high degree of pic values, 0.83 for vwa and 0.87 for tpox. these data were also compared with other reports from iranian and non - iranian populations (19, 20). in our study pic values for vwa and tpox were higher than those reported for the iranian population (19). in addition, the vwa and the tpox markers showed a high level of heterozygosity (0.78) and a lower level of heterozygosity (0.53), respectively. the pic values and ho for vwa locus were closer compared with the tpox locus. furthermore, our results for ho and pic measures of vwa and tpox loci were compared with other populations (2124). the highest ho for vwa and tpox was seen in portugal (21). although the lowest values of ho for vwa and tpox were reported in china (24). neither loci in our populations, arab and non - arab, were in the hardy - weinberg equilibrium. this is not unexpected, due to many sub - populations and too many consanguineous marriages (17, 18, 23). as reported previously, these loci have been found highly polymorphic with a large degree of variability in different populations (19, 21, 2529). reports from other populations indicated a high degree of variation in allele numbers of these str loci, which makes them particularly informative for forensics and paternity testing purposes (22, 3032). our results strongly support the application of vwa and tpox genetic markers for personal identity testing in the iranian population maryam jari collected samples, carried out the experimental studies, performed the statistical analysis and wrote the paper. arezu abdollahi and leila ahmadi collected samples, carried out the experimental studies, and maryam heidari helped to carry out the experimental studies. | objective(s):short tandem repeat (str) loci are the most informative dna genetic markers for attempting to individualize biological material for application in paternity and forensic cases.materials and methods : blood samples were collected and the total genomic dna was extracted. the dna samples were used for genotyping vwa and tpox str loci using pcr and polyacrylamide gel electrophoresis.results:this report presents allele frequency data and parameters of biological or legal interest, such as heterozygosity value, polymorphic information content (pic), genetic diversity index (gd) and population parameter () in arab and non - arab population of khuzestan province (iran) for the loci vwa and tpox. blood samples (n= 392 for vwa and n=308 for tpox) were collected from individuals unrelated throughout khuzestan province. the loci were genotyped using the polymerase chain reaction (pcr) followed by polyacrylamide gel electrophoresis (page) and silver staining. chi - square test showed that neither str loci were in agreement with the hardy - weinberg equilibrium.conclusion:the examined str loci in this study have proven a relatively high genetic variation in the iranian population. the data could be used for construction of a forensic genetic database for the iranian population. |
the influence of the physical activity on human health has been proven in a great number of previous researches, but unfortunately there is still a large part of the population that is not sufficiently physically active (1). diseases whose cause is hypokinesia represent a big problem of modern medicine. the sport, recreational physical activities are indispensable tool in compensation of lack of movement. for these reasons, many developed countries have created an extensive national strategy to promote physical activity among their citizens, which is a priority in the attempt to influence the reducing of enormous costs in health care in the long run. in adolescents, the health benefit from the physical activity includes : healthy growth and development of the bones, muscle and cardio - respiratory system, maintenance of the energy balance, avoidance of risk factors for cardiovascular disease, opportunities for social interaction and positive mental well - being, including higher self - esteem, lower anxiety, and lower stress (2, 3). given the fact that the physical activity declines by growing, the adolescence is a critical period for intervention. if, in early adolescence, there are habits for engaging in physical activity, the adolescents are more likely to be physically active in the adulthood (4). physical activity is a complex behavior influenced by several internal and external factors, such as socio - cultural, psychological - cognitive and the physical and social environment surrounding the individual. the explanation on how the factors influence the behavioral change is crucial in developing intervention strategies and educational programs that will contribute to increasing the levels of physical activity in young people (5). current guidelines recommend that all young people should participate in physical activity of at least moderate intensity for one hour per day. the results from the researchers conducted so far show that : the self - efficacy (confidence in own ability to perform specific behavior in certain situations), increasing the level of the perceived benefits towards physical activity, reducing the perceived barriers, increasing the social support from the closest ones (parents and peers), enjoyment in physical activity and access to the equipment and objects are factors associated with physical activity in adolescence (5, 6). in the republic of macedonia there are no many studies investigated the factors influencing the physical activity in adolescents from macedonian nationality (7), and the researches realized on macedonian adolescents from albanian ethnic community (8), who have their own social - cultural, religious and other specifics, are even fewer. on the other hand, in the world, a lot of researches have been realized on this topic in many countries (different geographical areas), but the question is if the results obtained from these researches can be also generalized for the population of the macedonian adolescents (9). because of everything stated above, this research has been realized in order to (a) determine the relationship of demographic, psychological, social and environmental factors with physical activity, (b) determine whether indicators of physical activity deffer by gender (c) and to make recommendations for interventions for the purpose of promoting active and healthy lifestyle in this group of population. our aim was to determine the relationship of demographic, psychological, social and environmental factors with physical activity and to determine whether indicators of physical activity differ by gender among macedonian adolescents from albanian ethnic community from 11 to 14 yr (n = 886). the research was realized on a sample of 886 macedonian adolescents from albanian ethnic community at the age from 11 to 14 yr. the sample was divided into two subsamples according to gender out of whom 427 (49.2%) male respondents and 441 (50.8%) female respondents. the average age of the respondents from both gender was 12.1 0.93 yr. the sample included students whose parents had given consent for their son / daughter to participate in the research, and who were psychologically and physically healthy and who regularly attended classes of physical and health education. measurements were conducted in mar, apr and may 2014, in standard school conditions at regular classes of physical and health education. a questionnaire was used to identify (i) demographic factors (ii) the physical activity, (iii) the sedentary behavior and (vi) the factors associated with physical activity. demographic factors : a ten - item investigator - developed questionnaire was used to collect demographic data. the following demographic data and participant characteristics were collected to describe the study sample : age, sex, number of family members, education of the father, education of the mother, age of the father, age of the mother, monthly income, whether the father is dealing with sport, whether the mother is dealing with sport. physical activity : the physical activity is assessed by using the instrument physical activity questionnaire (elementary school), which is construed by kowalski and crocker (10). it has been translated into macedonian and albanian and proofread by professionals and adapted for the purposes of this research. in many previous researches it has been determined that the reliability of the physical activity questionnaire instrument (elementary school) is high, ranging from.80 to.89. the validity of the instrument was determined in comparison with the accelerometer (css), which the respondents wore seven days and it was satisfactory (r =.39 to.46). sedentary behaviors : sedentary habits are assessed with a scale that is part of the hbsc study (11). the respondents were asked to answer a question, how long they spend in sedentary activity such as : watching tv (including watching video or dvds), computer work (playing computer games, chatting or surfing on the internet). the questions firstly refer to the working days (monday friday), and then to the non - working days. the respondent answers to each item fixed at three degree scale, ranked 1 h, 23 h, 4 h and more. perceived benefits are assessed by means of a 9-item scale, stating the reasons why some people are physically active. it is of the likert type of 5 degrees, ranked from 1 (completely disagree) to 5 (totally agree). the result is obtained as an average value of the responds to all items (12). perceived barriers are assessed by means of a 9-item scale, stating the reasons why some people are not physically active. it is of the likert type of 5 degrees, ranked from 1 (never) to 5 (very often). the result is obtained as an average value of the responds to all items (12). perceived exercise self - efficacy was assessed using the children s physical activity self - efficacy survey, an 8-item scale developed by garcia. very true to not at all true and a mean score, ranging from 15, was computed by averaging responses to the eight items. perceived sports competence was assessed using the physical ability subscale of the self - description questionnaire (14). very true to not at all true and a mean score, ranging from 17, was computed by averaging responses to the eight items. body image was assessed using a single item from the hbsc study (11). the question was like do you think your body is... parental support. levels of paternal and maternal support for being active were measured using an adapted version of a scale from the amherst health and activity study (15). five items assessed different aspects of parental support including encouragement, participation in joint activities, provision of transport, observation and praise. a mean score, ranging from 15, was computed by averaging responses to the five items (12). peer support for being active was measured using an abridged version of the parental support scales. two items assessed how often friends provided encouragement to be active and how often they took part in joint activities. a mean score, ranging from 15, was computed by averaging responses to the two items (12). physical activity enjoyment. the question of how much do you enjoy doing physical activity ? was asked, as well as asking respondents about their enjoyment of activity. neighborhood environment : perceptions of the local neighborhood were assessed using a question from the hbsc study (11). pupils were asked which of the following were present in the area where they live. a mean neighborhood perceptions score (range 13) was computed based on responses to the four items. the mean score neighborhood safety : two items, adapted from the amherst health and activity study student survey (12), were used to measure perceived neighborhood safety in relation to physical activity (walking or cycling). a mean neighborhood safety score (range 15) availability of local facilities : pupils were asked which of the following facilities they have in the area where they live : sports center, playing field, park, swimming pool, basketball courts or hoops, tennis courts. a total availability score (range 06) access to local facilities : pupils were asked how easy it is for them to get to the following facilities : sports center, playing field, park, swimming pool, basketball courts or hoops, tennis courts. a total access score (range 06) was computed based on the number of very easy or the normality of distribution of public variables is tested with the kolmogorov - smirnov method and log transformations were made when possible. the variables that satisfy the criteria of normality were analyzed by using parametric statistical procedures, while the variables that do not meet the criteria of normality are analyzed by using statistical procedures if nonparametric statistical procedures. independent samples t - tests and mann - whitney u tests were used to compare differences in pa for gender. comparison of means used a two - tailed hypothesis with the alpha levels set at p <.05. spearman s rho correlation was used to analyses the relationship between pa and potential correlates. the research was realized on a sample of 886 macedonian adolescents from albanian ethnic community at the age from 11 to 14 yr. the sample was divided into two subsamples according to gender out of whom 427 (49.2%) male respondents and 441 (50.8%) female respondents. the average age of the respondents from both gender was 12.1 0.93 yr. the sample included students whose parents had given consent for their son / daughter to participate in the research, and who were psychologically and physically healthy and who regularly attended classes of physical and health education. measurements were conducted in mar, apr and may 2014, in standard school conditions at regular classes of physical and health education. a questionnaire was used to identify (i) demographic factors (ii) the physical activity, (iii) the sedentary behavior and (vi) the factors associated with physical activity. demographic factors : a ten - item investigator - developed questionnaire was used to collect demographic data. the following demographic data and participant characteristics were collected to describe the study sample : age, sex, number of family members, education of the father, education of the mother, age of the father, age of the mother, monthly income, whether the father is dealing with sport, whether the mother is dealing with sport. physical activity : the physical activity is assessed by using the instrument physical activity questionnaire (elementary school), which is construed by kowalski and crocker (10). it has been translated into macedonian and albanian and proofread by professionals and adapted for the purposes of this research. in many previous researches it has been determined that the reliability of the physical activity questionnaire instrument (elementary school) is high, ranging from.80 to.89. the validity of the instrument was determined in comparison with the accelerometer (css), which the respondents wore seven days and it was satisfactory (r =.39 to.46). sedentary behaviors : sedentary habits are assessed with a scale that is part of the hbsc study (11). the respondents were asked to answer a question, how long they spend in sedentary activity such as : watching tv (including watching video or dvds), computer work (playing computer games, chatting or surfing on the internet). the questions firstly refer to the working days (monday friday), and then to the non - working days. the respondent answers to each item fixed at three degree scale, ranked 1 h, 23 h, 4 h and more. perceived benefits are assessed by means of a 9-item scale, stating the reasons why some people are physically active. it is of the likert type of 5 degrees, ranked from 1 (completely disagree) to 5 (totally agree). the result is obtained as an average value of the responds to all items (12). perceived barriers are assessed by means of a 9-item scale, stating the reasons why some people are not physically active. it is of the likert type of 5 degrees, ranked from 1 (never) to 5 (very often). the result is obtained as an average value of the responds to all items (12). perceived exercise self - efficacy was assessed using the children s physical activity self - efficacy survey, an 8-item scale developed by garcia. very true to not at all true and a mean score, ranging from 15, was computed by averaging responses to the eight items. perceived sports competence was assessed using the physical ability subscale of the self - description questionnaire (14). very true to not at all true and a mean score, ranging from 17, was computed by averaging responses to the eight items. body image was assessed using a single item from the hbsc study (11). the question was like do you think your body is... parental support. levels of paternal and maternal support for being active were measured using an adapted version of a scale from the amherst health and activity study (15). five items assessed different aspects of parental support including encouragement, participation in joint activities, provision of transport, observation and praise. a mean score, ranging from 15, was computed by averaging responses to the five items (12). peer support for being active was measured using an abridged version of the parental support scales. two items assessed how often friends provided encouragement to be active and how often they took part in joint activities. a mean score, ranging from 15, was computed by averaging responses to the two items (12). neighborhood environment : perceptions of the local neighborhood were assessed using a question from the hbsc study (11). pupils were asked which of the following were present in the area where they live. a mean neighborhood perceptions score (range 13) was computed based on responses to the four items. the mean score neighborhood safety : two items, adapted from the amherst health and activity study student survey (12), were used to measure perceived neighborhood safety in relation to physical activity (walking or cycling). a mean neighborhood safety score (range 15) availability of local facilities : pupils were asked which of the following facilities they have in the area where they live : sports center, playing field, park, swimming pool, basketball courts or hoops, tennis courts. a total availability score (range 06) was computed based on the number of access to local facilities : pupils were asked how easy it is for them to get to the following facilities : sports center, playing field, park, swimming pool, basketball courts or hoops, tennis courts. a total access score (range 06) was computed based on the number of very easy or the normality of distribution of public variables is tested with the kolmogorov - smirnov method and log transformations were made when possible. the variables that satisfy the criteria of normality were analyzed by using parametric statistical procedures, while the variables that do not meet the criteria of normality are analyzed by using statistical procedures if nonparametric statistical procedures. independent samples t - tests and mann - whitney u tests were used to compare differences in pa for gender. comparison of means used a two - tailed hypothesis with the alpha levels set at p <.05. spearman s rho correlation was used to analyses the relationship between pa and potential correlates. the mean age of students was 12.1 0.93 yr with 49.2% of the sample male and 50.8% female. scale items and cronbach s alpha are presented in table 2. on the basis of the obtained results, but taking into account the results of the previous researches applied the same scales of population at the same age, it can be concluded that the scales have satisfactory reliability (internal consistent) and can be applied in practice and scientific researches. characteristics of the sample internal consistency reliability of physical activity questionnaire, psychological and social variables in order to determine whether there are differences in physical activity, the sedentary habits, psychosocial factors, the quality and safety of the neighborhood, number, vicinity and access to sports facilities among male and female respondents, the mann - whitney u test is applied. from the review of table 3 which shows the results of applied analysis it can be seen that between male students and female gender statistically significant differences were determined in the variables : physical activity (p=0.001), television viewing - weekends (p=0.001), computer use - weekday (p=0.001), computer use weekends (p=0.011), homework (p=0.001), perceived barriers to physical activity (p=0.001), parents support (p=0.015), peer support (p=0.032), haw many of your friends are physically active (p=0.002), exercise self - efficacy (p=0.001), perceived physical competence (p=0.005), neighborhood environment (p=0.005). table 4 shows coefficients of correlation between physical activity and demographic, social, psychological variables, variables for assessment of the physical environment and sedentary habits among respondents of both genders. from review of the table it can be seen that in the respondents of both genders there is low statistically significant positive correlation (ranging from.12 to.28) determined between physical activity and demographic variables education of the father s education, the mother s education, monthly income of the family, whether the father is dealing with sport, whether the mother is dealing with sport. moderate positive statistically significant correlation (ranging from.27 to.38) has been determined between physical activity and variable perceived benefits towards physical activity, enjoyment in physical activity, enjoyment in the physical education class, perceived self - efficacy, confidence in their abilities, social support from parents, social support from peers, and the number of friends who exercise in respondents of both genders. in respondents of both sexes statistically significant correlation has not been established in variable perceived barriers, body image, the number of family members, age of parents, the variables for assessing the safety of the neighborhood, the number, the vicinity and access to sports facilities and sedentary habits (watching tv, computer work, studying). means and standard deviations for study variables grouped by gender and mann - whitney u tests correlations between physical activity and various factors using spearman s rho in order to determine how the demographic, psychological and social factors, variables for assessment of the physical environment influent multivariate, the hierarchical regressive analysis has been applied.. the first regressive equation explains 14.9% (p=0.001) of the total variable criterion variance with a statistically significant impact on the predictive variables : gender (=0.201, p=0.001), education of father (=0.137, p=0.022), education of mother (= 0.132, p=0.032), monthly income in the family (=0.152, p=0.001), whether the father is dealing with sport (=0.175, p=0.001), whether the mother is dealing with sport (=0.174, p=0.001). hierarchical regression analyses explaining pa note. partial r partial correlation coefficient ; standardized partial regression coefficient ; 1- value of - coefficient in the last equation analysis ; r multiple correlation coefficient ; r change the coefficient of determination ; significance. with the inclusion of the three social variables in the second step the amount of explained variance increases to 26.1% (p=0.001), but the coefficients of the linear relation in the equation for this system of 13 predictors with the level of physical activity is r=0.510 (f (13.491) = 13.309, p=0.001). the social support from parents (=0.202, p=0.001), social support from peers (=0.126, p=0.008) and the number of friends who do exercises (=0.141, p=0.001) are important determinants which statistically significant influence the criterion variable of physical activity. with the inclusion of the third block of psychological variables the amount of explained variance increases to 31.5% (p=0.001). out of the overall predictive system, statistically significant influence have the perceived benefits towards physical activity (=0.109, p=0.010), enjoyment in physical activity (=0.117, p=0.016) and perceived physical competence (=0.153, p=0.001). the public health impact of physical inactivity highlights the need to better understand patterns and determinants of physical activity participation in school - aged children. a large number of recent researches point to decreased physical activity during adolescence with further decrease in the early adulthood. however, less information is available regarding what constitutes these changes and patterns of determinants for population subgroups. in fact, understanding the factors that contribute to decrease of physical activity in adolescents is essential for development of effective programs and strategies to promote physical activity in this group of population. the level of physical activity in girls is lower than in boys (16 19). the results from our research also indicate that boys show significantly higher level of physical activity unlike girls. also, boys perceive a greater degree of self - efficacy, they have more confidence in their abilities, they perceive greater social support from parents and friends, they have more friends who exercise and spend more time on computer work. boys unlike girls consider that facilities and places where they can exercise and do sport near their settlement can be easily reached. girls unlike boys perceive a less barriers from physical activity, spend more time studying and watching tv in the weekend and perceive the neighborhood as a better place to live. the social enviroment where physical activity takes place has a critical impact on young people. family members, friends, teachers and coaches can have an important role in promoting physical activity among adolescents. social influence can function through different mechanisms including encouragement, modeling activity, joint action and practical support. the results from our research showed that many parents can influence on physical activity among adolescents from both genders. recent researches indicate that the parents role in promoting physical activity among their children can take various forms, such as the transfer of positive attitudes and values (20), participation in joint activities with children, organizing the activities for their children (21) and providing transportation to places where they can be physically active (6). the researches show that boys perceive greater social support from parents than girls, while social support from parents is important determinant on which depends whether and how many girls are dealing with physical activity. other features of the family, such as socio - economic status, education of parents can influence physical activity of adolescents. researches for the influence of socio - economic status on physical activity of children and adolescents are contradictory. the research realized within the study scottish health survey indicate that girls from families of lower socio - economic status have higher level of physical activity unlike the girls who come from higher socio - economic status (22). contrary to this research, the results of the research within the study health behavior of school - aged children (hbsc) indicate that adolescents from wealthier families are physically more active and are more likely to participate in vigorous exercise in their free time (12). the results of our research indicate that the monthly income of the family indirectly influence the physical activity. these differences may be due to several factors, such as costs associated with transportation to sports facilities, sports club membership, sports equipment and so on (23). this research does not determine the statistically significant connection between the number of family members and age of parents and physical activity. education of parents influences the physical activity, especially influent is the father s education. sports tradition in the family (parent as a model) has a major impact on physical activity among respondents of both genders. the results of the framingham heart study indicate that children of active parents are almost six times more likely to be physically active in terms of the children of inactive parents (24). furthermore, higher education of parents is associated with higher levels of physically activity of adolescents (25). moreover, the social support from their friends, especially the activity of the group (number of friends who exercise) is important determinant that can help increase physical activity among young people. this is confirmed in several previous researches realized for children, adolescents and preadolescents (26). the degree of perceived benefits to physical activity (belief in the benefits from the physical activity) is also important predictor of physical activity among respondents of both genders. it has been confirmed in several studies realized for preadolescents and adolescents (26, 27). the boys mostly believe that the physical activity will help them be healthier, stronger, and have more energy. girls mostly believe that the physical activity will help them be healthier, have more energy and look better. enjoyment in physical activity is also a strong predictor of physical activity among students of both genders. the concept of enjoyment is defined as a multidimensional construct consisted of factors associated with affect, competence, attitude, and cognition (28). according to scanlan and simons (29) enjoyment is an important factor for involvement in sport and physical activity and can contribute to increase the participation in the activity. rowland and freedson stated that providing enjoyable experience is a potential strategy for increasing the level of physical activity in youth (30). wallhead and buckworth found that enjoyment in physical education class is in relation with the motivational factors about the formation of the habit for active life outside of school, moreover, exercise is associated with physical engagement at physical education class (31). soini established statistically significant gender differences in the enjoyment of physical activity among 15 yr old adolescents (32). according to the research results of soini, boys enjoy more the physical activity unlike girls. in our research it is not determined statistically significant gender differences in the enjoyment of physical activity in this population group. self - efficacy is the belief in own abilities when attempting to fulfill a task. the self - efficacy is based on the idea of the importance of the subjective experience of the personal competence in realizing different goal, and not on the actual knowledge, abilities and skills. the self - efficacy is not assessed as a personal feature, but a belief in own capabilities to coordinate knowledge, abilities and skills to achieve the desired goal. personal factors (cognitive, emotional and biological), environmental factors and behavior have an interactive effect in the model of mutual determinism (33). the results of this research show that the self - efficacy and confidence in their abilities which are the construct of social - cognitive theory are positively associated with the physical activity among students of both genders. the results of other researches also show that the self - efficacy indirectly and directly affect the physical activity of children and adolescents (34 36). the statistically significant association between sedentary habits (watching tv, computer work, studying) and physical activity is not confirmed in this research. the fact that students from both genders much of their free time spend watching tv or working on computer is worrying. the world health organization and the commonwealth health organization, recommend that children and young people should not spend more than two h a day watching tv or using the computer (surfing, chatting or playing computer games). the research results show that the macedonian adolescents from albanian ethnic community, safety of the settlement, number, vicinity and access to sports facilities does not influence the physical activity. this is opposite to some previous researches realized for children and adolescents (37). on the basis of the above stated it can be concluded that it is necessary to prepare a national plan and program to promote physical activity in order to help young people to change unhealthy lifestyle habits and increase physical activity, and thus improve their health. these strategies, plans and programs should take into account the specifics of the environment, customs and cultural characteristics of the region. there is evidence that anyone who will increase the level of physical activity, even after long period of inactivity may have health benefits, regardless of age. changes can be made through a wide change in policy and practice, especially by increasing the intersectional cooperation and the adoption of new roles of different entities in their fields of competence. basically, it takes small changes in policy and practice, in order to promote and increase physical activity of young people. different approaches can be used : individual work, group work, workshops, conferences etc. the main promoter of these educational programs and strategies should be school, but also it should include several governmental and nongovernmental organizations, family and local government and state with wide media campaign. changes in school should be aimed at changing the curriculum which should include contents for physical activity and its importance including new forms of physical activity in the curriculum, improvement of the material base (buildings, exercise equipment etc.). respondents of both genders who have a higher level of self - efficacy, perceive greater benefits of physical activity, have greater confidence in their abilities, enjoy more physical activities, perceive greater social support from friends and parents have higher levels of physical activity. in this research it is not determined the significant relation between the number of family member and parents age and the physical activity. education of parents influences the physical activity among female respondents, while it is not confirmed for the male respondents. sports tradition in the family has a great impact on physical activity among respondents of both genders. an important relation is not determined between the sedentary habits (watching tv, computer work, studying), safety of the environment, the number, the vicinity and access to sports facilities and physical activity. the research results indicate the importance of preparing a national plan and program to promote physical activity in order to help young people change unhealthy lifestyle habits and increase physical activity, and thus improve their health. ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors. | background : the purpose of this study was to determine the relationship of demographic, psychological, social and environmental factors with physical activity and to determine whether indicators of physical activity differ by gender among macedonian adolescents from albanian ethnic community from 11 to 14 yr (n = 886).methods : research were conducted in 2014 in several primary schools randomly selected from tetovo and gostivar region of the r. macedonia. students completed a questionnaire which examined their level of participation in physical activity and sedentary behavior along with a number of potential correlates. hierarchical regression was used to explore the relationship between hypothesised factors and physical activity.results:the boys unlike the girls showed significantly higher levels of physical activity (p=0.001). respondents of both genders who perceive greater benefits from the physical activity (p=0.010). they have more confidence in their abilities (p=0.001), enjoy more in the physical activities (p=0.016), perceive greater social support from friends (p=0.008) and parents (p=0.001) and have higher levels of physical activity.conclusions:the results indicate the importance of developing a national plan and program to promote physical activity in order to help young people to change unhealthy lifestyle habits and increase the physical activity, thus improving their health. |
physicians often encounter questions from parents regarding the normal size of their child 's penis. aberrant growth of male external genitalia may be the first sign of underlying biophysiologic or psychosocial illness. medical consultations regarding these have associated medical, sexual, psychological, and social implications. morphological abnormalities of the penis can affect interpersonal relations and provoke emotional disturbances as the child grows into an adult. with relation to the genital size, it may be the child or parents who are suffering from a misconception, when all that is required is the knowledge of normal variation. at the same time, one must rule out micropenis, which is defined as a penis that is normal in terms of shape and function, but is > 2.5 sd smaller than the mean size in terms of length for age of the child. penile dimensions and normal variations in children have been previously reported, but similar data in indian population is sparse. it is important to establish reference values for penile dimensions in indian children as significant variations may exist among males from different races and ethnicities right from birth to childhood and adulthood. between october and december 2012, we conducted a cross - sectional study on 250 subjects with age ranging from birth to 10 years recruiting 25 subjects in each age group with 1-year intervals. informed consent from the parents in their own language was taken for examining their child, explaining the purpose of the study. we included healthy boys not suffering from any genital, endocrinological, nutritional, or psychosocial disease from among the relatives of other patients, boys admitted for disease unrelated to genitalia, and healthy children coming for routine immunization. a vernier calipers was used to measure : stretched length of flaccid penis from pubopenile vertex to the tip of the glans [figure 1 ]. penis was fully stretched till comfortably tolerated by the subjectdiameter at the mid - shaft on the transverse and dorsoventral axis (the mean was used in the data table) [figure 2]diameter at the corona on the transverse and dorsoventral axis (the mean was used in the data table). stretched length of flaccid penis from pubopenile vertex to the tip of the glans [figure 1 ]. penis was fully stretched till comfortably tolerated by the subject diameter at the mid - shaft on the transverse and dorsoventral axis (the mean was used in the data table) [figure 2 ] diameter at the corona on the transverse and dorsoventral axis (the mean was used in the data table). measurement of the length of stretched penis measurement of mid - shaft circumference all the measurements were taken by a single observer and taken twice to overcome the variability in measurements. the readings were found to be reproducible on examining the child second time in the majority of the cases, but in the case of difference between the two readings, the mean of the two was taken for data analysis. as the penis is not symmetrically cylindrical, diameters at the two mutually perpendicular axes at the corona and mid - shaft were found to differ by 0.5 - 1.5 mm and thus the mean of the two diameters was used in the tabulated data for each age group to nullify the difference. the diameters were multiplied by pi (= 3.14) to calculate circumferences at the mid - shaft and corona. the mean and standard deviation were calculated for each age group, with establishment of normal range for reference values with 2 sd. the height (in cm) and weight (in kg) of all the subjects were measured and the body mass index (bmi, kg / m) was calculated. data were tabulated for the mean stretched penile length and the mean circumferences at the corona and mid - shaft in all the age groups. to derive correlation of penile length with weight, height and bmi and to rule out the confounding factor of age, the pearson correlation coefficient was calculated individually for each age group. the data were compared with the similar studies conducted previously in other parts of the world, and the differences in the results were analyzed individually with each study using student 's t - test. all the penile dimensions increased, along with the height and weight of the child, in successive age groups. penile length increased gradually with the age, with a spurt between the ages of 3 and 4 years [figure 3 ]. the mid - shaft circumference [figure 4 ] showed a trend analogous to the penile length with a steep curve during the 3 - 4 year age group, which relatively flattens toward the end at 8 - 10 years of age, suggesting the relatively small increment in the penile dimensions in comparison with the other age groups in successive years [table 1 ]. coronal circumference followed a similar pattern of progression in size with age as observed in the penile length and mid - shaft circumference. correlation of penile length (cm) with age (years) correlation of mid - shaft circumference (cm) with age (years) the mean and normal range for all the penile dimensions in the various age groups along with pearson correlation coefficient of penile length with weight, height, and bmi the mean height and weight of the children in the study groups were in keeping with the normal range defined by the indian association of pediatrics. the pearson correlation coefficient values did not follow a regular trend for either weight, height, or bmi. some age groups showed statistically significant positive relation with penile length, some showed positive but not statistically significant correlation, and a few age groups even had a negative correlation with penile length [table 1 ]. the inconsistent pattern in the pearson correlation coefficient values in the various age groups was suggestive that the penile length did not have a direct correlation with weight, height, or bmi of the subjects. among all the age groups, the highest pearson correlation coefficient values for penile length with height (0.70), weight (0.77), and bmi (0.62) were found during infancy. upon individually comparing the results using student 's t - test with other studies conducted in the past, the values in this study were found to be statistically smaller from the most of the other studies (brazil, korea, and turkey) with a p < 0.01 during infancy, 4 - 5 years as well as 9 - 10 year age group. however, on comparing the results with the study conducted on bulgarian boys, the penile length was statistically smaller during infancy, smaller but not statistically significant in the age group 4 - 5 years and larger in the age group 9 - 10 years. the age - adjusted values of penile dimensions must be known in order to determine the abnormal penile size and to follow the treatment of underlying diseases. differences in penile anthropometry may exist between several populations owing to ethnic, geographical, genetic, and nutritional factors. the only other study in the literature for indian children that we could find was by vasudevan. their study comprised only of 135 newborn boys and they found the mean stretched penile length to be 3.57 cm and width to be 1.04 cm at birth. these values are marginally greater than our study group and may have been because of differences in growth parameters between north and south indian population. a study on taiwanese boys by wang. suggested that the average penile length increased with chronological age, which is similar to our findings. the mean penile length in the study conducted by gabrich. on brazilian children was 4.7 cm and 7.4 cm during infancy and at 10 year age, which are more than our study group having value 3.34 and 5.25 cm in the respective age groups. this may be due to comparatively higher values of average height and weight in the brazilian population. the penile length in the study conducted by analia. at the cleveland clinic was 3.55 cm during infancy, which is longer than 3.34 cm as measured in our study group, but at 10 years of age, it was 4.84 cm that is shorter in comparison to 5.25 cm as in our study group. this might be due to difference in the extent of stretching of penis and different methodologies adapted for measurements. korean boys found the mean penile length to be 3.5 cm during infancy and 5.6 cm at 10 years, which is slightly more than our study and similarly the penile length was comparatively more in turkish boys with values of 4.44 cm during infancy and 6.79 cm as found in the study by peyami. no direct correlation of penile length with either weight, height or bmi was found when all the age groups were evaluated. of all the age groups, height, weight and bmi had a strong positive correlation with penile length during infancy, which may suggest that birth weight and height may bear some influence on penile length of the child. found a significant positive correlation between the penile length and bmi in turkish children while in our study the correlation between the two variables was not found to be statistically significant. adolescent boys in indonesia found no significant variation in penile length in relation to bmi (pearson coefficient, 0.25). found penile shaft circumference to be 4.38 and 5.52 cm at 1 and 10 year age, and peyami. found the values to be 4.34 and 5.02, which are larger in comparison with values 3.05 and 4.78 noted in our study group. although there have been studies conducted in other parts of the world to measure penile length and shaft circumference in prepubertal age groups, we have additionally measured coronal circumference to take into account the size of the glans. these variables may be used in the future to analyze their effect on diseases affecting the morphology of penis like hypospadias, epispadias, etc. the size of the glans is a relevant criterion in hypospadias surgery, the smaller ones being more difficult to repair and may need local testosterone application to augment results. we used a vernier calipers in our study as it achieves highest accuracy in comparison with rigid tape, measuring ruler, and spatula used in various studies. although there is a theoretical risk of trauma to the child while taking the measurements with a vernier calipers due to its shape, we preferred it because of its accuracy and did not come across any traumatic event during our study. limitations of our study are that we took the mean of the two mutually perpendicular diameters for calculating the circumferences at corona and mid - shaft, which may not be the representative of true circumferences as penis is not uniformly cylindrical in shape. however, the method adapted for measurement was the same so inference with age could be derived. all the subjects were examined to exclude any genital, endocrinological, nutritional, or psychosocial disease but since no objective investigations were done to exclude these diseases, few of them may have had some confounding disease influencing the results. although all the measurements were taken twice by a single observer with the intend to overcome the variability in measurements, but if taken by two different observers might have reduced the bias or error. the penile length was 3.34 cm during infancy, 4.28 cm at 5 years, and 5.25 cm at 10 years, respectively. the mid - shaft circumference values were noted to be 3.05 cm during infancy, 3.86 cm at 5 years, and 4.78 cm at 10 years, respectively. coronal circumference had values of 3.29 cm during infancy, 4.11 cm at 5 years, and 5.05 cm at 10 years, respectively. the penile length increases with advancing age, but no direct correlation with body weight, height or bmi could be established. penile dimensions in north indian children were found to be statistically smaller in comparison with most of the previous studies conducted in other countries. | introduction : physicians frequently encounter questions by parents regarding the normal size of a child 's penis. we evaluated normal variations of penile dimensions, correlation of penile length with height, weight, and body mass index (bmi) of boys and analyzed the differences in penile dimensions from those reported from other countries.materials and methods : a cross - sectional study was conducted at our institution during october 2012-december 2012. a total of 250 subjects (birth to 10 years) were evaluated and divided into 10 groups with 1-year interval taking 25 children in each. penile dimensions measured twice by a single observer with vernier calipers included the length of flaccid penis fully stretched and diameters at mid - shaft and corona. diameters were multiplied by pi (= 3.14) to calculate circumferences. mean, standard deviation, and range were calculated. height, weight, and bmi were noted and statistically correlated with the penile length using the pearson correlation coefficient. data were compared with similar studies reported on other populations in the past and individually evaluated with every study using student 's t-test.results:the mean values for the penile length, mid - shaft circumference, and coronal circumference were 3.34, 3.05, 3.29 cm during infancy, 4.28, 3.86, 4.11 cm during 4 - 5 years, and 5.25, 4.78, 5.05 cm during 9 - 10 years, respectively. the penile length increased with advancing age in successive age groups, but it did not have a direct correlation with either height, weight, or bmi. penile dimensions in north indian children were found to be statistically smaller in comparison with most studies from other countries.conclusion:we provide the normal range and variations of penile dimensions in north indian children. |
oxidative stress plays a critical role in the initiation and development of diabetic nephropathy (dn) (baynes, 1991 ; forbes., 2008 ; giacco and brownlee, 2010 ; hinokio., 1999 ;, 2010 ; schnackenberg, 2002 ; singh., 2011 ; son., 2004 ; stanton, 2011). diabetes is associated with an increase in generation of reactive oxygen species (ros) in the glomerular and tubulointerstitial compartments of the kidney (forbes., 2008 ; kanwar., 2008 ; 2011 ;, 2010 ; koya., 2003 ; singh., 2011 ; stanton, 2011 ; vasavada and agarwal, 2005). although chronic hyperglycemia is sufficient to promote renal cell injury, data from experimental animal models as well as cultured cells indicate that a combination of growth factors, hormones and cytokines, in addition to high glucose concentrations, alter the redox status of the cells and contribute to cell damages in the diabetic kidney (abboud, 1997 ; campbell., 2011 ; gorin and block, 2013a ; 2013b ; kanwar., 2008 ; 2011 while multiple pathways lead to ros generation, i.e., mitochondrial electron transport chain, xanthine oxidase or uncoupled nitric oxide synthase, recent evidence demonstrates that the nox family of nadph oxidases constitutes a major source of ros in nonphagocytic cells, including renal cells (ago., 2010 ; barnes and gorin, 2011 ; bedard and krause, 2007 ; block., 2009 ; brown and griendling, 2009 ; brownlee, 2005 ; coughlan., 2009 ; craven., 2001 ; dikalov, 2011 ; gill and wilcox, 2006 ; gorin and block, 2013a ; 2013b ; gorin., 2005 ; griendling and fitzgerald, 2003 ; hwang., 2012 ; kiritoshi., 2003 ; kitada., 2011 ; lambeth, 2007 ; lambeth., 2007 ; lassegue and clempus, 2003 ; lassegue and griendling, 2010 ; lassegue., 2012 ; nishikawa., 2000 ; the present review will focus on the upstream regulators controlling the expression and activity of the nox enzymes as well as the downstream effectors targeted by nox - derived ros to initiate and propagate diabetes - induced glomerular and tubulointerstitial cell injury. to date, the nox family comprises seven members : nox1 - 5, and the dual oxidases (duox) duox1 and -2 (bedard and krause, 2007 ; geiszt, 2006 ; lambeth. gp91phox), and nox5 are the nox homologues that are predominantly expressed in glomerular cells (mesangial cells, glomerular epithelial cells or podocytes), glomerular endothelial cells and tubulointerstitial cells (tubular epithelial cells and interstitial fibroblasts) (gorin and block, 2013a ; 2013b). these nox homologues consist of six membrane - spanning regions with binding sites for nadph, flavin adenine dinucleotide (fad), and heme. the latter comprise electron transfer centers that pass electrons from nadph to oxygen forming superoxide (o2) and hydrogen peroxide (h2o2) (bedard and krause, 2007 ; brown and griendling, 2009 ; geiszt, 2006 ; geiszt., 2000 ; gorin and block, 2013a ; lassegue and griendling, 2010 ; lassegue., 2012 ; selemidis., 2008 ; shiose., the calcium - dependent homolog nox5 is found in the human kidney tissue and cells, but is not present in mice and rats (brandes., 2010 ; lassegue and griendling, 2010). the isoforms nox2 and nox1 require p22phox as an activating, stabilizing and/or regulatory subunit for binding to p47phox or noxo1 (bedard and krause, 2007 ; geiszt, 2006 ; selemidis., activation mechanisms for nox1 are similar to those of nox2, and involve complex formation with regulatory subunits upon agonist stimulation. although nox1 seems to primarily interact with the p47phox homolog noxo1 (nox organizer 1), the p67phox homolog noxa1 (nox activator 1), and small gtpase rac upon activation, it was reported that p47phox and p67phox can partially replace noxo1 and noxa1, respectively (bedard and krause, 2007 ; brandes and schroder, 2008 ; brandes., 2010 ; geiszt, 2006 ; lambeth, 2007 ; lassegue and griendling, 2010 ; selemidis., 2008 ; nox4 heterodimerization with p22phox enhances the enzyme activity but the oxidase does not required the regulatory subunits essential to other nox isoforms and is a constitutively active enzyme regulated primarily at the level of its expression in response to various stimuli, including mediators of dn such as high glucose (hg) (block., 2009 ; eid., 2009 ; 2013b) transforming growth factor- (tgf-) (bondi., 2010 ; cucoranu., 2005), angiotensin ii (ang ii) (block., 2008), insulin, insulin - like growth factor (igf - i), and advanced glycation end products (ages) or advanced oxidation protein products (aopps) (bedard and krause, 2007 ; lassegue and clempus, 2003 ; lassegue and griendling, 2010 ; lassegue., 2012 ; lee., 2007 ; new., 2012 ; selemidis., 2008 ; thallas - bonke., importantly, it is suggested that transcriptional events are implicated in the chronic control of nox4 protein expression (chai., 2008 ;, 2005 ; eid., 2009 ; 2010 ; 2013a ; hecker., 2009 ; moe., 2006 ; mittal., 2007 ; pedruzzi., 2004 ; serrander., 2007 ; wingler., 2001 ; 2005), whereas acute regulation of nox4 occurs via translational mechanisms without change in its mrna levels (block., 2008 ; 2009 ; regulatory proteins that enhance nox4 activity like polymerase delta - interacting protein 2 (poldip2) and the p47phox - related adaptor protein, tks (tyrosine kinase substrate with five sh3 domains) 5, have recently been identified (diaz., 2009 ; lyle., 2009 ; sedeek., 2009). a potential implication of rac in the control of nox4 function was suggested in endothelial and mesangial cells (chai. ; wu., 2007) ; however, it remains a controversial topic because, unlike nox2 or nox1, rac1 does not activate nox4 in transfected cells (bedard and krause, 2007). the activity of nox5 is not controlled by any other subunits and is strictly dependent on calcium binding to the enzyme through its ef hand domains (bedard and krause, 2007). it is important to mention that nox4 seems to produce a higher hydrogen peroxide to superoxide ratio than nox1, nox2 and nox5. recent studies strongly suggest that hydrogen peroxide formation occurs through nox4 third extracytosolic loop (e - loop) and that the structure of the e - loop may obstruct superoxide release as well as provide a source for protons, thus permitting rapid dismutation of superoxide to generate hydrogen peroxide (takac., 2011). although nox4 predominantly produces hydrogen peroxide, numerous studies in vascular or renal cells and tissue detected nox4-dependent superoxide production (ago., 2010 ; block., 2009 ; clempus., 2009 ; 2010 ; gorin., 2005 ; kuroda., 2005 ; 2010 ; liu., 2010 ; maalouf., 2012 ; peshavariya., 2007 ; the importance of nox4 in mesangial cell injury is supported by studies in experimental rodent models of diabetes as well as in vitro work in cultured cells exposed to hg. nox4 protein expression increases in the glomeruli, including the mesangium, and nox4-derived ros contribute to oxidative stress during the initial and chronic stages of diabetes (eid., 2009 ; 2010 ; etoh., 2003 ; fujii., 2007 ; 2010 ; the elevation in nox4 protein and ros generation are reversed by insulin treatment, confirming that hyperglycemia and hyperglycemia - induced mediators are responsible for these effects (etoh., 2003 ; gorin., our group provided the initial evidence that nox4-dependent ros generation mediates glomerular hypertrophy and mesangial matrix accumulation (gorin., 2005). we showed that inhibition of nox4 oxidase by administration of antisense oligonucleotides for nox4 significantly reduced glomerular enlargement as well as fibronectin accumulation in glomeruli from type 1 diabetic rats (gorin., 2005). recent studies using apoe / nox4 double knockout mouse or nox4 knockout mice on c57bl6/j background made type 1 diabetic with streptozotocin showed that genetic deletion of nox4 markedly attenuated diabetes - induced oxidative stress, mesangial matrix expansion as well as extracellular matrix protein fibronectin and collagen iv accumulation in the glomeruli (jha., 2014 ; thallas - bonke., it should be mentioned that both nox4 and nox5 expression are increased in human diabetic glomeruli (holterman., 2014). in cultured mesangial cells, glucose elicits a rapid upregulation in nox4 protein levels, including in the mitochondrial fraction, which is associated with an increase in cellular and mitochondrial ros production (block., 2009 ; eid., 2013b ; papadimitriou., 2014 ; shah., 2013). moreover, prolonged exposure of mesangial cells to hg has also been described to augment nox4 mrna and protein expression (etoh., 2003 ; fu., 2010 ; jeong., 2012). nox4 is required for hg - induced (acute or chronic) increase in ros production and accumulation of fibronectin in these cells (gorin., 2005). furthermore, nox4 participates to hg - mediated mitochondrial ros generation in mesangial cells (block., this contention is supported by the recent observation that ros generated by overexpression of nox4 are able to oxidize and affect the activity of mitochondrial proteins in cardiac myocytes (ago., 2010). moreover, nox4-derived ros have been reported to decrease mitochondrial function via disruption of complex i in endothelial cells (koziel., 2013). these findings suggest that mitochondrial electron transport chain may be a downstream effector of nox4. a short paracrine loop may exist, by which ros production by mitochondrial nox4 alters mitochondrial respiratory chain activity, thereby leading to more ros generation by the dysfunctional mitochondrial electron transport chain and alteration of mitochondrial function. recent work from our group identified important downstream targets of nox4-derived ros in the pathway linking hg to mesangial cell fibrotic injury (eid., 2013b). the study revealed the role of nox4 as a critical mediator of endothelial nitric oxide synthase (enos) uncoupling and decrease in nitric oxide (no) bioavailability induced by hg in cultured mesangial cells and in diabetes in vivo (eid. we demonstrate that ros derived from dysfunctional enos contribute to fibronectin expression in mesangial cells exposed to hg. the molecular mechanisms underlying this process involve the reaction of nox4-derived superoxide with no generated constitutively by functional enos resulting in the formation of peroxynitrite that subsequently uncouples enos, further promoting superoxide generation (eid., 2013b). in the diabetic milieu, nox4-dependent enos uncoupling, not only eliminates the protective effect of enos - derived no, but also converts the enzyme to a phlogistic mediator that further enhances ros generation and mesangial cell fibrotic response. a role for nox4 in peroxynitrite production and nos (enos or neuronal nos) uncoupling have been confirmed in other systems (de mochel., 2010 ; ito., 2013 ; siu., 2015) as well as in the diabetic kidney where nox4 deletion reduced peroxynitrite production in the glomeruli (jha., 2014 ; thallas - bonke., protein kinase c (pkc) is also a downstream target of nox4 in diabetic glomeruli. this is supported by the findings that the major isoforms of pkc that are increased with diabetes in the glomeruli can be normalized in nox4 knockout mice (thallas - bonke., 2014). we demonstrated that sestrin 2 attenuates hg - induced ros generation and mc mesangial cell fibrotic injury through blockade of nox4-dependent enos dysfunction / decline in no levels (eid. specifically, sestrin 2 counteracts nox4-mediated ros production via impeding the rapid upregulation of nox4 protein elicited by hg. the stress - inducible protein sestrin 2 is known to suppress ros and protect cells from oxidative stress but the mechanisms by which sestrin 2 exerts its antioxidant properties remain unclear. the characterization of sestrin 2 as an upstream negative regulator of nox4 unveiled a novel molecular mechanism by which sestrin 2 is able to inhibit oxidative stress. importantly, amp - activated protein kinase (ampk) functions as a mediator of sestrin 2 inhibitory effects on hg - stimulated and nox4-dependent enos dysfunction and extracellular matrix protein accumulation in mesangial cells (eid., 2013b). the function of ampk as a negative modulator of hg - mediated increase in nox4 expression in mesangial cells is further supported by a recent report (papadimitriou., 2014). it should be mentioned that the protective effects of sestrin 2/ampk is reciprocally blunted by hg. hence, hg promotes ampk inactivation via downregulation of sestrin 2, which in turn results in increased nox4 and nox4-dependent ros production followed by enos uncoupling and decrease in no bioavailability and enhancement of mesangial cell fibrotic response (eid. other studies have demonstrated that statins and rho kinase inhibitors prevent increased nox4 expression and oxidative stress in diabetic kidney, including glomeruli, concomitantly to the amelioration of mesangial matrix expansion and renal function, indicate that small gtpases rho and rac pathways (known targets of statins) may act as upstream modulators of nox4 expression and nox4-dependent ros release (fujii. micrornas also play a role in nox4 expression and serves as an endogenous silencer for nox4 gene in mesangial cells (fu. mir-25 seems to negatively regulate nox4 expression by directly targeting its 3 untranslated region (3-utr) (fu., 2010). the downregulation of microrna-25 (mir-25) by hg in mesangial cells or by hyperglycemia in diabetic kidney results in the relief of nox4 gene silencing that lead to increased nox4 expression and ros production (fu., 2010). similar functional relationship between mir-25 and nox4 was reported in the heart where downregulation of mir-25 results in increased nox4 expression and subsequent oxidative and nitrative stress (varga., 2013). thioredoxin - interacting protein (txnip) has been recently identified as an upstream regulator of nox4 protein expression in mesangial cells exposed to hg. txnip mediates hg - induced ros generation by nox4 in mesangial cells (shah., 2013). the mrna and protein expression of p22phox, the subunit required for full activation of nox4, are upregulated by glucose in mesangial cells as well as in glomeruli from diabetic animals (maeda., 2010 ; interestingly, nox4 and p22phox both contribute to hg - dependent oxidative stress and fibronectin or collagen iv accumulation as well as the expression of other markers of fibrotic injury in mesangial cells (xia. equivalent to what is seen for nox4, ampk activation is also able to counteract the induction of ros production by hg via blockade of a p22phox upregulation in mesangial cells (whiteside., 2009). these observations are consistent with the concept proposing that ampk act as a suppressor of oxidative stress via inhibition of nadph oxidase subunits expression in various biological systems including vascular tissues (mccarty., 2009 ; schuhmacher., 2011 ; because it is known that p22phox interacts with nox4 and enhances its activity, it is reasonable to think that in these cells, nox4 and p22phox may form a complex that accounts for hg - induced ros generation and the subsequent fibrotic response. ang ii elicits an acute increase in nox4 protein expression as well as a chronic prolonged upregulation of nox4 mrna and protein levels associated with enhanced ros production in these cells (block., 2008 ; fujii., 2010 ang ii also elicits an increase in mitochondrial abundance of nox4 protein and the oxidase contributes to ros production in mitochondria (lee. ang ii - induced ros generation in mesangial cells acts principally through nox4 as an upstream activator of extracellular signal - regulated kinase 1/2 (erk1/erk2), proline - rich tyrosine kinase-2 (pyk-2)/src/3-phosphoinositide - dependent protein kinase-1 (pdk-1), akt / protein kinase b (akt / pkb), and/or p70 s6 kinase (p70s6k) pathways that lead to cell hypertrophy and increased protein synthesis and/or fibronectin expression (barnes and gorin, 2011 ; block., 2008 ; more specifically, pyk-2 appears to act as a molecular scaffold binding to both pdk-1 and src, thereby allowing src to tyrosine phosphorylate and activate pdk-1, which in turn activate its downstream effectors, akt / pkb and p70s6k (block., 2008). / pyk-2 complex may favor the formation of signaling platforms bringing key intermediates into proximity, thereby facilitating the contact between nox4-derived ros and downstream effectors (block. importantly, the most proximal event physically tethering nox4 activation by ang ii to the rest of the signaling cascade is a redox - dependent posttranslational modification of src on cysteine residues induced by nox4-derived ros (block., 2008). it was reported that activation of rac1 by phospholipase a2 (pla2)-mediated arachidonic acid (aa) release appears to be implicated in nox4-dependent ros production and subsequent akt / pkb - mediated cellular hypertrophy in mesangial cell treated with ang ii (gorin. our recent work also placed nox4 as central mediator that control ang ii - induced redox signaling that lead to peroxynitrite - dependent enos dysfunction, decline in no bioavailability and mc fibrotic injury (lee., 2013a). the p22phox subunit is also required for the hypertrophic and fibrotic actions of ang ii in mesangial cells (block., comparable to what was observed in a myriad of cell types (barnes and gorin, 2011 ; bondi. 2005), tgf- specifically increases the expression of nox4 and ros production in mesangial cells. moreover, a role for nox4 in tgf--mediated ros generation and mesangial cell fibrotic injury was established (jha., 2014 ; p22phox is required for tgf--induced ros production in mesangial cells (xia., 2008). as described above for hg, ampk also acts as a negative regulator of tgf--dependent nox4 upregulation and subsequent ros production in these cells. pkc isozymes are positioned upstream and downstream of p22phox - containing nadph oxidases and ros production in mesangial cells exposed to hg or tgf- (kwan., 2005 ; furthermore, in mesangial cells, hg - induced oxidative stress involves autocrine tgf- stimulation of pkc and resultant generation of ros by p22phox - based nox oxidase via p22phox protein upregulation (xia., 2008). other studies with mesangial cells exposed to aopps place an nadph oxidase, distal to pkc activation, resulting in extracellular matrix protein overproduction and upregulation of tgf- (wei., 2009). the role of other nox catalytic isoforms or regulatory subunits in mesangial cell injury in the diabetic kidney has been less studied. a role for nox2 or nox1 is indirectly suggested by the observation that administration of apocynin significantly reduced glomerular fibronectin and collagen accumulation in type 1 diabetic rats (asaba., 2005). however, the involvement of nox2 in the mesangium is not clear as some studies indicate that nox2 is not detected in cultured human and rat mesangial cells whereas nox2 is detected in isolated mouse mesangial cells and in diabetic glomeruli (jones., 1995 ; liu., 2012 ; miyata., 2005). nox2 regulatory subunits p47phox and p67phox are systematically found in mesangial cells (jones., 1995 ; pleskova., 2006) and the expression and translocation of p47phox and p67phox to the membrane is increased in the diabetic glomeruli (kitada., 2003). hg causes an increase in p47phox protein expression in mesangial cells and downregulation of p47phox blunts high glucose - induced ros and extracellular matrix accumulation (hua., 2003 ; kwan., 2005 ; xia., 2006). importantly, diabetes - induced oxidative stress and glomerular injury are attenuated in p47phox - deficient type 1 diabetic akita mice (liu., 2012). moreover, deletion of p47phox attenuates diabetes - induced and hg - induced nox2 expression in glomeruli and mesangial cells, respectively (liu,., 2012). this indicates that p47phox - dependent activation of nox oxidase, likely nox2, is determinant for the promotion of dn. however, a recent study showed that glomerular mesangial matrix expansion and albuminuria are not attenuated in type 1 diabetic nox2 knockout mice (you., 2013). interestingly, an upregulation of nox4 is observed in these mice (you., 2013). together, these findings warrant a reassessment of the role of nox2 in dn. while nox1 is thought to promote the deleterious effects of glucose or ang ii in the vasculature (lassegue, 2001 ; lavrentyev and malik, 2009), its function in mesangial cell injury in the diabetic milieu has not been reported. indeed, a role for nox1 as a source of renal ros and mediator of dn has been challenged by the recent report showing that deletion of nox4, but not nox1, in a mice model of type 1 diabetes in apoe knockout mice resulted in renal protection from glomerular injury (jha., 2014). however, p47phox and p67phox recruitment to the membrane contributes to ang ii - mediated oxidative stress and mesangial cell growth (ding., 2007) and tgf- was reported to upregulate the expression of nox2, p22phox, p47phox and p67phox in mesangial cells (lee., 2003). aopps also increase p22phox, p47phox and nox4 protein expression (wei., 2009). aopps promote an increase in p47phox expression and translocation as well as its interaction with p22phox expression (wei., 2009). these events are associated with enhanced extracellular matrix protein synthesis. although pkc - dependent nadph oxidase activation mediates ages - induced mesangial cell fibrotic injury (thallas - bonke., 2008), the nox subunits implicated in these processes remain to be identified. in vivo studies suggested that induction of glomerular injury by ages is due to src homology 2 domain - containing transforming protein c1 (shc1) isoform p66-mediated nox4 expression (menini., 2007). figure 2 is an overview of the major redox pathways and signaling intermediates positioned upstream and downstream of nox enzymes that are stimulated by diabetic stimuli to promote glomerular mesangial cell injury. upregulation of nox4 protein expression in diabetic glomeruli including podocytes is accompanied by increased oxidative stress, loss of podocyte or foot process effacement as well as albuminuria in ove26 type 1 diabetic mice (eid., 2009 ; 2010 ; 2013a ; khazim., 2013 ; sharma., 2008). in cultured podocytes exposed to hg for prolonged time period, nox4 mrna and protein are augmented and nox4-derived ros play a key role in apoptotic cell death (eid., 2009 ; 2010 ; 2013a ;, 2014 ; khazim., 2013 ; piwkowska., 2011 ; sharma., hg had no effect on nox5 mrna expression but significantly increased nox4 mrna levels (jha., 2014). transgenic mice overexpressing human nox5 in a podocyte - specific manner exhibited early onset of albuminuria and podocyte foot process effacement (holterman., 2014). subjecting the mice to streptozotocin - induced type 1 diabetes further exacerbated these changes (holterman. a sequential regulation of nox oxidases by cytochrome p450 of the 4a family (cyp4a) was recently identified in podocytes in which 20-hydroxyeicosatetraenoic acid (20-hete) generation by cyp4a mediates the stimulatory effect of hg on nox4 and nox1 expression and the resultant ros production at later time points (eid., 2009). in the presence of hg concentrations, nox4 promotes podocyte cell death via activation of p53- and puma - dependent apoptotic pathway (eid., 2010). the oxidative stress triggered by hg appears to be exacerbated by the fact that the ros generated by nox4 affects the balance between oxidants and antioxidants by decreasing the activity of key antioxidant enzymes such as glutathione peroxidase (gpx) and catalase (piwkowska., 2011). importantly, inactivation of ampk - activated protein kinase (ampk) by hg accounts for the increase in nox4 mrna and protein expression as well as subsequent ros production and podocyte apoptosis (eid., 2010). ampk activators, e.g. aicar (5-aminoimidazole-4-carboxamide-1-riboside) or adiponectin, significantly reduced nox4 expression, oxidative stress and podocyte injury in vitro or in vivo (eid., 2010 ; piwkowska., 2010 a recent report indicates that mammalian target of rapamycin complex 1 (mtorc1) is an upstream positive regulator of nox4 expression and subsequent ros production and injury in podocytes exposed to hg or in vivo in glomeruli from ove26 type 1 diabetic mice (eid., 2013a). the same report showed that ampk exerts its protective actions by inhibiting mtorc1 pathway via tuberin activation (eid. although podocytes also express nox2, p22phox, p47phox and p67phox (greiber., 1998 ; nistala., similar to nox4, nox1 protein levels are increased in glomeruli from ove26 mice (eid., 2009 ; 2010). nox1 protein is upregulated by hg in cultured podocytes and, as for nox4, mtorc1 positively regulates nox1 expression (eid., 2013a). while ang ii- or tgf--induced oxidative stress mediates podocyte injury (campbell., 2011 ; nistala., 2008 ; ziyadeh and wolf, 2008), very little is known regarding the role of the nox oxidases in the podocyte dysfunction promoted by these agonists or the other major mediators of dn. similar to what is observed in mcs, ang ii - dependent increase in nadph oxidase activity is associated with the upregulation of nox4, nox2, rac and p22phox expression in podocytes (nistala., 2008 ; whaley - connell., 2008). stimulation with ang ii upregulates nox5 expression in human podocytes and nox5 is required for ang ii - induced ros production as well as altered podocyte cytoskeletal dynamics (holterman., 2014). nox4 was detected within the mitochondria in podocytes and a recent report suggested that tgf--induced mitochondrial nox4 upregulation via the sma and mad homologue (smad) 2/3 pathway is responsible for ros production, mitochondrial dysfunction, and apoptosis in podocytes (block., 2009 ; das., 2014 ; yu., 2014a). note that in human podocytes, nox5 mrna expression is enhanced following treatment with tgf- (jha., 2014). figure 3 is an overview of the major redox pathways and signaling intermediates positioned upstream and downstream of nox enzymes that are stimulated by diabetic stimuli to promote glomerular mesangial cell and podocyte injury. similar to glomeruli, tubules from type 1 diabetic rats show an increase in nox4 mrna and protein expression and downregulation of tubular nox4 levels with in vivo administration of antisense oligonucleotides reduces diabetes - mediated ros production and extracellular matrix protein synthesis in the renal cortex that is mainly composed of tubular epithelial cells (etoh., 2003 ; fujii., 2010 ; gorin., the importance of nox4 as a major source of ros in the tubular compartment was further supported by a recent study in type 1 diabetic apoe / nox4 double knockout mice or nox4 knockout mice on c57bl6/j background showing that nox4 genetic deletion markedly reduced ros production in renal cortex (jha., 2014 ; noteworthy, nox4 was also shown to be responsible for diabetes - induced ros production in mitochondria isolated from cortex (jha., importantly, the function of nox1 as a generator of ros in tubules is weakened by the observations that ros production is not affected in the renal cortex of type 1 diabetic apoe / nox1 knockout mice (jha., 2014). interestingly, nox4 protein expression is increased in renal cortex but is unchanged in medulla from type 2 diabetic mice (sedeek., 2010). increased nox4 expression in diabetic tubules correlates with an augmentation in p22phox levels (etoh. whilst the levels of nox2 and p47phox are not affected in renal cortex from type 2 diabetic mice (sedeek., 2010), nox2 is increased in the cortex from type 1 diabetic rats (gorin., 2005). a role for nox2 is challenged by a recent report showing that tubulointerstitial injury is not ameliorated in type 1 diabetic nox2 knockout mice (you., 2013). exposure of cultured renal proximal tubular epithelial cells to hg leads to the upregulation of nox4 protein expression but seems to have no effect on nox2, nox1, p22phox or p47phox expression (ford. furthermore, nox4-dependent ros production is required for glucose - induced increase in fibronectin accumulation and tgf- expression in these cells (ford., 2013 ; sedeek., the profibrotic action of the oxidase is corroborated by the finding that overexpression of nox4 in tubular cells causes a robust increase in fibronectin synthesis (new.,, nox5 has been shown to be express together with nox1, nox4 and nox2 but its role in hg - induced ros production and cell injury was not studied (yu., 2014b). a potential upstream regulator of nox4 expression in the tubules is protein kinase c- since diabetes - induced nox4 expression and oxidative stress are attenuated in renal cortex from pkc--null mice kidney (ohshiro., 2006). interestingly, pkc seems to be positioned both upstream and downstream of nox4 since it was recently reported that the increase in the major isoforms of pkc in tubules was blunted in the renal cortex from diabetic nox4 knockout mice (thallas - bonke., 2014). a role for matrix metalloprotease a disintegrin and metalloprotease1 7 (adam17) as upstream regulator of nox4 was recently established in vivo and in vitro. pharmacological inhibition of adam17 attenuated diabetes - mediated nox4 upregulation, oxidative stress and matrix protein accumulation in ove26 mice kidney cortex (ford., 2013). in cultured tubular epithelial cells, adam17 activity is required for hg - induced increase in nox4 protein expression, ros generation and fibrotic injury (ford., 2013). similar to podocytes, ampk activation attenuates hg - mediated nox4 protein upregulation in tubular epithelial cells, supporting the concept that the kinase acts as a suppressor of oxidative stress (lee. p38mapk has been also identified as a critical downstream effector of nox4 in the signaling pathway linking hg and tubular cell injury (sedeek., 2010). ang ii has been shown to utilize nox4 as a mediator of its injurious effects in tubular epithelial cells. chronic angiotensin ii treatment upregulates nox4 expression and induces epithelial - to - mesenchymal transition in renal epithelial cells through nox4-dependent ros production and resultant src / caveolin - mediated activation of epidermal growth factor receptor / erk signaling pathway (chen., 2012 ; peng., 2009 ; xu., 2010 furthermore, ang ii upregulates nox4 expression in the mitochondrial fraction of renal tubular cells and nox4 is required for ang ii - mediated ros mitochondrial production (kim., ros derived from nox4 contribute to ang ii - dependent apoptosis in these cells (kim., 2012). ang ii enhances the expression of nox2, p22phox and p47phox subunits along with nox4 in tubular cells (hannken., 1998 ; xu., 2010). the effects on p22phox, p47phox and nox4 appear to be mediated by lectin - like oxidized low - density lipoprotein receptor-1 (hannken. another report shows that hg induces p22phox expression and oxidative stress via angiotensin ii type 1 (at1) receptor activation, further underlining the important role of ang ii as a regulator of nox signaling in tubular cells (takao., 2011). furthermore, a p22phox - containing nadph oxidase is critical for ang ii - mediated tubular cell hypertrophy through induction of p27kip1, a factor promoting cell cycle arrest (hannken., 1998). as in mesangial cells, src and erk pathways appears to be a primary target of nox4-derived ros and plays a key role in the resultant fibrotic or hypertrophic response in tubular cells. ampk also inhibits ang ii - induced increase in nox4 expression and tubular epithelial - to - mesenchymal transition (emt) (lee., 2013b). although it is clear that oxidative stress is implicated in tgf--mediated tubular cell injury (rhyu., 2005), there is a deficit of causal evidence supporting the role of the oxidase or other nox enzymes in tgf- signaling in tubular cells. stimulation of tubular cells by tgf- results in an increase in nox4 protein expression, an effect prevented by ampk activation (lee., 2013b). conflicting results exist concerning the regulation of other nox subunits by tgf- in these cells. on one hand, tgf- has no effect on nox2, p22phox and p47phox expression and that only p67phox seems to be required for the deleterious actions of the cytokine in renal tubular cells (zhang., 2009). on the other hand, tgf- was shown to upregulate p22phox (lee. nox4-derived ros also serve as signal transducer for the fibrotic effects of igf - i in renal tubular epithelial cells via akt / pkb and mtorc1/p70 signaling pathways activation (new., figure 4 is an overview of the major redox pathways and signaling intermediates positioned upstream and downstream of nox enzymes that are stimulated by diabetic stimuli to promote tubulointerstitial cell injury. the information regarding the roles of hyperglycemia - mediated oxidative stress or nox - derived ros in interstitial cell injury, particularly in the activation of renal fibroblast into myofibroblasts, remains very limited. there is indication that exposure of kidney fibroblasts to hg upregulates nox4 and nox2 mrna and protein expression and that this is associated with an increase in ros generation and hypertrophy (williams and gooch, 2014). calcineurin a- (cna) maintains basal nox4 and nox2 expression and is required for hg - induced increase in nox4 and nox2 expression in these cells (williams and gooch, 2014). it seems that cna promotes nox4 and nox2 transcription via activation of the nuclear factor of activated t cells (nfat) (williams and gooch, 2014). a functional link between nox4 or nox2 and ros generation and hypertrophy is suggested by the fact that expression of nox4 or nox2 in cna deficient kidney fibroblasts (where nox4 and nox2 are downregulated) is able to restore the stimulatory effects of hg on these biological outcomes (williams and gooch, 2014). nox4 is the predominant nox homolog implicated in kidney myofibroblast differentiation and expression of fibronectin and other fibrotic markers in response to tgf- (barnes and gorin, 2011 ; bondi., 2010). however, unlike cardiac and lung myofibroblasts where nox4 acts upstream of transcription factor smad2/3, nox4 is positioned downstream of smad3 and proximal to erk in the pathway linking tgf- to the fibrotic response in renal fibroblasts (barnes and gorin, 2011 ; cucoranu. these results are in agreement with what is reported in pulmonary vascular smooth muscle cells, where tgf--induced proliferation occurs through a nox4-dependent pathway downstream of smad3 (sturrock., 2006). small gtpase rhoa and its downstream target rho kinase (rock) have been identified as upstream positive regulators of nox4 expression and activity in the pathway linking tgf- to kidney myofibroblast differentiation (manickam., 2014). interestingly, rhoa / rock seems to act via the increasing the expression of poldip2, a newly discovered nox4 enhancer protein (manickam., 2014). moreover, the data suggest that besides enhancing nox4 activity poldip2 is also able to regulate the expression of the oxidase. it is apparent from this review that the in vivo and in vitro experimental evidence support a fundamental role for nadph oxidases of the nox family, and especially the homologue nox4, in the pathogenesis and pathophysiology of dn. the consequence of these observations is the consideration of the nox homologs and their associated subunits as relevant therapeutic targets for the treatment of dn. there has been recently a considerable effort for the generation and development of agents able to inhibit the nox enzymes in a homolog - specific manner (altenhofer., 2012 ;., 2011 ; laleu., 2010 ; lambeth., 2008). among these inhibitors, the small - molecule dual nox4 and nox1 inhibitors from the pyrazolo pyridine chemical series, referred as to gkt136901 and gkt-137831, have drawn considerable attention. preclinical studies performed with these inhibitors in experimental animal models indicate that they effectively attenuate the pathological changes observed in renal complication of type 1 and type 2 diabetes, atherosclerosis, ischemic retinopathy, liver fibrosis and idiopathic pulmonary fibrosis (aoyama., 2012 ; carnesecchi., 2011 ; di marco., 2014 ; gorin and block, 2013a ; 2013b ; gray., 2013 ; hecker., 2014 ; jha., 2014 ; jiang., 2012 ; 2014 ;, 2010 ; sedeek., 2013 ; vendrov., 2010 ; gkt137831 have recently been successfully used in a phase 1 clinical trial and is currently being evaluated in a phase 2 clinical trial in patients with type 2 diabetes and albuminuria (clinical-trials.gov reference number nct02010242). beside the direct inhibition of nox4 and others nox oxidases, the present review suggests that adjunct therapies targeting the agonists or signaling intermediates that regulate the expression or function of nox subunits and subsequent ros production as well as the downstream targets of nox oxidases implicated in the pathological processes should be considered for the treatment of diabetic complications. in regards to the observation reported above, such strategies could involve the use of pkc inhibitors, agents that disrupt the age signaling, inhibitors of the renin - angiotensin system (angiotensin - converting enzyme inhibitors and ang ii receptor blockers), statins (that inhibit rac1 and rho), rho / rho kinase inhibitors, ampk activators (i.e. metformin), sestrin - mimicking small molecules or peptides sestrin analogues / agents that stimulate sestrin activity or expression. importantly, the small molecules nox inhibitors that are currently available affect nox activity without altering the expression of the oxidases. therefore, the utilization of agents that modulate nox expression or that target downstream effectors of the oxidases as adjunct therapy to nox allosteric inhibitors may allow a more effective neutralization of the enzyme due to the blockade of the sustained increase in nox protein levels promoted by chronic hyperglycemia or other mediators of dn. | oxidative stress has been linked to the pathogenesis of diabetic nephropathy, the complication of diabetes in the kidney. nadph oxidases of the nox family, and in particular the homologue nox4, are a major source of reactive oxygen species in the diabetic kidney and are critical mediators of redox signaling in glomerular and tubulointerstitial cells exposed to the diabetic milieu. here, we present an overview of the current knowledge related to the understanding of the role of nox enzymes in the processes that control mesangial cell, podocyte and tubulointerstitial cell injury induced by hyperglycemia and other predominant factors enhanced in the diabetic milieu, including the renin - angiotensin system and transforming growth factor-. the nature of the upstream modulators of nox enzymes as well as the downstream targets of the nox nadph oxidases implicated in the propagation of the redox processes that alter renal biology in diabetes will be highlighted. |
in birds and some lizards, females are heterogametic with a zw karyotype, while males are zz homogametes. the molecular basis for sexual differentiation in birds is unknown : arguments exist for doses of z masculinizing chicks and for w information feminizing. asw was identified as a tandemly repeated gene conserved on avian w chromosomes that is expressed in early female development and appears to be an inactive form of avian z - encoded hint. hint is a dimeric enzyme that hydrolyzes amp linked to lysine, whose enzyme activity is required for regulation of the cdk7 homologous kin28 kinase in yeast. of 16 residues most conserved across all life forms for amp interactions, 15 are sexually dimorphic in birds, that is, altered in the female - specific asw protein. genomic and expression data suggest that asw may feminize chicks, dominantly interfering with hint function by heterodimerization. we consider whether positive cooperativity could explain how hint heterodimerization with an inert enzyme might reduce specific activity by more than 50% and provide data sufficient to reject this model. instead, we hypothesize that asw carries a signal for mislocalization and/or proteolysis, and/or dominantly suppresses the remaining hint active site to function as a dominant negative. molecular modeling suggests that asw and hint can heterodimerize and that gln 127, an asw - specific alteration for trp123, dominantly interferes with the hint active site. an extra dose of hint in zzw chicks, and thus more hint homodimer, may partially overcome the feminizing influence of asw and lead to the observed intersexual characteristics of zzw triploids. dimorphic sexes are the norm in animals, although there are rotifer species that consist solely of parthenogenetic females, corals that produce long - lived clones that reproduce by fragmentation, and some lizards and fish that spin - off parthenogenetic lines that survive for multiple generations. among the vast number of animal species that reproduce with males and females, sexual differentiation flies and worms are among the invertebrates that determine sex by doses of x - chromosomal information. within the vertebrates, there are several mechanisms that account for sexually dimorphic development. because temperature controls sexual development in crocodiles, many turtles and some lizards, environmental sex determination (esd) has been proposed to be the primordial vertebrate mechanism for sexual differentiation. some reptiles and most birds and mammals have chromosomal sex determination (csd) systems and these systems involve remarkably different chromosomes and genes. csd strategies, then, may have evolved to program genetically the developmental routines that occur in response to temperature in the vertebrates that use esd. the natural history of vertebrates suggests that there were multiple solutions to the problem of csd. nearly all mammals have the xx female and xy male system, with xxy individuals being male and xo individuals female. these observations led to a search for the testis - determining factor on the y chromosome culminating in isolation of the sry gene, which is sex - determining in humans and mice. beyond sry, there are several autosomal genes including the sry - related sox9 gene, mis, sf1, wt1 and dmrt1 that contribute to sexual organ formation, alterations of which can cause male - to - female sex reversal in xy males. because there are rodent species with no sry gene and no y chromosome, any of these genes or the x - linked dax1 gene might be considered candidates for a sex - determining gene in the xx rodents such as mole voles whose body plan is not so different from that of mice. in birds, males are the homogametic sex, with two z chromosomes, and females the heterogametic sex, with one z and one w chromosome. z and w are not related to mammalian x or y chromosomes and, furthermore, it is not known if the w chromosome confers femininity and/or if doses of the z chromosome confer masculinity. in mammals and invertebrates, diploid animals of genotype xo and xxy were crucial in establishing that doses of x confer femininity in invertebrates and that presence of y confers masculinity in mammals. diploid birds with one or three sex chromosomes were last reported 70 years ago, but triploid birds with zzz and zzw karyotypes have been observed. while zzz triploids seemed like normal males that produced abnormal sperm, zzw animals appeared female on hatching and then developed some male gonadal and behavioral characteristics as they matured. these studies suggest that the dosage versus determining factor argument is a false dichotomy in birds : doses of z chromosome appear to masculinize while presence of the w appears to feminize. according to theory, a pair of autosomes can evolve into sex chromosomes by mutation of a control gene. if a control gene confers sexual development by dosage, it might become lost from the alternative sex chromosome. dmrt1 is a candidate dosage - dependent gene for masculinity in birds and alligators on the basis of z - linkage, conservation, and gene - expression patterns. alternatively, if a control gene confers sexual development as a dominant determining factor, it may have evolved as an allele of a gene on the opposite sex chromosome. this latter mechanism is thought to relate sry to the x - linked sox3 gene in mammals. histidine triad (hit) enzymes are a superfamily of nucleotide hydrolases and transferases that contain a catalytic motif related to the sequence hishishis (where represents a hydrophobic amino acid) and act on substrates containing a nucleoside monophosphate. branch 1 of the hit superfamily includes the ubiquitous hint enzymes plus two enzymes with a more phylogenetically resstricted distribution, namely aprataxin, which is lost in humans with ataxia - oculomotor apraxia and the scavenger mrna decapping enzyme dcps. while dcps enzymes are specific for hydrolysis of cap structures such as 7megpppg, prototypical hint enzymes such as rabbit hint and yeast hnt1 hydrolyze adenosine 5'-monophosphoramide substrates such as amp - lysine to amp plus lysine. loss of this enzymatic activity renders yeast cells temperature - sensitive for growth on galactose medium and hypersensitive to mild mutations in the yeast homolog of mammalian cdk7, that is, kin28, the kinase component of general transcription factor tfiih. loss of hnt1 enzymatic activity also renders cells hypersensitive to mutations in the cyclin h homolog ccl1, the mat1 homolog tfb3, and to cak1, the activating kinase for kin28, all of which lead to destabilized kin28 complexes and a likely increase in concentration of kin28 monomers. consequently, it was suggested that a kin28 monomer is the likely target of hint regulation, potentially because it is post - translationally adenylylated and is a protein substrate of the lysine - deadenylylating activity of hint. indeed, two - dimensional electrophoretic analysis of kin28 is consistent with kin28 being subject to a post - translational modification in addition to phosphorylation that appears to be controlled by hnt1 genotype (a. krakowiak and c.b., unpublished results). finally, it is important to note that hint is a dimer with two identical purine nucleoside - monophosphate - binding sites per dimer. the amino - acid residues that have remained most constant throughout evolution are those that form the dimer interface and make direct contact with amp. chickens and many other orders of birds are dimorphic for hint - related genes on their sex chromosomes. the chicken z chromosome contains the locus for a typical hint gene, predicted to encode a polypeptide 83% identical to rabbit hint. however, on the gene - poor, female - specific w chromosome, the asw (avian sex - specific w - linked) gene was identified, encoding a predicted asw protein with striking similarity to hint with the specific exception of residues involved in amp recognition. as shown in figure 1, the degree to which the nucleotide - binding site was altered is remarkable. although human biochemists can depress activity more than 10-fold in fhit and hint with a single active - site his mutation, hens apparently continued to peck at the sequence of hint, altering all four of the absolutely conserved his residues. in all, 15 of 16 normally conserved residues identified in immediate proximity to the adenine base, the ribose and the 5 ' phosphate are altered in asw. asw is absent in the ratites, emu and ostrich, which have indistinguishable sex chromosomes, but is tandemly repeated approximately 40 times on the w chromosome of all the non - ratite birds examined. confirming the subtractive manner in which asw was cloned, both groups found that asw mrna is highly expressed in the female urogenital ridge at the stages preceding and during sexual differentiation. additionally, hint mrna is expressed at levels about two - fold greater in males than in females, in developing chicks more than in adult chickens, and at a message level one - seventh to one - tenth the level of asw in stage-29 females. asw was cloned a third time as a message that is increased in cells lacking the splicing factor asf / sf2. surprisingly, asf / sf2 did not affect splicing of the asw message but rather destabilized the mrna. involvement of asf / sf2 in the regulation of asw mrna is interesting because sex - specific splicing of doublesex mrna in drosophila, a sex - determining gene for which the mammalian dmrt genes were named, is mediated in part by asf / sf2. although the initial cloning of asw pre - dated knowledge that hint is an enzyme, both groups suggested that asw might function by a dominant - negative mechanism through heterodimerization with hint, thereby inducing female development. heterodimerization was also discussed in the review literature, also without awareness that hint function depends on enzymatic activity. genomic and expression data suggest that the purpose of asw is titration of hint function. how might this work ? in the simplest case, a normally dimeric enzyme with two active sites that is produced as a heterodimer with one good and one bad active site would be expected to have 50% of the specific activity of the homodimer. this is not a scenario for dominant negativity and, in fact, such a scenario does not explain why asw is produced at all. if asw were simply an inert dimerization partner for hint, a 50% reduction in hint cellular specific activity could be obtained if there were no asw genes on the w chromosome. males would have two doses of hint and females one dose, such that twice as much hint dimer could be made in males as in females. furthermore, if asw were simply a loss - of - function allele, given the paucity of genes on the w chromosome, sex - chromosome theory suggests that such a gene would be lost. the repeated and highly expressed nature of the gene suggests that it has evolved to be dominantly interfering - the challenge is to determine the mechanism of dominance over the z - encoded hint. one might expect a hint - asw heterodimer to have substantially less than 50% of the activity of a hint homodimer if hint homodimers showed cooperativity with respect to substrate binding and/or hydrolysis. for example, if the first nucleotide substrate were to bind weakly but the presence of the first bound substrate promoted efficient binding of a second substrate, then a heterodimer containing one functional and one nonfunctional active site would retain only one low - affinity binding site and be severely defective. this mechanism can be largely excluded, however, because sigmoidal substrate saturation kinetics were not observed with amp - nh2. the data for both rabbit and yeast hint enzymes indicate that the dimer 's two crystallographically defined nucleotide - binding sites bind nucleotide independently and fit well to a single submicromolar km for each enzyme (figure 2). as cooperativity is difficult to invoke in this system, we consider that for asw to titrate hint enzyme activity by heterodimerization, asw must carry a signal for mislocalization and/or proteolysis, and/or somehow alter the hint active site. we therefore constructed a molecular model of the proposed hint - asw heterodimer by superimposing the chicken hint sequence on the determined x - ray structure of rabbit hint, and threading and minimizing the asw sequence on the opposing monomer. this analysis suggested : first, that hint and asw do retain sufficient sequence identity at the dimer interface to form a heterodimer ; second, that asw has an insertion sequence at the bottom of the dimer that could be a site of alternative localization or proteolysis ; and third, that gln127, a residue that asw has substituted for trp123 of hint, may interfere with the function of his114 in hint across the dimer interface. although altered specificity of a putative hint - asw heterodimer is conceivable, the simplest enzymatic mechanism for dominant interference is that gln127 from asw depresses activity from the hint active site. the dimer interface of hint is formed by antiparallel interactions between helix 2 and its symmetry mate and strand 4 and its symmetry mate. these sequences are contiguous in the primary sequence of hint and form the region of greatest identity with asw (figure 1). apart from the curious substitution of 15 of 16 nucleotide - proximal residues in asw, the most dissimilar region of asw consists of a five amino - acid insertion between strands 1 and 2 and substitution of gly - ala - pro (asw) for asp - glu - ser (hint) at the amino - terminal end of helix 2 the bulky amino acids in the asw insertion (pro - leu - trp - thr - arg), which in hint is an extremely tight turn, may be a handle for altered localization or proteolysis. cellular localization of green fluorescent protein (gfp) fusions to hint and asw was investigated in male chick embryo fibroblasts. gfp - hint was found to be distributed in the cytoplasm and the nuclei, though somewhat concentrated in the nuclei with respect to the gfp control. though localization of hint in asw - overexpressing cells was not examined, if hint has extranuclear (that is, cdk7-exclusive) functions, then excluding hint from the cytoplasm may be an important function of asw. the crystal structure of nucleotide - bound forms of rabbit hint showed that the carboxy - terminal amino acids of each hint monomer extend across the dimer interface and are buried near the opposing nucleotide. the trp residue in the extreme carboxy - terminal trp - pro - pro - gly motif is buried near his114, the final his of the hit motif, of the opposing monomer. his114 corresponds to his98 of fhit, which was localized in low barrier hydrogen - bonding distance to the - bridging oxygen of a bound apppa analog in co - crystal structure with fhit his96asn. on the basis of mutagenesis of the conserved his residues in fhit and the location of these his residues in crystal structures of hint and fhit, a catalytic role was proposed for his114. the structures show that his51 is in the same plane as his114, with the n of his 51 positioned to accept a hydrogen bond from the n of his114. this geometry was considered critical for the enzymatic activity of hint and fhit. as shown in figure 1, asw sequences contain a substitution of gln127 for trp123 of hint. as shown in figure 3, gln127 of asw is predicted to alter the conformation of hint his114 such that his114 is not positioned for productive hydrogen bonding, dominantly depressing hydrolase activity from the hint active site. in short, loss of the amp - binding site is predicted to account for negativity of asw. gln127 and possibly insertion sequences that may have a role in stability or localization are predicted to make asw dominant. identification of sex - linked asw and hint genes in birds has raised new experimental questions. it will be interesting to learn whether asw and hint heteromultimerize and what the in vitro and in vivo stabilities of heterooligomers are with respect to the homodimers. it will be interesting to determine whether asw homodimers display any binding to hint substrates, what degree of hint enzyme activity is retained by the putative hint - asw heterodimer, and whether gln127 is required for depression of hint enzymatic activity in hint - asw heterodimers. turning to genetic analysis, if asw has a significant role in feminization of birds, then viruses that increase expression of asw may promote female development in zz eggs, potentially in a manner that requires gln127. if hint is part of the z chromosome that works by gene dosage, then viruses that direct expression of hint may promote male or intersexual development in zw eggs as was seen with zzw triploids. finally, if it is true that hint enzyme activity is important in making zz chicks male or if hint inhibition is important in making zw chicks female, then it will be interesting to learn whether hint is involved in establishment or maintenance of sex in other animals. we thank charles j. cole of the american museum of natural history for helpful discussions. the primary sequences of rabbit and chicken hint are aligned with chicken and quail asw. sixteen residues involved in amp interactions, including 14 identified from hint - nucleotide co - crystal structures plus his110 and trp123, are boxed. note that 15 of 16 nucleotide - interacting (boxed) residues are chromosomally dimorphic (pink / blue) and that the dimer interface, which is located primarily in helix 2 and strand 4, is highly conserved. substrate concentration - dependent hydrolytic rates (per monomer) for rabbit hint (filled circles) and yeast hnt1 (open triangles) indicate that hint hydrolases, although dimeric, have a single km for substrate. reproduced with permission from. (a) hint dimer with conformations of his114 and trp123 as determined crystallographically and a model of adenosine 5'-monophosphoramide substrates in ball - and - stick representation. (b) proposed structure of an asw (pink)-hint (blue) heterodimer with predicted conformation of asw gln127 extending into the vicinity of hint his114 and the bound hint substrate. (c) stereo view of a close - up superposition of the hint homodimer (green) depicted in (a) with the asw - hint heterodimer (pink and blue) depicted in (b). note that asw gln127 (in pink) is proposed to alter the conformation of hint his114 (in blue) such that catalysis from the hint active site is depressed. | in birds females are heterogametic with a zw karyotype, while males are zz homogametes but the molecular basis for sexual differentiation in birds is unknown. genomic and expression data suggest that asw may feminize chicks, dominantly interfering with hint function by heterodimerization. |
it starts with bleeding gums, but can finally lead to tooth loss in case of negligence. periodontal disease ranges from mild gingivitis to severe disease that results in the destruction of the supporting tissue and alveolar bone loss. previous epidemiological studies in developing countries have shown that the periodontal status of the population that determined by the presence of gingival inflammation, dental plaque and dental calculus was worse compared to countries with higher developmental levels.other epidemiological studies [35 ] suggested that subjects in developing countries did not necessarily have a higher prevalence and severity of period on titis than subjects in industrialized nations even though they may have more dental plaque, calculus and gingival inflammation. previous studies have reported general conclusions regarding the assessment of periodontal status, as they were based on pre - selected population samples. miyazaki reviewed the community periodontal index of treatment needs (cpitn) data according to the previous classification of this index from independent surveys conducted in 79 countries and reported little difference in the prevalence of periodontal pockets between developing and industrialized countries. very few epidemiological studies that have been carried out in african countries showed low extent and severity of periodontal pockets or attachment loss, while more studies and reports are available from eastern european and scandinavian countries. similar investigations have not been carried out on a wide spectrum of population samples in greece during the last decades. comparisons with the 1985 findings indicate that severe periodontal diseases may be declining in greece, whereas gingivitis may be increasing. in order to test the characterization of prevalence and severity of periodontal disease, significance of differences between full - mouth examination and partial recording protocols for this reason, the mentioned protocols for assessment of periodontal status include several periodontal indices, such as russell s index, periodontal disease index, gingival periodontal index, gingival bone count index, extent and severity index and community periodontal index. therefore, the purpose of the present study was to examine the periodontal condition of a sample of adults in three isolated regions in a city in greece and estimate the association of demographic, behavioural and clinical factors with periodontal disease. in the present study, 640 individuals, 300 males and 340 females, 2069 years of age were selected. all the selected subjects were inhabitants of three isolated villages in n. w achaia, one of the biggest municipalities in greece, who visited a private practitioner for their regular dental follow - up which was organized by the greek dental association for all the greek population annually. the whole population of the mentioned villages was estimated as 1,023 inhabitants according to the local authorities. the majority of the inhabitants worked in the capital of achaia, patra and attended the local university. inco - operation between the greek dental association and the local authorities, they emphasized the necessity and importance for an annual dental follow - up. an additional motivation for participation was the distribution of printed instructions on proper oral hygiene aspects to the individuals examined. the sample was divided into five groups based on age ; namely, group i (120 individuals), 20 to 29 years old ; group ii (130 individuals), 30 to 39 years old ; group iii (140 individuals), 40 to 49 years old ; group iv (130 individuals), 50 to 59 years old ; group v (120 individuals), 60 to 69 years old. the participants of the study underwent an oral clinical and physical examination and filled a similar self - administered questionnaire that included several epidemiological variables. the oral health study presented here took place between may and october 2011 at the mentioned private practice. the present study was not an experimental one. in greece, only experimental studies must be reviewed and approved by authorized committees (such as the greek dental association, ministry of health). all participants were informed about the evaluation to which they would be submitted and gave their informed consent for participation. if an index tooth was absent, the closest distal tooth was used as a substitute. in the absence of a tooth distal to the index tooth, molar or premolar index teeth were never replaced by the examination of an anterior tooth. in individuals with extensive destruction of the cemen to - enamel junction due to dental caries, erosion, abrasion, presence of prosthetic restoration or dental calculus, the location of the junction was estimated according to the location of adjacent teeth. individuals who had undergone a previous periodontal treatment, conservative or surgical, within the previous six months were excluded from the study sample. edentulous adults (n= 0) were excluded from the study due to the impossibility of evaluating the periodontal conditions in this population. two independent physical examinations were performed, one blinded and one non - blinded to the medical history. just before the physical examination, participants filled in a similar self - administered questionnaire that included several epidemiological variables such as age, gender, educational level (primary, secondary, college, university), smoking status (current regular smoker, occasional smoker and non - smoker), dental visits (frequency and reasons for the last dental visit), oral hygiene habits (tooth brush frequency, use of dental floss / mouthwash) and data regarding their variables of their general health. socioeconomic status of the sample was excluded from the study because of the current economic crisis in greece, as it could be a negative motivation for the individuals in order to participate. on the other hand, the study sample consisted of individuals who were permanent inhabitants in isolated areas and no significant differences regarding their socieconomic status would be expected. one well - trained and calibrated dentist who was also registered as an active member. in the hellenic society of periodontology (hsp) and european federation of periodontology (efp) performed the examinations of the participants and the clinical measurements. the clinical measurements concerned the following teeth (according to ramfjord index) : 16, 21, 24, 36, 41 and 44. the reason that ramfjord index was used is that it represents a simple, easy, reproducible and representative as possible periodontal index, especially in isolated societies with a low - level of oral hygiene and it is one of the most appropriate indices for partial - mouth recording protocols. the variables that were measured clinically were the following : assessment of dental plaque and dental calculus (0= no calculus, 1= supra - gingival calculus, 2= subgingival calculus). presence or absence of supra - gingival plaque was recorded after disclosing soft deposits using erythromycin solution (3%) for a period of 30 seconds ; the teeth and gingival were dried with compressed air while dental unit light was used as the light source for the inspections. dental plaque and calculus indices were recorded from the mesio - buccal site of each index tooth. the plaque index (pli), which was used was a modification of the one determined by le.scores 2 and 3 were difficult to be distinguished from each other and therefore were combined into a single category (i.e., 0= no dental plaque, 1= plaque detected after running the probe across the cervical area of the index tooth, 2= plaque that could be detected visually).probing depth (pd) assessmentclinical attachment level (cal)location of the free gingival margin (fgm) relative to the cemento - enamel junction(cej), fgm - cejbleeding on probing (bop) assessment of dental plaque and dental calculus (0= no calculus, 1= supra - gingival calculus, 2= subgingival calculus). presence or absence of supra - gingival plaque was recorded after disclosing soft deposits using erythromycin solution (3%) for a period of 30 seconds ; the teeth and gingival were dried with compressed air while dental unit light was used as the light source for the inspections. dental plaque and calculus indices were recorded from the mesio - buccal site of each index tooth. the plaque index (pli), which was used was a modification of the one determined by le. scores 2 and 3 were difficult to be distinguished from each other and therefore were combined into a single category (i.e., 0= no dental plaque, 1= plaque detected after running the probe across the cervical area of the index tooth, 2= plaque that could be detected visually). probing depth (pd) assessment clinical attachment level (cal) location of the free gingival margin (fgm) relative to the cemento - enamel junction(cej), fgm - cej bleeding on probing (bop) the above mentioned indices (pd), (cal), (fgm - cej) and (bop) were recorded at six sites on each index tooth. a william s probe (pcp10-se, hu - friedy mfg., chicago, il, usa) color coded at 1, 2, 3, 5, 7, 8, 9, 10 mm was used for periodontal examinations and pd and cal were measured to the nearest millimeter. mean cal (averaged across sites in each individual)was used to describe the cumulative amount of periodontal destruction. prevalence of periodontal destruction was determined as the percent of individuals who showed a 4.06.0 mm or higher than 7.0 mm cal at their most severely affected sites. the extent of mild, moderate and severe periodontal destruction was estimated by the percent of sites per individual with a cal of 2.04.0 mm, 4.06.0 mm and higher than 7.0 mm. the reason why the mentioned classification was not based on the american academy of periodontology, is that, as mentioned in an isolated population, the oral hygiene level is poor enough and the presence of deep pockets and severity of clinical attachment loss could be expected extensively. a randomly chosen sample of 128(20%) individuals was re - examined clinically by the same dentist in order to establish the intra - examiner variance. after consideration of the code numbers of the double examined participants no differences were recorded between the 1 and 2 clinical assessments. it is obvious that in this case the in traexaminer consistency of clinical recordings per sextant was not estimated by k index. the individual was the statistical unit. for each participant, the mean values of the clinical parameters ; namely, the dental plaque, dental calculus, pd, cal and fgm - cej were calculated. multiple linear regression analysis was used in order to assess the association of the mean cal with demographic, behavioural and clinical parameters. associations were assessed by adding each independent variable to the fully adjusted model and testing whether the explained variable was increased significantly. tooth brushing frequency was classified into three levels (daily, occasionally, never). frequency of dental visits was dichotomized (yearly or more frequently vs. less frequently or never). gender and smoking were classified as dichotomous variables and age, mean plaque and mean calculus scores (averaged across sites in each individual) were used as continuous variables. the final multivariate model contained variables that were significant after adjusting for all other variables in the model. the variables included in the final model for mean cal were then included in regressions using mean pd and mean fgm - cej as dependent variables. the purpose for using the above mentioned procedure was to assess whether the association of the independent variables with the mean cal was related to variations in mean pd and/or to variations in the location of the fgm relative to the cej. the data analysis was performed using the statistical package of spss ver.17.0 (spss inc., chicago, il, usa). a p value less than 5% (p 5 mm). the extent of a higher than 4 mm cal that was assessed was 11.8% in a vietnamese population and 20% in a younger adult population showing a higher level than that of the present study. according to the above observations, the mean cal and the percentage of sites / individual with cal 2 mm in the present study are comparable to those mentioned studies that had regular access to dental care and preventive dentistry, while are less severe that those reported for populations in the european and other countries. those differences could be attributed to several factors such as the heterogeneous population samples, the different interests that have been showed by the population samples regarding the value of oral hygiene and the need for a regular dental follow - up, the origin of the sample collected (such as the dental hospital or private practice), the fact that most of the reviewed studies assessed the periodontal conditions using full - mouth clinical examinations with four or six sites per tooth, while others used two facialsites per tooth in half of the dentition. in addition, the sample of the present study concerned individuals who sought dental treatment in a private dental practice and we could not consider it as random one. if it is supposed that a site with cal 46 mm presents moderate periodontitis and that rates greater than 7 mm are consistent with a diagnosis of advanced periodontitis, according to table 2 which shows the prevalence of individuals with at least one affected site, at least 68.2% of the 60 to 69-year - old individuals showed sites with moderate periodontitis and at least 27.6% of the subjects of the same age group showed advanced periodontitis. however, the final model showed that age, gender and dental plaque were the most important factors associated with gingival recession ; whereas, age, gender and calculus were the factors that were associated with cal. in a study by corraini., multivariate analysis identified dental plaque, calculus and age as risk indicators for cal 5 mm.. found that an older age and a high percentage of sites with calculus were significant positive predictors of a high percentage of sites with cal 4 mm while the same parameters were statistically significant predictors of a high percentage of sites with cal 7 mm. similar results regarding age and cal were observed in a study by holtfreter. and also a study by wang.. however, bouchard. found that age and gender were powerful independent predictors of cal. in the present study, the prevalence of pocket depth increased with age up to the 40 to 49-years - old age group for a pd of 46 mm and then decreased, while for severe pockets (7 mm) the prevalence increased with age up to the 5059-years - old age group. previous studies have recorded higher prevalence rates of probing depths such as 75% (5 mm), 69.7%(4 mm), 25.3%(6 mm) and 11.3% (> 5.5 mm), while other studies have recorded lower prevalence rates of probing depths such as 30% (4 mm), less than 5 % (6 mm) and 43.3% (4 mm). similar findings with higher prevalence rates have been recorded and assessed ; 84%(4 mm) among 3039-year - old individuals and 93% (pd 4 mm) among 50 - 59-year - old individuals while in another study 76.9% (4 mm) was detected among 35 - 44-year - old individuals and 87.7% (4 mm) among 65 to 74-year - old individuals. the extent of the pocket depths in the present study was lower than the pocket depths in a study of a vietnamese population and another study including young adults, while it was higher than the pocket depths in a study in the amazon rain forest. as mentioned, gingival inflammation was determined by the percentage of sites per individual that showed bop. according to the results of the present study, gingivitis increased significantly with age except in the 60 to 69-year - old age group. however, the above - mentioned finding was not in agreement with a previous study performed by ronderos.. bop (57.8%) was lower than a study in the usa, which was estimated as 82.8%, while in another study in brazil, it occurred in 97.9% of the samples. similar observations were recorded in a study by silva - boghossian. in brazil, whereas in a study in greece, bop was 16.2%. according to the mentioned observations, the present study suggests that even without prior therapeutic or preventive period on talintervention, subjects with extensive dental plaques, gingivitis and calculus do not present severe loss of period on taltissue. this finding is in agreement with a previous study which showed that destructive periodontal disease is not necessarily an inevitable consequence of long - standing gingival inflammation. in addition, the present study does not support that periodontal disease is more extensive among poor societies and populations with minimal access to dental care and preventive dentistry, situations that are common in isolated villages / towns. bop was not associated with cal, pd or gingival recession, while previous studies showed that bop was significantly associated with cal and increased pd, but not with gingival recession. this finding regarding age is in agreement with observations of other studies in several age groups. this relationship between the occurrence of gingival recession and age could be attributed to the longer period of exposure to factors that cause gingival recession and the cumulative effects of the lesion itself. regarding the role of dental plaque and gingival inflammation in the development of gingival recession, previous studies have showed that gingival inflammation was the most frequent precipitating etiological factor of gingival recession. they suggested that a localized inflammatory process causes the breakdown of connective tissue destruction and have also reported that gingival recession was associated with a high level of dental plaque while a study by goutoudi. revealed that gingival margin recession was associated with both high inflammatory and plaque scores. in addition, a significant association between gingival recession and periodontal disease was recorded. a study by slutzkey and levin showed a negative correlation between dental plaque on the buccal tooth aspect and gingival recession, while loe. emphasized the role of poor oral hygiene, dental plaque and calculus in gingival recession. pd was significantly associated with age and gender, but not dental plaque or calculus. found that the prevalence of periodontal pockets greater than 6 mm was associated with gender. the multivariate analysis indicated an association between dental plaque and gingival recession and this association was independent of calculus. furthermore, tobacco consumption, educational level, tooth brushing frequency and reasons for dental visit were not significantly related to the mean attachment loss. bergstrm, bokor - bratic and many investigators have shown the effects of smoking on the periodontium and periodontal health. these studies have shown that cigarette smoking is clearly established as one of the most significant risk factors in the development and the progression of periodontal disease. regarding the role of dental hygiene habits, it is known that proper use of tooth brush and use of dental floss are the most effective tools for removal of dental plaques from tooth surfaces, especially in terproximal surfaces. similarly, a regular dental follow - up can prevent the development, extent and severity of all forms of periodontal diseases. the role of educational level regarding oral hygiene habits is also known, since more educated individuals have less gingival inflammation or other forms of periodontal diseases, which might be attributed to the fact that more educated individuals have realized the value and importance of preventive dentistry and oral hygiene procedures, applying proper habits and standards of oral hygiene, and following regular dental check - ups. similar observations have been recorded in other studies, indicating that the level of education is the most important contributor to periodontal destruction. recorded that low educational subjects exhibited significantly more periodontal attachment loss and had significantly fewer healthy gingival units, while zini. showed that a lower level of education was associated with severe chronic periodontitis. periodontal disease of this sample was mainly associated with gingival inflammation and presence of dental plaque and calculus. however, the majority of the individuals showed cal and gingival recession rather than deep pockets. despite the extensive gingival inflammation and poor oral hygiene of the sample, it did not present destructive forms of periodontal disease. study results showed that a strong need exists for improvement of the population s self - awareness of oral hygiene and better oral health education mainly in rural regions of greece. in addition, it is important to focus more on periodontal aspects of dental care and effective prevention programmes and better control of periodontal disease are required. | objective : the aim of the present study was to examine the periodontal condition of an adult population in three isolated regions in greece and to determine the association of periodontal disease with several demographic, behavioral and environmental factors.materials and methods : the study population consisted of 640 individuals, aged 20 to 69 years from three isolated regions. the following indices were assessed : pocket depth (pd), clinical attachment level (cal), dental plaque, calculus and bleeding on probing (bop). statistical analysis was accomplished by multiple linear regression model which was used to assess the association between the mean clinical attachment loss and clinical, demographic and behavioral parameters.results:the samples of the study showed high levels of dental plaque, dental calculus and bop. the final multivariate model showed that age (p=0.000), gender (p=0.016) and presence of calculus (p=0.000) were associated with the mean clinical attachment loss. age (p=0.000), gender (p=0.000) and dental plaque (p=0.027) were associated with gingival recession, while age (p=0.018) and gender (p=0.000) were associated with probing depth. bleeding on probing, dental plaque, toothbrush frequency, level of education, tobacco consumption and reasons for dental visits were not associated with the mean clinical attachment loss.conclusion:periodontal disease consists of a complicated destructive condition of the periodontal tissue with a. multi - factorial etiology. oral hygiene instructions and a regular dental follow - up could play a significant role in the prevention of periodontal disease. |
apteronotidae is the gymnotiformes family with the largest number of formally described species : 86 species divided into 15 genera (de santana, 2007 ; de santana and vari, 2009, 2010a, b ; albert and crampton, 2009 ; de santana and crampton, 2010). the species of this family are found in rivers from panama to northern argentina, including rivers that flow into the pacific ocean (eastern colombia), the orinoco, maracaibo, magdalena, guyana shield, the amazon, and the paran - paraguay and san francisco basins (mago - leccia, 1994). intra- and interspecific variations in size and shape of the head have been observed, probably related to trophic specialization and/or aggression between males (cox - fernandes, 1998 ; albert, 2001 ; cox - fernandes., 2002). a few species of the order gymnotiformes have been cytogenetically analyzed and have shown highly diverse karyotypes, with differences in both chromosome structure and number (artoni., 2000). among the studied members of this order, the chromosome number varies from 2n = 24 in apteronotus albifrons (howell, 1972 ; almeida - toledo., 1981 ; mendes., 2012) to 2n = 54 in gymnotus carapo, g. mamiraua, g. inaequilabiatus and g. paraguensis (lacerda and maistro, 2007 ; milhomem., 2007, 2008, 2012a, 2012b ; scacchetti., although apteronotidae is the gymnotiformes family with the largest number of described species, chromosomal information is available only for apteronotus albifrons, which has 2n = 24 chromosomes (howell, 1972 ; almeida - toledo., 1981 ; mendes., 2012) reportedly, apteronotus albifrons from the parana river also has b chromosomes, which were found as microchromosomes (mendes., 2012). here, we made the cytogenetic characterization of three additional apteronotidae species, sternarchorhamphus muelleri, parapteronotus hasemani and sternarchogiton preto, in an effort to increase the amount of chromosomal information available for representatives of this family, to allow comparative analyses and provide new insights into the possible mechanisms underlying the diversification of these species. we analyzed two males of species sternarchorhamphus muelleri (mpeg 22759) from the anequara river (abaetetuba - pa ; 14042.6 s and 490016.6 w), 15 specimens (10 males and five females) of parapteronotus hasemani (idsmictio 059, idsmictio 0637, idsmictio 0746, idsmictio 0747, idsmictio 0754, idsmictio 0758, idsmictio 01001, idsmictio 01869, idsmictio 02047, idsmictio 02056, idsmictio 02057, idsmictio 02058, idsmictio 02095, idsmictio 02129, mpeg 22757) from the rivers of the reserva de desenvolvimento sustentvel mamirau (rdsm) - am (3250,2 s e 645126,6 w), and five specimens of sternarchogiton preto (one male, one female and three of unidentified sex ; mpeg 22758) collected in the caripetuba river (abaetetuba - pa ; 13723,49s e 485533w) (figure 1). the specimens were deposited in the instituto de desenvolvimento sustentvel mamirau (idsm) and the museu paraense emlio goeldi (mpeg). map of the collection sites of the sternarchorhamphus muelleri, parapteronotus hasemani and sternarchogiton preto specimens (reserva de desenvolvimento sustentvel mamirau, am). apteronotidae specimens preserved in 70% ethanol : (a) sternarchorhamphus muelleri, (b) parapteronotus hasemani, and (c) sternarchogiton preto (source : laboratrio de citogentica ufpa). (1978) and analyzed by conventional staining (giemsa), c - banding (sumner, 1972), ag - nor (howell and black, 1980), cma3 (schweizer, 1980), and dapi (pieczarka., 2006) staining. fluorescent in situ hybridization (fish) was performed with telomeric probes (all telomere, oncor) and 18s rdna probes obtained from species prochilodus argentus (hatanaka and galetti jr, 2004) and labeled with biotin or digoxigenin by nick translation. hybridization was detected with avidin-(cy3 or fitc) or anti - digoxigenin-(cy3 or fitc). the species sternarchorhamphus muelleri was found to have a karyotype of 2n = 32 (28m / sm+4st / a) and a fundamental number (fn) of 60. c - banding showed the presence of constitutive heterochromatin (ch) in the centromeric regions of most chromosomes, except for pairs 7, 11, 12, 13, 14 and 16, where the banding was almost imperceptible (suggesting that there was relatively little ch). pairs 1, 3 and 10 had pericentromeric heterochromatic blocks, and pair 15 had a heterochromatic block in the proximal region of the long arm. a nucleolar organizer region (nor) was found in the interstitial region of the long arm of pair 15, and showed heteromorphism between the homologs (figure 3a, b). karyotypes submitted to conventional staining and c - banding : a and b - sternarchorhamphus muelleri, c and d - parapteronotus hasemani ; and e and f- sternarchogiton preto. abbreviations : m - sm = metacentric - submetacentric ; st - a = subtelocentric - acrocentric. the karyotype of parapteronotus hasemani was 2n = 52 (36m / sm+16st / a) and fn = 88. pairs 1, 22 and 23 also had heterochromatic blocks in the proximal regions of their long arms. pairs 2, 3, 5 and 6 had large heterochromatic blocks that spanned nearly the entire length of their short arms. a nor was found on the distal short arm of pair 3 ; it coincided with a secondary constriction and showed heteromorphism between the homologs (figure 3c, d). sternarchogiton preto had a 2n = 52 (38m / sm+14st / a) karyotype and fn = 90. ch was observed in the centromeric region of most chromosomes, except for pairs 5, 9, 12, 16 and 20, where the banding was almost imperceptible. in addition, pair 1 had a heterochromatic block throughout the long arm, pair 2 had a heterochromatic block in the proximal region of the long arm, and pair 3 had a heterochromatic block on the long arm. pairs 4, 8, 21, 23 and 24 had heterochromatic blocks on their short arms. a nor was observed in the distal region of the long arm of pair 3, within the heterochromatic block, and showed heteromorphism between the homologs (figure 3e, f). the three studied species showed similar results in the fluorochrome, cma3 and dapi staining, as well as fish analysis with 18s rdna and telomere repeat probes. cma3 labeled the nors in all three species, showing that the ribosomal genes were interspersed with gc - rich sequences. dapi staining revealed that the ch was at - rich, and fish with 18s rdna probes showed that these sites were localized in a single pair per species, coincident with the nor. finally, fish using the telomeric probes did not indicate the presence of any interstitial telomeric sequences (figure 4a l). fluorescent staining of sternarchorhamphus muelleri (first column), parapteronotus hasemani (second column) and sternarchogiton preto (third column) metaphases, respectively, with cma3 (a, b, c), dapi (d, e, f), 18s rdna probe (g, h, i), and telomeric probe (j, k, l). white arrows indicate cma3-marked nors ; yellow arrows indicate nors marked by 18s rdna probe. family apteronotidae is not only more diverse in its number of genera, species and morphological forms, it also displays the greatest variation in diploid number among the gymnotiformes, ranging from 2n = 24 (16m / sm+8st / a) in apteronotus albifrons (howell, 1972 ; almeida - toledo., 1981 ; mendes., 2012) to 2n = 52 in parapteronotus hasemani (36m / sm+16st / a) and sternarchogiton preto (38m / sm+14st / a) (this work). sternarchorhamphus muelleri (2n = 32 ; 28m / sm+4st / a) has an intermediate diploid number (this work). parapteronotus hasemani and sternarchogiton preto have the same diploid number (2n = 52), but differ in their karyotypic formulae. this difference can be explained by chromosomal inversions, which can change the morphology of chromosomes while maintaining the diploid number (alves., 2003 ; de oliveira., 2006). karyotypes of 2n = 52 are also found in other apteronotidae species (sternarchorhynchus cramptoni, s. oxyrhynchus, platyurosternachus macrostomus, apteronotus bonapartii ; unpublished data), while 2n = 50 is seen in two species of genus adontosternarchus (a. clarkae and a. balaenops ; unpublished data). although they share the same diploid number, p. hasemani and s. preto display considerable differences in their c - banding patterns and the location of their nors. the karyotype of sternarchorhamphus muelleri (2n = 32) is quite different from those of the other two species in chromosome number and morphology, ch distribution and nor location. such events are believed to explain the reduction of 2n in genus ancistrus (loricariidae) (alves. have been suggested for other groups of fishes (cipriano., 2008 ; margarido and moreira - filho, 2008). the location of the nors in this species is quite different from that seen in other species, as it typically appears in the interstitial region of the long arm of an acrocentric chromosome. this variation in the nor - bearing chromosome may have arisen, in this case, through a pericentric inversion that moved the nor to the middle of the chromosome arm. (2010) used chromosome painting to show that the degree of chromosomal rearrangement between karyotypes of two cryptic species of gymnotus cf carapo (2n = 40 and 2n = 42) was much greater than that estimated using classical cytogenetics (milhomem., 2008). thus, the pericentric inversions and centric fusions that were believed to differentiate the karyotypes of these species may be an underestimate of the actual degree of genomic rearrangement at work. ch was found in the centromeric regions of virtually all chromosomes of the analyzed species. dapi staining revealed that this ch is at - rich, which is consistent with reports concerning other gymnotiformes (milhomem., 2007, 2008, 2012a, b ; silva., 2008, 2009 ; cardoso., 2011). the three species analyzed in the present study showed differing ch patterns, with several additional blocks in non - centromeric regions. processes related to the dynamics of repetitive dna, such as amplifications and translocations, might have been involved in the development of these blocks. (2004) showed that in arabidopsis the heterochromatin is determined by transposable elements and related to tandem repeats. transposons have been found in the heterochromatin of several fish groups, including cichla kelberi (teixeira., 2009), hisonotus leucofrenatus (ferreira., 2011), and antarctic fishes of the suborder notothenioidei (ozouf - costaz., 2004). furthermore, transposons have been associated with the karyotypic variation observed in erythrinus erythrinus (cioffi., 2010), and the formation of the y chromosome in chionodraco roseofuscus (capriglione., 2000). souza. (2009) analyzed species of genus peckoltia (siluriformes : loricariidae) and proposed possible homeologies among some pairs with similar c - banding patterns, morphologies and nor localizations. in the karyotypes of p. hasemani, s. muelleri and s. preto, we identified chromosomes (pair 1 in the first two species and pair 2 in the latter) that resembled one another in chromosome morphology, size and hc distribution (figure 5). possible homeologies among sternarchorhamphus muelleri, parapteronotus hasemani and sternarchogiton preto (conventional giemsa staining and c - banding). heteromorphisms in nor size (by 2-fold or more) have been described in some gymnotiformes species, and the nors of eigenmannia sp and e. virescens were found to be larger than those of the other examined species. tandem duplications, unequal crossovers involving repeated regions, and/or accidental duplications might explain these variations (foresti., 1981). the nor regions of the tested species stained positive with cma3, revealing that the ribosomal genes are interspersed with gc - rich sequences (pends., 1993). our results resemble those found in other gymnotiformes and additional fish species (artoni and bertollo, 1999 ; milhomem., 2007, 2008, 2012a, b ; silva., 2008, 2009 ; fish with telomeric probes failed to show any evidence of interstitial telomeric sequences (itss). this may reflect the absence of chromosomal rearrangements involving the telomeres (silva., 2009). alternatively, telomeric sequences away from the chromosomal ends may have undergone sequence changes that hinder probe hybridization (albun., 1996). accumulating evidence suggests that variability in diploid number and karyotype formula can be explained by chromosome rearrangements, such as fusions (which can decrease the diploid number) and inversions. according to the phylogenies of alves - gomes. (1995) and albert (2001), the family gymnotidae, represented by gymnotus and electrophorus, occupies the basal position among the other gymnotiformes. the species in this family have diploid numbers ranging from 34 to 54, with a higher occurrence of 2n above 50 (for review, see milhomem., 2012a, b). thus, we believe that the higher diploid numbers (2n = 50 or 52) are basal in apteronotidae and that the karyotypes of apteronotus albifrons and sternarchorhamphus muelleri underwent rearrangements that decreased their 2n values. their ch patterns are also quite variable, showing additional blocks that are characteristic to each species. the nors varied in size and location on the chromosome, but all three species had a single nor. together, the results presented herein add new information that may prove valuable in future studies of this group, facilitating taxonomic identification, and increasing our understanding of the chromosomal evolution and phylogenetic relationships of the apteronotidae. | cytogenetic studies were carried out on samples of parapteronotus hasemani, sternarchogiton preto and sternarchorhamphus muelleri (apteronotidae, gymnotiformes) from the amazon basin. the first two species exhibited both a 2n = 52 karyotype, but differed in their karyotypic formulae, distribution of constitutive heterochromatin, and chromosomal location of the nor. the third species, sternarchorhamphus muelleri, was found to have a 2n = 32 karyotype. in all three species the dapi and chromomycin a3 staining results were consistent with the c - banding results and nucleolar organizer region (nor) localization. the 18s rdna probe confirmed that there was only one pair of ribosomal dna cistron bearers per species. the telomeric probe did not reveal interstitial telomeric sequences (its). the karyotypic differences among these species can be used for taxonomic identification. these data will be useful in future studies of these fishes and help understanding the phylogenetic relationships and chromosomal evolution of the apteronotidae. |
acute suppurative thyroiditis (ast) accompanied by an abscess is a rare clinical case. possible reasons for the infrequency of ast include the gland 's abundant blood supply and lymphatic drainage and the antimicrobial action of iodine 1. ast is found primarily in children. according to miyauchi., the most common mechanism for thyroid infection is the transmission of infective organisms via a pyriform sinus fistula 2. mainstream management of ast is antimicrobial therapy, directed against the likely bacterial pathogens. here a 65yearold japanese man was admitted complaining of painful swelling in the neck and with high fever. stent placement in left superficial femoral artery for arteriosclerosis obliterans (aso) had been performed 45 days before this episode in our hospital. on examination, his height, body weight, and body mass index values were 156 cm, 53.3 kg, and 21.9 kg / m, respectively. a swollen mass was palpable on his left neck with erythematous change, hot feeling, and tenderness. laboratory data revealed white cell count : 12,500 (40008000)/l, creactive protein (crp) : 26.0 (0.30.6) mg / dl, blood urea nitrogen (bun) : 34 (921) mg / dl, serum creatinine (cr) : 6.89 (0.20.9) mg / dl, free t4 : 1.69 (0.91.8) ng / dl, thyroidstimulating hormone (tsh) : 0.52 (0.343.5) iu / ml, hba1c : 5.7 (ngsp : < 6.5)%, and postprandial blood glucose : 123 (< 200) mg / dl. neck ultrasonography revealed a wellcircumscribed large cystic mass with debris (56 51 32 mm) in left thyroid gland (fig. 1a and b). computed tomography also revealed a lowdensity lesion in left lobe of thyroid gland (fig. thus, he was diagnosed as suffering from acute suppurative thyroiditis (ast) in infected thyroid cyst. intravenous administration of meropenem 1 g / day was started. on the fourth hospital day the aspirated pus from thyroid cyst and blood were sent for microbial culture and sensitivity test. 2). second computer tomography revealed incidental aortic dissection (stanford type b). thoracic endovascular aneurysm repair (tevar) surgery was performed for aortic dissection on the 60th hospital day. 2). four months after discharge, neck ultrasonography showed resolution of the supportive thyroiditis (fig. (a, b) wellcircumscribed large cystic mass with debris in left thyroid gland in neck at ultrasonography. (c) an abscess in the left lobe of thyroid gland in neck ct (arrow). mepm, meropenem ; vcm, vancomycin ; sbt / abpc, sulbactam / ampicillin ; ampc, amoxicillin ; tevar, thoracic endovascular aneurysm repair. (a) cystic area in the left lobe decreased in volume after drainage and antibiotics. (b) thyroid gland was almost cured with heterogenous scarred upper middle of left lobe. acute suppurative thyroiditis (ast) is a rare infectious disease of the thyroid gland. analysis of 1309 thyroid operations performed during twentytwo years revealed 117 instances of thyroiditis, but only six of these were suppurative 3. pyriform sinus fistulae related to third and fourth branchial pouch anomalies were one of the causes of ast 4. in the present case, there were two atypical aspects : (i) the onset at middle age and (ii) pyriform sinus fistula was not found. no invasive infectious sign on surrounding tissue of the thyroid was revealed by neck ultrasonography. two possible precipitants may be susceptible to infection in hemodialysis and stent placement for aso. some recognized causes of ast are neoplastic thyroid nodule, subacute thyroiditis, hashimoto thyroiditis, and penetration of the thyroid gland by foreign bodies such as fish bones and trauma 5, 6, 7. underlying diseases including tuberculosis 8, diabetes mellitus 9, and human immunodeficiency virus infection 10 appear to increase the risk for developing of thyroid abscess. although diabetes is recognized to lead to poor immune response, in this case, the glycemic control (as represented by hba1c) was very good. hemodialysis patients are at risk for infections 11. according to the dialysis patient death registry and the reports on sepsisrelated mortality compared to that of the general population, sepsis mortality was from 100 to 300 times higher for chronic dialysis patients than that for the general public 12. wang., hypothesized that this association includes increased susceptibility to infection, the presence of comorbidities such as diabetes, and repetitive exposure to pathogens during hemodialysis 13. mh, nk, ya, hu, kt, and th : were responsible for correction of the article and supervision. | key clinical messageacute suppurative thyroiditis (ast) accompanied by an abscess is a rare clinical case. hemodialysis patients are at risk for infections. sepsis mortality was from 100 to 300 times higher for chronic dialysis patients than that for the general public. thus, special care should be taken against infection in patients under hemodialysis. |
bonding of the orthodontic attachments by using light cure adhesive has gained more popularity in orthodontic practice today. it gives the opportunity for the operator to manipulate the bracket position to the right position and then start the polymerization with a visible light source, usually at 450 470 nm.1 the most disadvantage of light cure system is time consuming where each bracket needs 40 seconds for curing. many attempts were done in the last decade to decrease the curing time either by introducing new light sources or enhancements of the conventional halogen source. oesterle in a study of light curing and setting times, recommended using a 40 second total light curing time (20 seconds on the mesial side and 20 seconds on the distal side of the brackets). frost tried to reduce the curing time by using an elliptical light guide. another trail was done by bishara.4 they tested the standard cured light guide with an 11 mm diameter and 4 mm mini turbo light guide and found no significant differences in shear bond strength between them. evans investigated the shear bond strengths of orthodontic brackets by using different light guides (power slot and turbo tip) and found that the light guides could significantly reduce the curing times without affecting the shear bond strength. many authors thought to introduce another light source like laser or plasma arc light and finally the micro - xenon light. blankenau compared the degree of polymerization by argon laser and conventional light - curing unit and found that the polymerization with argon laser produced an even greater polymerization than halogen light. in contradict to the concept which states that laser may produce more heat generation, powell stated that, at recommended curing times ; in vitro pulp - chamber temperature increases from argon lasers were significantly lower than those of the conventional curing lights. featheringham8 compared 3 different visible light curing systems ; the argon laser at 4, 6, 8 and 10 seconds, plasma arc curing unit at 2, 4, 6 or 8 seconds and conventional halogen light at 40 seconds. he concluded that the curing by plasma arc at 4 seconds resulted in significantly higher mean shear bond strength than curing with the plasma arc at 2 seconds or the argon laser at 4 seconds, argon at 8 seconds showed higher bond strengths than it did at 4 seconds. cacciafesta investigated the polymerization with micro - xenon light of a resin - modified glass ionomer. they found no statistically significant differences between the bond strength of control group cured with halogen light and those of groups cured with xenon light for 10, 5 and 2 seconds respectively. they concluded that the micro - xenon light enabled the clinician to reduce the curing time without affecting the shear bond strength. recently, staudt tested the shear bond strength with a high - power halogen light curing source. they concluded that the high power halogen light seemed to be cost - effective solution to reduce curing time to 23 seconds. laser source, plasma arc light and micro - xenon light are very expensive and need special equipments that may not be available in dental office. therefore, introducing a new light source capable of reducing the curing time with reasonable economic cost is a premium goal for the dental field. it is important to evaluate the degree of polymerization of polymeric adhesives, since inadequate polymerization results in inferior physico - mechanical properties.11,12 orefice monitored the monomer conversion of dental composites during visible light irradiation in situ by infrared spectroscopy and evaluated the microhardness of the composites with different degrees of conversion. ft- raman spectroscopy is also one of the methods that can be utilized to obtain structural information for opaque materials such as enamel, dentin and related polymeric composites.14 the main objective of this study was to use ft - raman spectroscopy technique to measure the degree of polymerization of a dental composite resin cured with a new light - curing source designed at the national research center of egypt. in this study, a new metal - halide light curing source was adopted for photo polymerization of the dental adhesive used. this light source is composed mainly of a metal - halide lamp for special applications (blv, steinhoring, germany). the energy consumption of this lamp is 150w and current 1.8a, with power density 1500 mw /cm. the advantages of metal - halide lamp are very high color temperature, high color stability over the entire life time, long life time (average life time 6000 hours) and special light spectrum as shown in figure 1. a conventional halogen light source for dental cure, power density 0.6 w / cm (caulk dentsply, milford, de, canada) was used as a reference light curing unit for comparison with the newly introduced metal halide source., lincolin, il, usa) is a composite resin - based adhesive commercially available. it is used in this study to evaluate the efficiency of the new metal - halide lamp for polymerization of the composite. three sizes cylindrical specimens of the composite were prepared (2x2, 3x3 and 6x3 mm, diameterx height) using closed - end teflon moulds. the open end was filled with the uncured composite to an excess with slight pressure (to avoid air bubbles). then, the specimen was light cured by a direct contact with the light source under investigation for the following periods of time : 2, 4, 8, 12, 20 and 40 seconds. the complete hard set specimen was released from the mould, ground into fine powder and packed in nmr glass tube (5 mm internal diameter) ready for ft - raman spectroscopy measurements. each specimen of a certain irradiation time was prepared three times to obtain three raman spectra. processing the samples and recording their spectra occurred within 24 hours from the beginning of the light curing at room temperature (211c). uncured composite specimen was filled directly into the nmr glass tube and underwent raman analysis. all the ft - raman measurements were carried out by using the nexus 670 ftir ft- raman spectrometer (nicolet, usa). this emits continuous - wave laser energy at wave length of 1064 nm (9398 cm) and has a maximum power level of approximately 1.5w at the sample. the installed detector in the ft - raman module is in - gaas, which is an air- cooled detector. the used sample configuration is 180 reflective with fully motorized sample position adjustment, with an nmr - tube sample holder. all the ft - raman spectra were collected in the spectral range from 3701 to 98 cm and 46 scans. the laser power was 0.7 w. the spectra were processed using the computer program omnic esp, 5.2a (nicolet, usa). the dp of the composite can be determined by comparison of the ratio (r) of the reacting aliphatic c = c bonds to the unreacting aromatic c = c bonds before and after curing. the aliphatic and aromatic c = c bonds have characteristic raman scattering peaks located at 1638 and 1610 cm, respectively as well as the infrared absorption peaks but with higher intensities. the peak height (h) and underlying area (a) was calculated for each peak, using a standard baseline technique with aid of computer program provided with the spectrometer. the ratio (r) was determined twice by means of the peaks heights and areas for confidential results. the percentage dp of each specimen was calculated using the following equation.1517 the obtained data were subjected to analysis of variance (anova) according to snedcor and co - chran.18 duncan s multiple range test was used to compare between means of treatments according to walter and duncan19 at probability 5%. in this study, a new metal - halide light curing source was adopted for photo polymerization of the dental adhesive used. this light source is composed mainly of a metal - halide lamp for special applications (blv, steinhoring, germany). the energy consumption of this lamp is 150w and current 1.8a, with power density 1500 mw /cm. the advantages of metal - halide lamp are very high color temperature, high color stability over the entire life time, long life time (average life time 6000 hours) and special light spectrum as shown in figure 1. a conventional halogen light source for dental cure, power density 0.6 w / cm (caulk dentsply, milford, de, canada) was used as a reference light curing unit for comparison with the newly introduced metal halide source. alpha - dent (dental technologist inc., lincolin, il, usa) is a composite resin - based adhesive commercially available. it is used in this study to evaluate the efficiency of the new metal - halide lamp for polymerization of the composite. three sizes cylindrical specimens of the composite were prepared (2x2, 3x3 and 6x3 mm, diameterx height) using closed - end teflon moulds. the open end was filled with the uncured composite to an excess with slight pressure (to avoid air bubbles). then, the specimen was light cured by a direct contact with the light source under investigation for the following periods of time : 2, 4, 8, 12, 20 and 40 seconds. the complete hard set specimen was released from the mould, ground into fine powder and packed in nmr glass tube (5 mm internal diameter) ready for ft - raman spectroscopy measurements. each specimen of a certain irradiation time was prepared three times to obtain three raman spectra. processing the samples and recording their spectra occurred within 24 hours from the beginning of the light curing at room temperature (211c). uncured composite specimen was filled directly into the nmr glass tube and underwent raman analysis. all the ft - raman measurements were carried out by using the nexus 670 ftir ft- raman spectrometer (nicolet, usa). this emits continuous - wave laser energy at wave length of 1064 nm (9398 cm) and has a maximum power level of approximately 1.5w at the sample. the installed detector in the ft - raman module is in - gaas, which is an air- cooled detector. the used sample configuration is 180 reflective with fully motorized sample position adjustment, with an nmr - tube sample holder. all the ft - raman spectra were collected in the spectral range from 3701 to 98 cm and 46 scans. the laser power was 0.7 w. the spectra were processed using the computer program omnic esp, 5.2a (nicolet, usa). the dp of the composite can be determined by comparison of the ratio (r) of the reacting aliphatic c = c bonds to the unreacting aromatic c = c bonds before and after curing. the aliphatic and aromatic c = c bonds have characteristic raman scattering peaks located at 1638 and 1610 cm, respectively as well as the infrared absorption peaks but with higher intensities. the peak height (h) and underlying area (a) was calculated for each peak, using a standard baseline technique with aid of computer program provided with the spectrometer. the ratio (r) was determined twice by means of the peaks heights and areas for confidential results. the obtained data were subjected to analysis of variance (anova) according to snedcor and co - chran.18 duncan s multiple range test was used to compare between means of treatments according to walter and duncan19 at probability 5%. the typical ft - raman spectra recorded for the composite before and after photoinitiation by the metal - halide light source are shown in figure 2. the spectra reveals a scattering raman peak at 1638 cm corresponding to the reacting aliphatic c = c stretching of the vinyl group in the acrylates and methacrylates.12,20 the spectra show a remarkable decrease in the intensity of this peak as a result of the exposure to the new light source. the scattering peak at 1610 cm referring to the unreacting aromatic rings in the bis - gma molecule is commonly used as an internal reference for precise quantitative analysis.15,17,21 for more confidential data, the ratio of the peaks 1638 to 1610 cm (r) was calculated twice, by means of peaks height and peaks area. tables 1 and 2 give the mean values of the percentage degree of polymerization (dp%) of the resin cured by each light source calculated by the peaks height and peaks area, respectively. these values are plotted against the time of exposure in figures 3 and 4, respectively. both figures exhibit the same behavioral pattern of dp% with curing time either the composite was halogen - cured or metal - halide cured. there is a rapid increase in the dp% with increasing the curing time up to 12 seconds, followed by gradual increase up to 40 seconds for any sample size. it could be noticed that the smallest sample size (2x2 mm) had always the highest values of dp%. the results of statistical analysis of the data given in tables 1 and 2 revealed that the samples cured by the metal - halide source produced higher polymerization rate values than those cured by the halogen source. the polymerization rate was directly proportional to the exposure time and inversely proportional to the sample size irrespective to the light source used. it could be noticed that the polymerization rate values of the samples given in table 2 were higher than their corresponding values given in table 1. this observation can be explained by the fact that the area of the peak is supposed to be more representative to the actual scattering of the peak than its height. therefore, we can rely on the results reported in table 2 conveniently. from this table, the newly introduced metal - halide source produced satisfactory polymerization (51.40%) of the composite in a rather short curing time, 8 seconds. there was no significant difference in the polymerization values between the samples cured by the metal - halide source for 12, 20 and 40 seconds (59.19, 59.35 and 61.68%, respectively). results in both tables 1 and 2 revealed that for all sample sizes, curing with metal - halide light for 12 seconds achieved polymerization rates comparable to or even higher than that produced by 40 seconds halogen curing. this finding is practically important since the thickness of the adhesive under the bracket is too thin, so 12 seconds of metal - halide curing is helpful to obtain enough polymerization for bonding brackets in short time. the overall dp value data of the resin produced by exposure to each light source irrespective to the exposure time (from 2 to 40 seconds) or sample size are given in table 3. the statistical evaluation of the data using an independent sample (duncan test) demonstrated that the metal - halide source produced a significant increase in the overall polymerization in comparison with that produced by conventional halogen light source. vibrational spectroscopy, such as ft - infrared and ft - raman, provides key information on the structure of molecules. the position and intensity of the feature in the vibrational spectrum can be used to study the molecular structure or to determine the chemical identity of the sample. vibrations that are strong in an infrared spectrum (involving strong dipole moments) are usually weak in a raman spectrum. likewise, non - polar functional group vibrations that give very strong raman bands usually result in weak infrared signals. for example, oh- or nh- stretching vibrations and the vibration of carbonyl groups are usually very strong in an ft - ir spectrum and are usually weak in raman spectrum. however, the stretching vibrations of c = c such as in dental composites are very strong in the raman spectrum.22,23 the results of this investigation revealed that there were statistically significant differences between the degree of conversion values of metal - halide source and halogen source. the lamp of metal - halide source is completely different from the halogen lamp at many aspects ; the power density of the metal - halide is 1500 mw / cm while for halogen light is only 600mw / cm. the halogen lamp has a limited effective lifetime approximately 100 hours due to degradation of the bulb s components by high heat generated.12,2426 the metal - halide lamp has several advantages such as space saving design, economic due to long lifetime (average lifetime 6000 hours), high color stability over the entire life time, axial arc, and ability to produce good light beam and uv protection. according to fujibayashi who stated that the quality of light polymerization was not exclusively due to light intensity but also due to the narrow absorption peak of the initiator system (camphorquinone). the absorption curve of cam - phorquinone extends between 360 and 520 nm, with the maximum at 465 nm.12,28 for metal - halide source, most photons emitted were within the maximum absorption curve of camphorquinone ; while the photons emitted by conventional halogen light were around the optimal spectrum range. according to the results of many investigations comparing new light sources reached to an accepted degree of conversion in reduced time as, fast halogen light 1015 seconds, plasma arc light 69 seconds and led light 20 seconds with the conventional halogen light 40 seconds.12,29,30 they concluded that led was the most suitable as a good alternative to the halogen light, where the plasma arc light were bulky and usually incorporated a noisy cooling fan, both plasma arc light and fast halogen light were more expensive than the conventional halogen light. they preferred the led because it was smaller, cordless and did not require cooling fan and had an estimated life over 10000 hours.31 in comparison with the previously mentioned sources, the metal - halide source is not expensive like the plasma arc light or fast halogen light, and it has long life period 6000 hours near led. moreover, it has the ability to produce higher degree of conversion in markedly reduced curing time (12 seconds). therefore, the new metal- halide source can be recommended for orthodontic field since bonding of 20 brackets needs 800 seconds with conventional halogen light while with new source requires only 240 seconds. the new metal - halide light source produces comparable degree of polymerization in a significant shorter curing time and therefore can be recommended for clinical use. | objectivesto use the ft - raman spectroscopy for evaluation the degree of polymerization of dental composite as a result of photo curing with a new light source in comparison to the conventional halogen light.materials and methodsin this study a new light source, based on a metal - halide lamp (topspot g12) was developed at nrc - egypt for curing dental composites. two groups of 108 composite samples each were cured using both the new light source and a conventional halogen source, as a control source. different samples sizes (2x2, 3x3 and 6x3 mm2) were cured for different periods of time (2, 4, 8, 12, 20, and 40 seconds). the spectroscopic data were analyzed statistically by anova and duncan s multiple range test (p <.05).resultsthe results showed that the samples cured by the new metal - halide source produced higher polymerization rates than those cured by the halogen source. the polymerization rate was directly proportional to the exposure time and inversely proportional to the sample size, irrespective to the light source used. the results also showed that 12 seconds of metal - halide light curing produced polymerization rate comparable to or even higher than that produced by 40 seconds halogen light curing.conclusionsthe new light source produced a satisfactory degree of polymerization in a remarkable shorter curing time and it can be recommended for clinical use. |
cancer is a heterogeneous genetic disease of somatic cells arising from accumulated genetic changes on cancer genome resulted in alterations of gene expression, unregulated cell growth and triggering formation of malignant neoplasm. systematic genomic approaches have been applied to dissect tumorigenic pathways for diagnostic and prognostic applications and to search for potential cancer genes as therapeutic targets. these technologies revealed a global view of cancer genomic aberrations including loss of heterozygosity (loh) and comparative genomic hybridization (cgh) analyses to identify chromosome aberrations as well as microarray and proteomic analyses to profile the alteration of cancer gene expression. it has been proposed that there are four to seven somatic aberrations occurred at the rate - limiting steps during epithelial tumor progression including six categories of essential alterations in cell physiology that collectively perturb regulatory circuits of normal cell proliferation and homeostasis leading to malignant growth (1). since multiple signaling pathways might be disrupted at different points in different cancers and since aberration of mutator genes could promote the genome instability during cancer development, the patterns of genetic aberrations tend to be non - random but differ between cancers of different tissues and of different subtypes from the same tissue. toward accelerating our understanding of tumorigenesis for better management of cancer patients, genomic approaches for systematically measuring somatic altered cancer genome and gene expression should be critical for clinical applications such as diagnosis, prognosis, classifying cancer subtypes and options of therapeutic treatment (2,3). however, identification of putative cancer gene is still hampered by the difficulty of further refining the precise aberrant region owing to the low resolution of chromosome alterations detected by cgh and the large deletions of loh detected by microsatellite markers (4). in addition, the noisy data of chromosome aberrations and inconsistent results of altered gene expression detected by microarray and proteomic experiments, further demonstrated an emergent need of integrating qualified cancer genomic and expression data for developing new and effective cancer therapeutic targets. (58) human hepatocellular carcinoma (hcc) is the sixth most common cancer worldwide and the third most common cause of cancer death with prevalent areas in asia and sub - saharan africa. (9) although recent studies suggested increase of hcc incidence in western countries, > 80% of the hcc cases occurred in above endemic areas are owing to exposure of major risk factors such as hepatitis viruses, mycotoxins and alcohol abuse. (10) since hcc progression is usually asymptomatic resulted in poor prognosis and low 5-year survival rate (1215%), comprehensive molecular genetic studies will be important for improving clinical management of hcc. previous studies by our group and others have already conducted experiments of genome - wide loh, cgh, microarray and proteomic analyses (1114). comprehensive analysis further allowed us to reveal two major genetic pathways, genome stable and instable pathways, in hcc progression. we therefore selected hcc as a cancer model to construct a public accessible integrated database, oncodb.hcc, with user - friendly and graphically displayed interfaces as useful resources for facilitating researches in hcc tumorigenesis. as indicated in figure 1, the chromosome view interface demonstrates the main features of the database. the hcc data are constructed based on physical maps of human and rodent genome sequences from ensembl and illustrated in several aspects including cancer genomic aberration data of loh and cgh studies, altered gene expression in transcriptomes and proteomes analyses, genes with experimental data in hcc tissues reported in pubmed articles and the qtls of rodent hcc models. the interactive interface further allows users to display chromosome regions defined by physical position, cytogenetic bands and sequence - tagged site (sts) markers. in expression view, a total of 9785 genes can be searched by using gene i d and gene description showing individual gene data including 9162 genes displayed the detail altered expression profiles of individual arrays reprocessed from re - analyzed stanford hcc microarray data and experimental data of the gene extracted from published articles in pubmed. to demonstrate data reliability in our database, aurka, a gene reported to be up - regulated in majority of hcc tissues (15), was selected as positive control to perform semi - quantitative rt pcr experiments in 45 pairs of hcc tissues. as indicated in figure 2a, the aurka is highly up - regulated and the expression profiles are almost identical to that of re - analyzed stanford hcc microarray data. the chromosome view of oncodb.hcc : (a) indicated the chromosomal region displayed in cytogenetic position ; (b) demonstrated the expression intensity of genes along the physical positions of chromosome in terms of patient number by selecting gene expression cut - off value (default value = 1) in re - analyzed stanford hcc microarray data ; (c) showed the microarray / proteomic expression results from references ; (d) indicated the positions of genes collected from wet - lab experimental results ; (e) revealed the comparative maps and the syntenic regions of mouse and rat hcc qtls ; (f) displayed the loh frequencies and minimum deletion regions (mdr) in positions of microsatellite markers ; and (g) the cytogenetic locations of cgh results. all genes and markers were annotated and hyperlinked against physical maps of genomes in ensembl. the up - regulated genes and gain / amplified chromosomal regions were displayed in red series color. in contrast, the down - regulated genes and loss / deleted chromosome regions were displayed in green series color. experimental validation of representative genes in the hcc gene set. for each gene, the semi - quantitative rt pcr results in 45 hcc pairs are presented in gel images (left), in quantified expression profiles after normalization with -actin expression as internal control (upper right) and in comparison with expression profiles of the gene in re - analyzed stanford hcc microarray data from expression view of oncodb.hcc (lower right). (a) aurka is a positive control for hcc data process ; (b and c) genes selected from criteria of significant expression difference in at lease three independent microarray / proteomic studies ; and (d f) genes selected with criteria of at least 2-fold expression difference in at least 70% of paired arrays in re - analyzed stanford hcc microarray data. stringent criteria were applied to select a set of 614 hcc genes with significant altered expression in hcc tissues (supplementary files in oncodb.hcc). among them, 446 genes were supported with more than two independent studies in terms of altered expression including 145 concordantly up - regulated, 176 concordantly down - regulated and 125 genes with mixed up-/down - regulated expression in hcc tissues. the concordant expression of hcc genes could serve as potential biomarkers for hcc diagnosis. in addition, there were 234 genes with limited experimental data in hcc and 256 genes located within recurrent chromosome aberration regions. in addition to aurka, we further selected 30 out of 234 genes with limited wet - lab experiments in hcc for experimental validation. the validated results were demonstrated in terms of concordant expression of gene up- or down - regulation in 45 pairs of hcc tissues by rt pcr analysis and in comparison to 57 pairs of hcc samples of re - analyzed stanford hcc microarray data (figure 2 and supplementary files in oncodb.hcc). a near perfect concordant result except ckap2 (96.7%, 29/30 genes) in altered gene expression of hcc was obtained for 12 genes selected based on three independent microarray and/or proteomic reports and for 18 genes selected based on at least 2-fold expression changes within 70% patients in re - analyzed stanford hcc microarray data. to provide additional genetic support and refine the hcc loci for targeting cancer genes, the qtls of rodent hcc models identified via linkage studies were integrated into oncodb.hcc based on comparative maps of rodent genomes in ensembl. the results of 35 rodent hcc qtls were displayed according to the relative positions of human chromosomes (figure 1e). comparative mapping of hcc loci demonstrated that over 45% rodent qtls (8 of the 12 mouse hcc qtls and another 8 of the 23 rat hcc qtls) are located in the major aberrant loci of human hcc (table 1). among 16 syntenic qtls located in 12 hcc loci, 10 qtls in 6 loci are potentially located in gain / amplified regions and 6 qtls in 6 loci are located in loss / deleted regions. while the critical regions of hcc loci existed in comparative genomes of human and rodents, the hcc loci could be effectively narrowed down due to the scrambled structure of genomes by comparing human and rodent syntenic regions. we narrowed down two human hcc loci to 2 mb and another 6 hcc loci in between 4 and 10 mb. interestingly, the comparative mapping of hcc loci allowed us to split three human major hcc aberration regions 1q, 4q and 8p2123 into two smaller regions and to conclude that at least two putative cancer genes located on the same arm of above three human hcc chromosomes. hcc loci and putative cancer genes by comparative mapping strategy number of references with significant loh in the region defined by original authors. g : cgh gain ; l : cgh loss ; and a : cgh amplification. as indicated in figure 1, the chromosome view interface demonstrates the main features of the database. the hcc data are constructed based on physical maps of human and rodent genome sequences from ensembl and illustrated in several aspects including cancer genomic aberration data of loh and cgh studies, altered gene expression in transcriptomes and proteomes analyses, genes with experimental data in hcc tissues reported in pubmed articles and the qtls of rodent hcc models. the interactive interface further allows users to display chromosome regions defined by physical position, cytogenetic bands and sequence - tagged site (sts) markers. in expression view, a total of 9785 genes can be searched by using gene i d and gene description showing individual gene data including 9162 genes displayed the detail altered expression profiles of individual arrays reprocessed from re - analyzed stanford hcc microarray data and experimental data of the gene extracted from published articles in pubmed. to demonstrate data reliability in our database, aurka, a gene reported to be up - regulated in majority of hcc tissues (15), was selected as positive control to perform semi - quantitative rt pcr experiments in 45 pairs of hcc tissues. as indicated in figure 2a, the aurka is highly up - regulated and the expression profiles are almost identical to that of re - analyzed stanford hcc microarray data. the chromosome view of oncodb.hcc : (a) indicated the chromosomal region displayed in cytogenetic position ; (b) demonstrated the expression intensity of genes along the physical positions of chromosome in terms of patient number by selecting gene expression cut - off value (default value = 1) in re - analyzed stanford hcc microarray data ; (c) showed the microarray / proteomic expression results from references ; (d) indicated the positions of genes collected from wet - lab experimental results ; (e) revealed the comparative maps and the syntenic regions of mouse and rat hcc qtls ; (f) displayed the loh frequencies and minimum deletion regions (mdr) in positions of microsatellite markers ; and (g) the cytogenetic locations of cgh results. all genes and markers were annotated and hyperlinked against physical maps of genomes in ensembl. the up - regulated genes and gain / amplified chromosomal regions were displayed in red series color. in contrast, the down - regulated genes and loss / deleted chromosome regions were displayed in green series color. experimental validation of representative genes in the hcc gene set. for each gene, the semi - quantitative rt pcr results in 45 hcc pairs are presented in gel images (left), in quantified expression profiles after normalization with -actin expression as internal control (upper right) and in comparison with expression profiles of the gene in re - analyzed stanford hcc microarray data from expression view of oncodb.hcc (lower right). (a) aurka is a positive control for hcc data process ; (b and c) genes selected from criteria of significant expression difference in at lease three independent microarray / proteomic studies ; and (d f) genes selected with criteria of at least 2-fold expression difference in at least 70% of paired arrays in re - analyzed stanford hcc microarray data. stringent criteria were applied to select a set of 614 hcc genes with significant altered expression in hcc tissues (supplementary files in oncodb.hcc). among them, 446 genes were supported with more than two independent studies in terms of altered expression including 145 concordantly up - regulated, 176 concordantly down - regulated and 125 genes with mixed up-/down - regulated expression in hcc tissues. in addition, there were 234 genes with limited experimental data in hcc and 256 genes located within recurrent chromosome aberration regions. in addition to aurka, we further selected 30 out of 234 genes with limited wet - lab experiments in hcc for experimental validation. the validated results were demonstrated in terms of concordant expression of gene up- or down - regulation in 45 pairs of hcc tissues by rt pcr analysis and in comparison to 57 pairs of hcc samples of re - analyzed stanford hcc microarray data (figure 2 and supplementary files in oncodb.hcc). a near perfect concordant result except ckap2 (96.7%, 29/30 genes) in altered gene expression of hcc was obtained for 12 genes selected based on three independent microarray and/or proteomic reports and for 18 genes selected based on at least 2-fold expression changes within 70% patients in re - analyzed stanford hcc microarray data. to provide additional genetic support and refine the hcc loci for targeting cancer genes, the qtls of rodent hcc models identified via linkage studies were integrated into oncodb.hcc based on comparative maps of rodent genomes in ensembl. the results of 35 rodent hcc qtls were displayed according to the relative positions of human chromosomes (figure 1e). comparative mapping of hcc loci demonstrated that over 45% rodent qtls (8 of the 12 mouse hcc qtls and another 8 of the 23 rat hcc qtls) are located in the major aberrant loci of human hcc (table 1). among 16 syntenic qtls located in 12 hcc loci, 10 qtls in 6 loci are potentially located in gain / amplified regions and 6 qtls in 6 loci are located in loss / deleted regions. while the critical regions of hcc loci existed in comparative genomes of human and rodents, the hcc loci could be effectively narrowed down due to the scrambled structure of genomes by comparing human and rodent syntenic regions. we narrowed down two human hcc loci to 2 mb and another 6 hcc loci in between 4 and 10 mb. interestingly, the comparative mapping of hcc loci allowed us to split three human major hcc aberration regions 1q, 4q and 8p2123 into two smaller regions and to conclude that at least two putative cancer genes located on the same arm of above three human hcc chromosomes. hcc loci and putative cancer genes by comparative mapping strategy number of references with significant loh in the region defined by original authors. g : cgh gain ; l : cgh loss ; and a : cgh amplification. oncodb.hcc is the first attempt to establish a detail bioinformatic resource of one tumor genome by integrating genomic data of chromosome aberrations, altered gene expression, experimental data of genes in hcc tissues and qtls of rodent hcc models. three important advantages were revealed after data integration in oncodb.hcc : first, data integration from independent studies containing aberrant consequences from levels of dna, rna and protein could avoid possible pitfalls of data inconsistency from a single genomic approach and provide lines of evidence to conclude somatic aberrations. second, due to the heterogeneity nature of hcc tumorigenesis, successful gene validation in oncodb.hcc is critical for revealing significantly altered hcc genes for signatures of somatic aberrations in dissecting tumorigenic pathways and in clinical applications. finally, integrated genomic data in oncodb.hcc could narrow down and prioritize critical cancer genes and regions for positional cloning and molecular studies of cancer genes in hcc. the quality of integrated data in oncodb.hcc was experimentally supported by successful validation of altered expression in selected genes with limited wet - lab experimental studies in hcc. therefore, the open access oncodb.hcc should serve as a valuable resource for hcc research community. the oncodb.hcc is the first comprehensive integration of cancer genomic data in one prevalent cancer with experimental validation and available freely to the research community. the future perspectives for oncodb.hcc are to further integrate other newly emerging tumorigenic factors such as epigenetic modulations, point mutations and microrna alterations in genome - wide aspects. in addition, commercial available 300k or 500k high density snp chips are potentially useful to reveal high density novel genomic alterations in hcc genome. in conclusion, the comprehensive oncodb.hcc is an invaluable resource for better understanding the tumorigenic mechanisms and developing useful information in clinical applications. oncodb.hcc could serve as a bioinformatics resource that is applicable to other prevalent human cancers for dissecting tumorigenic pathways and the foundation of tumor systems biology. | the oncodb.hcc () is based on physical maps of rodent and human genomes containing quantitative trait loci of rodent hcc models and various human hcc somatic aberrations including chromosomal data from loss of heterozygosity and comparative genome hybridization analyses, altered expression of genes from microarray and proteomic studies, and finally experimental data of published hcc genes. comprehensive integration of hcc genomic aberration data avoids potential pitfalls of data inconsistency from single genomic approach and provides lines of evidence to reveal somatic aberrations from levels of dna, rna to protein. twenty - nine of 30 (96.7%) novel hcc genes with significant altered expressions in compared between tumor and adjacent normal tissues were validated by rt pcr in 45 pairs of hcc tissues and by matching expression profiles in 57 hcc patients of re - analyzed stanford hcc microarray data. comparative mapping of hcc loci in between human aberrant chromosomal regions and qtls of rodent hcc models revealed 12 syntenic hcc regions with 2 loci effectively narrowed down to 2 mb. together, oncodb.hcc graphically presents comprehensive hcc data integration, reveals important hcc genes and loci for positional cloning and functional studies, and discloses potential molecular targets for improving hcc diagnosis and therapy. |
total tobacco cessation is the only treatment that improves the symptoms and reduces the risk of amputation in buerger 's disease. surgical revascularization is often ineffective because the distal target vessels are often involved in this diffuse segmental disease. calcium channel blockers, anticoagulants, thrombolytics, prostaglandin analogs, sympathectomy, adrenalectomy, spinal cord stimulators, omental transfers, and stem cell treatment have all been tried with limited success in decreasing rest pain and avoiding amputation. a 28-year - old male had been suffering from acute right foot pain at rest and coldness with dysesthesia, cyanosis on the right 1st toe, and claudication for 3 weeks. the ankle - brachial pressure index (abi) was 0.6 on the right leg. as he was a heavy smoker, the patient 's hands and feet were usually cold, and the symptoms became severe when he was exposed to a cold environment. he was free of other diseases, including autoimmune disease, hypercoagulable state, and diabetes mellitus. computerized tomography (ct) angiography showed segmental obstructions of the below - knee popliteal artery, which showed intraluminal thrombosis, and in the posterior tibial artery. 1). we decided to operate because of the severity of the symptoms and the short segmental obstruction with thrombus. we performed endarterectomy and vein patch angioplasty of the obstructed popliteal artery with the greater saphenous vein. however, the arteriotomy site was closed with patch angioplasty because of the occurrence of a large amount of back bleeding. after the first operation, the patient 's symptoms did not improve and the patient requested reoperation to alleviate the symptoms. we performed vein patch angioplasty in the segmental obstruction of the posterior tibial artery 2 weeks after the first operation. after the operation, the patient 's symptoms improved and he was discharged on postoperative day 15. the lumen was near - totally or totally occluded by organized thrombi that presented as recanalized vessels. remarkable chronic inflammatory cells such as lymphocytes and plasma cells were observed in the recanalized vessel wall with less inflammation in the walls of the blood vessels. ct angiography showed diffuse posterior tibial artery obstructions in the patch angioplasty of the posterior tibial artery in the upper direction (fig. a pre - procedural angiogram showed severe stenosis in the upper part of the vein patch angioplasty of the posterior tibial artery. the possibility of stenosis and obstruction always exists in the blood vessels of patients with buerger 's disease due to inflammatory reactions. angioplasty of the posterior tibial artery was performed using a balloon catheter (4 - 40 mm, ever cross ; ev3 endovascular inc., angiography after this procedure demonstrated a good final angiographic result and the absence of critical stenosis. improved blood circulation was observed in the ct angiography performed after 1 year (fig. this patient quit smoking from before the first operation until the time of this report. for 2 months after the endovascular procedure, warfarin was used. from after the first operation until the present, aspirin and clopidogrel (plavix) have been used. buerger 's disease is defined as a nonatherosclerotic segmental disease that is characterized by occlusive, inflammatory, and thrombotic changes. several different criteria, such as those suggested by shionoya and olin, have been proposed for the diagnosis of buerger 's disease. typical angiographic findings were distal vessel disease with no presence of arterial wall calcification and the development of a rich typical subcutaneous network of collaterals, usually referred to as corkscrew collaterals. tobacco use, in any of its forms, plays a central role in the pathogenesis and progression of the condition. clinical presentation usually begins with ischemia of the distal small arteries and veins of the legs, arms, feet, and hands, which is manifested by claudication of the corresponding extremities. progression of the disease is typically characterized by calf claudication and, eventually, ischemic pain at rest. the prognosis for patients with buerger 's disease with respect to limb loss is significantly worse than that for patients with either atherosclerosis or the various forms of necrotizing immune arteritis. at present, the only proven strategy to prevent the progression of the disease and avoid amputation, in some cases, is the complete discontinuation of cigarette smoking or any other use of tobacco in any form. nevertheless, many patients continue to have a very poor quality of life owing to the persistence of symptoms such as intermittent claudication, raynaud 's phenomenon, or even amputations. the risk of amputation is highly correlated with continuing to smoke ; the amputation rate varies from 42% to 5% at the cleveland clinic foundation according to tobacco use. at the end of a 5-year follow - up, the median prevalence of amputation in patients with buerger 's disease, based on the most recently reported series, is reported to be as high as 24.4% for minor amputations and 8.6% for major amputations, for a total amputation rate of 33% for patients treated conservatively. surgical revascularization for patients with buerger 's disease is possible only in a few cases, as a result of diffuse distal involvement with no distal runoff vessels available for bypass surgery. consequently, surgical procedures are technically challenging, with very low feasibility and patency rates. endovascular treatment for buerger 's disease is not generally reported in the medical literature as either feasible or effective. graziani. reported that an endovascular procedure is safe, technically feasible, and effective, independent of the continued or non - use of tobacco, which leads us to argue that the procedure alone achieved these high rates of clinical success, the decreased rates of amputation, and the maintenance of clinical improvement during midterm follow - up periods. although the physiopathology of the disease is characterized by the presence of inflammation arteritis and associated thrombosis, this did not preclude or affect the results of the primary angioplasty of the treated lesions. in our case, the patient was suspected of having a subacute form of buerger 's disease on the basis of the microscopic findings. it seems that inflammatory changes in blood vessel obstruction were in progress after the patient underwent patch angioplasty of the segmental obstruction. we resolved this by endovascular intervention, and we have been observing the patient 's progress for about a year, but no strictures or other abnormalities have been found. finally, although tobacco cessation is one of the most important steps for a significant reduction in disease progression, we believe that an endovascular procedure can be an effective treatment, even in addition to more conservative and surgical management, in patients with buerger 's disease and critical limb ischemia. | surgical revascularization for patients with buerger 's disease is possible only in a few cases, due to the diffuse segmental involvement and the lack of distal runoff vessels available for bypass surgery. we encountered a case of resting pain in the right foot, coldness with dysesthesia, and cyanosis on the right 1st toe. the patient was treated with an endovascular intervention after vein patch angioplasty failed due to an inflammatory reaction of buerger 's disease. we suggest that an endovascular procedure can be an effective treatment, even in addition to more conservative and surgical management, in patients with buerger 's disease and critical limb ischemia. |
as a service to our authors and readers, this journal provides supporting information supplied by the authors. such materials are peer reviewed and may be re - organized for online delivery, but are not copy - edited or typeset. technical support issues arising from supporting information (other than missing files) should be addressed to the authors | we have investigated the interaction of peptides containing phosphohistidine analogues and their homologues with the prototypical phosphotyrosine binding sh2 domain from the eukaryotic cell signalling protein grb2 by using a combination of isothermal titration calorimetry and a fluorescence anisotropy competition assay. these investigations demonstrated that the triazole class of phosphohistidine analogues are capable of binding too, suggesting that phosphohistidine could potentially be detected by this class of proteins in vivo. |
diogenes syndrome is a rarely reported syndrome of extreme self - neglect, social withdrawal and domestic squalor. other synonyms for diogenes syndrome include severe self - neglect syndrome, aged recluse and social breakdown of the elderly. herein, we report a case of diogenes syndrome presenting with an unusual mask of dirt resembling a carapace. a 55-year - old lady with crusted plaques over head, neck and trunk since 3 months was brought to the hospital by her relatives. the onset of neglect coincided with the awareness of a lump in her right breast. there were adherent crusts over scalp, large dark brown confluent thick crusts forming a mask resembling a carapace over face and ears sparing the perioral area [figure 1a and b ]. dark brown dirt like papules were found on neck, lower back, intermammary and inframammary area. there was poor oral hygiene and halitosis.a 6 8 cm solitary, firm, non - tender, mobile mass was present in her right breast [figure 2 ]. (a and b) mask of dirt resembling a carapace complete blood counts, fasting blood sugar, liver and renal function tests were within normal limits. a skin biopsy revealed hyperkeratosis, increase in basal pigmentation and focal vacuolar changes [figure 3a ]. pas stain showed few budding fungal yeast forms in the stratum corneum [figure 3b ]. (a) hyperkeratosis with yeasts in stratum corneum (h and e 40). (b) pas - positive budding yeasts (pas 40) right breast lumpectomy specimen had a biphasic pattern composed of glands and cellular stroma on histopathological examination. the glands showed leaf - like projections due to underlying proliferation of cellular spindle - shaped stromal cells. three months after removal of the lump the patient returned for follow up with a clean face and renewed interest in social activities. the term diogenes syndrome was first coined by clark., in 1975, to describe elderly patients with severe self - neglect, poor personal and domestic hygiene. it is named after the cynical greek philosopher diogene de sinope who advocated minimizing needs and a natural way of life. nutritional deficiencies of iron, folate, vitamin b12, vitamin c, vitamin d and serum proteins may be seen. mental illness, withdrawal and denial of need in old age may be triggered by various stressful situations. the cutaneous manifestations of neglect (dermatitis passivata) arise from accretion of keratin and dirt and resemble a carapace. sparing of the perioral area could have resulted from her habit of wiping the perioral area after meals. vigorous rubbing with soap and water can cause complete clearance of lesions arising from neglect. terra firma forme dermatosis presents with dirt like patches which can be cleared with isopropyl alcohol but not soap and water. it can be differentiated by the absence of neglect and presence of adequate hygiene. though dermatitis passivata may be commonly seen in clinical practice, awareness about diogenes syndrome with its wider health and psychosocial implications is needed. our patient improved with the manual removal of crusts, psychiatric counseling and removal of the lump. our case is unusual in its mask of dirt resembling a carapace and the presence of a breast lump being a possible stressor. diogenes syndrome presenting with an unusual mask like carapace and responding to clinical and psychological interventions was seen. | diogenes syndrome is characterized by extreme self - neglect, social withdrawal, and poor personal and domestic hygiene. we report a case of diogenes syndrome presenting with dermatitis passivata. an unusual mask of dirt resembling a carapace, onset of neglect after awareness of a breast lump and resumption of personal grooming and social activities after removal of the lump and counseling were seen. |
emphysematous pyelonephritis (epn) is a severe necrotizing, gas - forming infection of the renal parenchyma and surrounding tissue. it commonly occurs in patients with uncontrolled diabetes mellitus or obstruction within the urinary tract. epn has been associated with a high mortality rate, usually due to sepsis, and has therefore been treated with early, aggressive therapy such as nephrectomy. with improvement of minimally invasive intervention strategies, antibiotics, and general supportive care, however, there has been a paradigm shift in the treatment of epn towards an approach that preserves renal function. spontaneous subcapsular hematoma (sch) has been reported in association with malignant and benign tumors, vascular disorders and approximately 7% of cases are associated with kidney infection. there have been a few reported cases of epn with spontaneous sch both locally and internationally,,,,. however, all of these cases initially presented with epn and had a subsequent diagnosis of sch. in this report, we describe a case of epn that initially presented as a spontaneous sch in a diabetic patient that was successfully treated with percutaneous drainage and antibiotics. a 57-year - old woman with a history of diabetes mellitus and hypertension presented to our emergency room with 2 days of sudden - onset left flank pain. she had no history of recent trauma, hematological abnormalities, liver disease, or kidney disease, with the exception of acute pyelonephritis (apn) that required hospitalization 4 years previously. her complete blood count and blood chemistry levels taken 6 months prior to this presentation were all within normal limits. her medication list included anti - diabetics (metformin 850 mg, glimepiride 2 mg, and vildagliptin 50 mg), antihypertensives (cilnidipine 10 mg), and acetaminophen (650 mg 3 times daily) for intermittent leg pain. her vital signs at the time of admission were as follows : blood pressure 96/52 mmhg, heart rate 118 beats / min, respiratory rate 28 breaths / min, and body temperature 37.3c. on physical examination, she was mentally alert, but appeared acutely ill. palpation of her abdomen revealed diffuse abdominal tenderness, especially on the left side, with severe left costovertebral angle tenderness. her laboratory data showed a white blood cell count of 23,010/l (82% neutrophils and 12% band - neutrophils), hemoglobin of 11.9 g / dl, platelet count of 76,000/l, high - sensitivity c - reactive protein of 29.89 mg / dl, urea nitrogen of 48.3 mg / dl, creatinine of 3.44 mg / dl, and prothrombin time (international normalized ratio) of 1.32. urinalysis demonstrated 1019 white blood cells and many red blood cells per high - power field. protein (2 +) and occult blood (4 +) were also detected, and her urine specific gravity was 1.02. spot urine chemistry showed a protein / creatinine ratio of 1.79 (g / g), and the fractional excretion of sodium was calculated to be 0.52%. a noncontrast computed tomography (ct) scan of the abdomen revealed a crescenteric high - density fluid collection of 2.2 cm in diameter surrounding the left kidney with extension into the infrarenal space. the patient was afebrile on presentation, although her initial laboratory results suggested the presence of urinary tract infection. therefore, blood and urine culture were obtained and empiric antibiotic treatment was initiated (intravenous ceftriaxone 2 g / d). additionally, she was given intravenous fluids to maintain her hydration status, as well as insulin for glucose control. a urologist was consulted regarding surgical management of her sch. however, because the patient s vital signs were stable after initial medical management, it was decided instead to observe her closely while treating conservatively with antibiotics, hydration, and bed rest. the patient developed a fever of 38.2c within 24 hours of admission, but it subsided within 1 day. on day 4 of admission, she continued to have pain in her left flank, left lower quadrant, and left upper quadrant. both urine and blood cultures revealed growth of extended - spectrum -lactamase (esbl) negative escherichia coli. additional laboratory studies showed a white blood cell count of 12,790/l, platelet count of 130,000/l, hemoglobin of 9.5 g / dl, and creatinine of 1.11 mg / dl. on hospital day 7, a repeat ct scan was performed with the use of contrast dye enhancement. newly noted air shadows, consistent with epn of the left kidney with parenchymal necrosis were observed (fig. 2). there was a subtle interval increase in the size of the sch of the left kidney and an interval increase in perirenal and infrarenal haziness and fluid collection. percutaneous drainage (pcd) of both the intrarenal air and perinephric hematoma was performed using an 8.5 fr pigtail catheter. although her clinical symptoms and laboratory values showed improvement, imaging studies showed progression to epn. therefore, the patient s antibiotics were changed to intravenous piperacillin / tazobactam (tazocin) 4.5 g every 8 hours. the initial pigtail drainage was dark and bloody in color, with approximately 150 ml of fluid collected on the 1 day. culture of the drainage fluid also revealed growth of esbl - negative e. coli, which was the same pathogen identified in the blood and urine cultures. a diagnosis of epn with sepsis due to esbl - negative e. coli and concomitant sch was made. the patient did not have any subsequent fever, her platelet count was within normal limits (405,000/l), and her left flank pain improved over time. a follow - up ct scan performed on hospital day 12 showed mildly decreased epn and a smaller hematoma on the side where the catheter had been placed, but mildly increased hematoma and air shadows on the side without drainage (fig. follow - up ct scan performed on hospital day 29 showed decreased air on the right anterior side but residual air on the posterior side (fig. we subsequently removed the anterior catheter and repositioned the posterior catheter so that the tip was located within the air pocket in the left posterior pole. the patient s clinical symptoms and laboratory data continued to show improvement and she was discharged home on hospital day 34 with oral antibiotics (ciprofloxacin 500 mg every 12 hours). at her outpatient follow - up appointment 11 days after discharge, almost no drainage was observed from the pigtail catheter. the catheter was removed, and follow - up ct scans showed almost no air pockets and markedly decreased hematoma. the patient was placed on oral antibiotics for 1 more month. spontaneous sch without a history of trauma or malignancy is rare, and sch due to infection accounts for 1 month, but resolved after 2 months treatment. you reported a case of apn with sch that was successfully treated with intravenous antibiotics alone, and kim and choi reported cases of apn with sch that were successfully treated with pcd and antibiotics. our patient also showed rapid improvement of clinical symptoms and decreased intrarenal air and hematoma with appropriate infection control and drainage. in conclusion, our case was a rare example of epn initially presenting as spontaneous sch that was successfully treated with pcd and antibiotic therapy. even though infection is a rare cause of sch, a high index of suspicion must be maintained as timely diagnosis and treatment may be crucial in preserving renal function in the patient. | emphysematous pyelonephritis (epn) is a life - threatening infection characterized by the formation of gas. complications of epn include septic shock, acute renal failure, and disseminated intravascular coagulation. spontaneous subcapsular hematoma (sch) has also been reported as a rare complication of epn, although there have been no reports to date of this occurring prior to the presentation of epn. we report a case of epn that initially presented as spontaneous sch. the patient was admitted for left flank pain, and initial computed tomography revealed sch without any air shadows. laboratory findings and clinical symptoms suggested the presence of urinary tract infection and the patient was started on antibiotics. fever developed 24 hours after admission. on follow - up computed tomography 7 days later, epn was newly observed, and a percutaneous drain was inserted. blood, urine, and drainage fluid cultures all revealed growth of extended - spectrum -lactamase - negative escherichia coli. |
inflammation in patients with end - stage renal disease (esrd) receiving hemodialysis (hd) is associated with malnutrition and cardiovascular diseases and resulted in poor clinical outcome. pentraxin 3 (ptx3) is a multifunctional soluble receptor that modulates the innate immunoinflammatory response and it belongs to the pentraxin - superfamily that includes c - reactive protein (crp) [2, 3 ]. plasma ptx3, similar to crp, is considered to be an inflammatory marker of endothelial dysfunction and is also linked to increasing cardiovascular mortality risk. however, because different cell types and organs produce ptx3 and crp [2, 3 ], ptx3 and crp may be involved in different pathophysiologic mechanisms. the role of ptx3 has been demonstrated through experiments using ptx3-deficient or ptx3-overexpressing mice, and ptx3 may exert tissue - protective and anti - inflammatory effects [3, 69 ]. however, the functional role of human ptx3 in vivo is still under discussion. boehme and colleagues first described that the ptx3 levels of hd patients with esrd are higher than healthy subjects or esrd patients without receiving hd. it was interesting that spontaneous production of ptx3 in whole - blood samples from hd patients was significantly higher than that in samples from healthy subjects. otherwise, the mechanism underlying the production and the pathophysiological role of ptx3 in hd patients has not been elucidated fully. to address this issue, we focused on not only baseline ptx3 levels with a single measurement but also annual change in ptx3 levels in patients receiving chronic dialysis, because regularly receiving hemodialysis and continuously uremic condition for many years modify vascular status depending on several factors such as inflammation and oxidative stress [10, 11 ]. here we show that the annual decline in ptx3, but not high - sensitive crp (hscrp), is a risk of the incidence of vascular access trouble that is a critical and specific complication for hd patients. this study is the first to focus on the annual change of pentraxins in a hd cohort. this study was approved by the ethics committee of namegata general hospital, namegata, ibaraki, japan, and nonhospitalized patients who regularly received hd were enrolled in this observational cohort study. exclusion criteria were the presence of clinical signs of acute infection, active vasculitis, active hepatitis, and hiv at the time of evaluation and willingness to participate in the study. of the 89 subjects initially enrolled in the study during 2011, 4 subjects died, and another 9 subjects did not complete exact one - year follow - up, primarily because they transferred to other hospitals, thus yielding a total of 76 patients who completed the study. clinical demographic data of the subjects at baseline are given in table 1. the patients received regular hd treatment 3 times / week and all of them have internal arteriovenous fistula (avf) or arteriovenous graft (avg) for hemodialysis. blood was drawn from the arterial needle before starting a hd session both in baseline year (july 2011) and in the next year (july 2012) with exact one - year interval. body mass index (bmi) was taken on a dialysis day immediately after a dialysis session. vascular access troubles were defined as the need for catheter intervention or reoperation to remedy occlusion or stenosis of avf or avg or as the replacement of a permanent vascular catheter due to the occlusion of avf or avg from baseline year (july 2011) to two years later (the end of 2013). the patient, who died or transferred to other hospitals after measurement in next year, was censored at the time of death or lost to follow - up. finally, the mean observed period in all subjects was 2.12 years. blood samples were placed in chilled tubes containing ethylenediaminetetraacetic acid (2 mg / ml) or no anticoagulants and centrifuged at 5500 g for 10 min at 4c ; the obtained plasma or serum, respectively, was stored at 80c until analysis. plasma concentrations of ptx3 were determined by using a commercial human ptx3 enzyme - linked immunosorbent assay (elisa) kit system (perseus proteomics, tokyo, japan). serum concentrations of hscrp were determined by using the nephelometric n - latex crp ii kit (siemens diagnostics, erlangen, germany). serum level of intact parathyroid hormone was determined by using elecsys immunoassay systems (roche diagnostics, basel, switzerland). 2-microglobulin was determined by latex coagulation analysis kit (eiken chemical, tokyo, japan). other biochemical parameters were evaluated by using an autochemical analyzer (ci 16200, toshiba, japan). comparisons between groups were appropriately assessed by the mann - whitney u test after normality test. cox - regression models were performed to assess the risk of incidence of vascular access troubles ; these models included age, sex, bmi, dialysis - periods, and presence of diabetes mellitus (dm)., we observed 45 male patients, 31 patients with dm, 45 patients with age over 65 years, 42 patients with dialysis - period over 5 years, and 44 patients with bmi less than 22 kg / m. we next examined differences in baseline levels of pentraxins (i.e., ptx3 and hscrp) among subpopulations based on sex, presence of dm, age, dialysis - period, or bmi. it was shown that there were higher levels of ptx3 in patients with dialysis - period less than 5 years or bmi over 22 kg / m than the others (table 2). however, levels of hscrp are comparable among the subpopulations divided by sex, presence of dm, age, dialysis - period, or bmi (table 2). because increased level of ptx3 would be dependent on length of dialysis - period as shown in table 2, we observed the annual changes in levels of ptx3 and hscrp from baseline to the next year in this study population (figure 1). the mean change was + 0.010 ng / ml / year or + 0.032 mg / dl / year in ptx3 or hscrp, respectively. to know the pathophysiological implications of annual change in pentraxin levels in hd patients, we examined whether pentraxins can predict the incidence of vascular events by utilizing cox - regression model. the number of patients with vascular access trouble during follow - up years (from 2011 to the end of 2013) was twenty. table 1 shows the characteristics of the patients with stratification by the absence or presence of vascular access events. the level of ptx3 in baseline year in the patients with vascular access troubles is comparable to that without vascular access troubles as well as levels of hscrp (figure 2(a)). interestingly, we observed the inverse trend for annual changes of parameters between hscrp and ptx3 which is suggesting that annual decline in ptx3 would be related to the incidence of vascular access troubles (figure 2(b)). consistent with this result, table 3 shows that annual decline in ptx3 is a significant risk for the incidence of vascular access troubles (hazard ratio ; 0.732 per + 1 ng / ml / year in ptx3, p = 0.039) by cox - regression model with adjustment for covariants including dialysis - period and bmi which is crucially involving the level of ptx3, while hscrp is not significant risk (hazard ratio ; 3.605 per + 1 mg / ml / year in hscrp, p = 0.597). ptx3 is produced by various types of cells and increases rapidly in response to primary local inflammation and innate immunity [8, 9, 12, 13 ]. in patients with chronic kidney disease and those with esrd receiving hd, increased, but not increasing, level of ptx3 is strongly predictive of poor clinical outcome and may be independent risk factors of mortality. the subjects in our study had higher level of ptx3 at baseline (mean sd ; 4.6 2.2 ng / ml) than that of healthy control (0.76 0.2 ng / ml) referred to in the previous observation. moreover, plasma level of ptx3, but not hscrp, in patients with longer dialysis - period over five years is higher than that in those with shorter dialysis - period (table 2). therefore, we expected that annual change of ptx3 levels while undergoing chronic dialysis has distinguishing role from that of hscrp. recently, notable results were described in ptx3-deficient murine experiments ; the elevation of ptx3 during cardiovascular diseases has recently been postulated to be a compensatory response to protect the body from inflammation [3, 7, 8 ]. human ptx3 prevents cells from becoming apoptotic by inhibiting the activation of factor h or by eliminating apoptotic cells quickly before they can secrete proinflammatory factors in vitro. in contrast, serum levels of crp are well recognized as reflection on the generation of proinflammatory cytokines in vivo, which cause malnutrition and cardiovascular diseases via several canonical pathways and result in poor prognosis. taken together, one explanation for high level of ptx3 in patients with long dialysis - period is that hd patients with higher ptx3 live longer because they avoid overproduction of inflammatory factors. although cross - sectional studies with a single measurement of pentraxins are available [4, 9 ], few previous cohort studies focus on the annual changes in ptx3 levels in patients receiving chronic dialysis. to address this complicated accumulation of knowledge about ptx3, a cross - sectional study with a single measurement of ptx3 might not be suitable to assess causality in the observed associations. therefore, we conducted a longitudinal cohort study in hd patients to know whether annual changes of pentraxin levels could be a risk of the incidence for vascular event in hd patients. consequently, we here show that annual decline in ptx3 is a risk of the incidence of vascular access trouble with adjustment for physical status represented by bmi that is well - known confounding factor for increased ptx3 [1517 ] (table 2). though the involvement of ptx3 in the patency of avf was previously mentioned only briefly, our study is the unique observation to support compensatory response of ptx3 to protect the body from vascular thrombotic event. ptx3 is produced by various stimuli including lipopolysaccharide and cytokines such as tumor necrosis factor and interleukin-1 [9, 12 ]. moreover, the promoter region of human ptx3 contains binding sites for the redox - sensitive transcription factors including nuclear factor b, which is critically involved in the regulation pathway of inflammatory mediators in innate immunity [13, 19 ]. increased oxidative stress occurs in hd patients and is dependent on many factors including aging, loss of residual renal function, uremic conditions, and receiving regular hd all of which were represented in our study cohort. though we could not determine what the major factor for annual change in ptx3 in this hd cohort was, it was speculated that excessive oxidative - stress would be one of the causes for annually changing ptx3. recently, some report suggested a crucial protective role of ptx3 in thrombotic diseases. in acute myocardial infraction, depletion of intracellular ptx3 in neutrophils correlates with increased plasma levels and with platelet - neutrophil aggregates in vivo. these phenomena can be explained as follows : ptx3 is released from neutrophils via several stimuli and binds to p - selectin on activated endothelial cells and activated circulating platelets and dampens their proinflammatory and prothrombotic actions, thus contributing to its cardioprotective effects in human. in contrast, several prospective studies show that hscrp levels are positively associated with the incidence of myocardial infraction, stroke, and venous thrombosis [23, 24 ]. in thrombophilic condition such as essential thrombocythemia and polycythemia, high rate of thrombotic event is observed in the high crp levels or low ptx3 levels. altogether, it is considered that ptx3 antagonizes the thrombotic function of crp likely through a reduction of vascular inflammation. in this research, by serial measurements of levels of ptx3, here we show that the annual decline in ptx3, but not in hscrp, is a risk of the incidence vascular access troubles that is a critical complication for hd patients. this is the noteworthy study to focus on the annual change in pentraxins in a hd cohort and to support the evidence for the function of ptx3 to protect bodies from vascular thrombotic events. | pentraxin 3 (ptx3), a multifunctional modulator of the innate immunoinflammatory response, is higher in patients undergoing hemodialysis than healthy control. our study focused on annual change in ptx3 levels in patients with chronic hemodialysis, because regularly undergoing hemodialysis for many years modifies vascular inflammatory status. to demonstrate whether annual change in ptx3 is associated with vascular events, we measured blood levels of pentraxins (ptx3 and high - sensitivity c - reactive protein (hscrp)) at baseline and in the next year in 76 hemodialysis patients and observed 20 patients with vascular access troubles during follow - up years. the annual decline in ptx3, but not hscrp, is a significant risk of the incidence of vascular access trouble that is a critical and specific complication for hemodialysis patients (hazard ratio ; 0.732 per + 1 ng / ml / year in ptx3, p = 0.039). this study is the first to focus on the annual change of pentraxins in a hemodialysis cohort. |
the safdgs family member recruitment and data collection procedures were reported previously (23). in brief, probands were mexican americans with t2 dm with low income, and all first-, second-, and third - degree relatives of probands were invited to participate in the study. a variety of metabolic, hemodynamic, anthropometric, and demographic variables were collected from about 700 individuals drawn from 39 large mexican american families. diabetes status was defined by the 1999 criteria of the world health organization (i.e., fasting glucose levels 126 mg / dl and/or 2-h glucose levels 200 mg / dl). of the individuals examined at safdgs, gfr values were estimated as described previously (5) by the two recently recalculated gfr prediction equations (24) : the recalculated simplified four - variable modification of diet in renal disease (gfr - mdrd) study equation (25) and the cockcroft - gault (gfr - cg) equation (26) adjusted for body surface area and corrected for the bias in the mdrd data. the quantitative trait values of triglycerides (tgl) and albumin - to - creatinine ratio were log transformed and used in the association analyses since their raw data were nonnormally distributed. the institutional review board of the university of texas health science center at san antonio approved all procedures, and all subjects gave informed consent. exons and 2-kb putative promoter region of irs1 gene were screened for dna sequence variants in 32 individuals, who contributed positively to the linkage, by direct sequencing of pcr products amplified from genomic dna isolated from peripheral blood leukocytes. sequencing was performed using abi prism big dye terminator cycle sequencing kit (applied biosystems [abi ]) and a capillary sequencer (model 3730xl ; abi). genotyping of all the snps was performed using the taqman assay (abi), which was carried out on a geneamp pcr system 9700 (abi), and fluorescent signals were detected on an abi prism 7700 sequence detector (abi). to assure accuracy of the genotyping, coded blind replicate samples from 50 subjects the genotypic data were checked for mendelian consistency using the program simwalk2 (27). we performed association analysis in our complex pedigree - based data using the measured genotype approach (mga) within the variance components (vc) analytical framework (28,29). the vc - based approach accounts for the nonindependence among family members. in this analytical technique, vcs are modeled as random effects (e.g., additive genetic effects and random environmental effects), whereas the effects of measured covariates such as age and sex are modeled as fixed effects on the trait mean. the marker genotypes were incorporated in the mean effects model as a measured covariate, assuming additivity of allelic effects (2830). the effect of this measured genotype (i.e., association parameter) together with other covariate effects and vcs were estimated by maximum likelihood techniques. for association analysis between genotypes and gfr, age and sex terms in addition to other covariates were used in consideration of both biological and/or statistical significance. for gfr - cg analysis, age, age, sex, diabetes, duration of diabetes, systolic blood pressure (sbp), and antihypertensive medications were used as covariates. for gfr - mdrd, age, sex, bmi, diabetes, duration of diabetes, sbp, and antihypertensive medications were used as covariates. the hypothesis of no association is tested by comparing the likelihood of a model in which the effect of the measured genotype is estimated with a model where the effect of the measured genotype was fixed at zero. twice the difference in the log - likelihoods of these models yields a test statistic that is asymptotically distributed, approximating a distribution with one degree of freedom. before mga was performed, the quantitative transmission disequilibrium test (31) was used to examine hidden population stratification. given the ld patterns among the examined 13 snps, we obtained a p value after adjusting for multiple testing using an effective number of snps calculated following the method of li and ji (32). for a statistical threshold of 0.05, the required experiment - wide significance threshold, using the effective number of snps (which was 7.66) to adjust for multiple testing was 0.00667. because the issue of correction for multiple testing is not straight forward with regard to multiple correlated traits, there were no additional attempts to correct for multiple testing because the corresponding models are not independent. the bayesian quantitative trait nucleotide (bqtn) analytical technique was used to analyze the irs1 snp and gfr data further, with the use of the program solar. this technique has been detailed elsewhere (33). given complete snp data for a gene, this statistical technique can be used to identify the sequence variants that are either potentially functional or that exhibit the highest disequilibria with such potential functional sites. the bqtn model is a simple extension of the classical variance component model, which aims to disentangle the genetic architecture of a quantitative trait. because a candidate gene may contain a number of snps that could generate several possible competing models of quantitative trait nucleotide (qtn) action, we used a bayesian model selection / model averaging approach to analyze the snp data simultaneously to estimate the probability that each snp has a direct effect on the phenotype (33,34). to determine whether the associated snp(s) found by the qtn analysis can account for our reported linkage of gfr at chromosome 2q3537, we combined the qtn analysis with our identity - by - descent based variance component linkage analysis. if a variant, or set of variants, in the irs1 gene contributes to the observed linkage signal, linkage analysis conditional on a fixed effect, mga of the polymorphism will yield a lod score near zero. alternatively, if the associated polymorphism is in less than complete ld with the true functional site, linkage analysis will yield a reduced, but nonzero lod score. the plasmids carrying irs1 gene with glycine at codon 972 (wild type) and arginine at codon 972 (mutant) were provided by dr. plasmids were transfected into human kidney mesangial cells using the fugene hd transfection reagent as recommended (roche diagnostics). cells (9095% confluence) were washed twice with pbs, and media were replaced with 0.45 ml of opti - mem i (invitrogen). precomplex of the plasmid dna (0.5 g / well) with fugene (0.5 l / well) in opti - mem was mixed (50 l / well) and incubated at room temperature for 5 min. dna and fugene complexes were added to each well and incubated at 37c with 5% co2. cells were then incubated for 6 h followed by addition of 0.5 ml of fresh media with 34% serum to a final concentration of 17%. after 48 h of transfection, cells were made quiescent by serum deprivation for 24 h, followed by treatment with insulin (100 m) for 0, 5, and 10 min. polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with the following antibodies : anti - irs1 (upstate), antiphospho - irs1 (tyr 941) (santa cruz), anti - akt, antiphospho - akt (thr 308) (cell signaling), and antiactin (sigma). exons and 2-kb putative promoter region of irs1 gene were screened for dna sequence variants in 32 individuals, who contributed positively to the linkage, by direct sequencing of pcr products amplified from genomic dna isolated from peripheral blood leukocytes. sequencing was performed using abi prism big dye terminator cycle sequencing kit (applied biosystems [abi ]) and a capillary sequencer (model 3730xl ; abi). genotyping of all the snps was performed using the taqman assay (abi), which was carried out on a geneamp pcr system 9700 (abi), and fluorescent signals were detected on an abi prism 7700 sequence detector (abi). to assure accuracy of the genotyping, coded blind replicate samples from 50 subjects the genotypic data were checked for mendelian consistency using the program simwalk2 (27). we performed association analysis in our complex pedigree - based data using the measured genotype approach (mga) within the variance components (vc) analytical framework (28,29). the vc - based approach accounts for the nonindependence among family members. in this analytical technique, vcs are modeled as random effects (e.g., additive genetic effects and random environmental effects), whereas the effects of measured covariates such as age and sex are modeled as fixed effects on the trait mean. the marker genotypes were incorporated in the mean effects model as a measured covariate, assuming additivity of allelic effects (2830). the effect of this measured genotype (i.e., association parameter) together with other covariate effects and vcs were estimated by maximum likelihood techniques. for association analysis between genotypes and gfr, age and sex terms in addition to other covariates were used in consideration of both biological and/or statistical significance. for gfr - cg analysis, age, age, sex, diabetes, duration of diabetes, systolic blood pressure (sbp), and antihypertensive medications were used as covariates. for gfr - mdrd, age, sex, bmi, diabetes, duration of diabetes, sbp, and antihypertensive medications were used as covariates. the hypothesis of no association is tested by comparing the likelihood of a model in which the effect of the measured genotype is estimated with a model where the effect of the measured genotype was fixed at zero. twice the difference in the log - likelihoods of these models yields a test statistic that is asymptotically distributed, approximating a distribution with one degree of freedom. before mga was performed, the quantitative transmission disequilibrium test (31) was used to examine hidden population stratification. given the ld patterns among the examined 13 snps, we obtained a p value after adjusting for multiple testing using an effective number of snps calculated following the method of li and ji (32). for a statistical threshold of 0.05, the required experiment - wide significance threshold, using the effective number of snps (which was 7.66) to adjust for multiple testing was 0.00667. because the issue of correction for multiple testing is not straight forward with regard to multiple correlated traits, there were no additional attempts to correct for multiple testing because the corresponding models are not independent. the bayesian quantitative trait nucleotide (bqtn) analytical technique was used to analyze the irs1 snp and gfr data further, with the use of the program solar. this technique has been detailed elsewhere (33). given complete snp data for a gene, this statistical technique can be used to identify the sequence variants that are either potentially functional or that exhibit the highest disequilibria with such potential functional sites. the bqtn model is a simple extension of the classical variance component model, which aims to disentangle the genetic architecture of a quantitative trait. because a candidate gene may contain a number of snps that could generate several possible competing models of quantitative trait nucleotide (qtn) action, we used a bayesian model selection / model averaging approach to analyze the snp data simultaneously to estimate the probability that each snp has a direct effect on the phenotype (33,34). to determine whether the associated snp(s) found by the qtn analysis can account for our reported linkage of gfr at chromosome 2q3537, we combined the qtn analysis with our identity - by - descent based variance component linkage analysis. if a variant, or set of variants, in the irs1 gene contributes to the observed linkage signal, linkage analysis conditional on a fixed effect, mga of the polymorphism will yield a lod score near zero. alternatively, if the associated polymorphism is in less than complete ld with the true functional site, linkage analysis will yield a reduced, but nonzero lod score. the plasmids carrying irs1 gene with glycine at codon 972 (wild type) and arginine at codon 972 (mutant) were provided by dr. plasmids were transfected into human kidney mesangial cells using the fugene hd transfection reagent as recommended (roche diagnostics). cells (9095% confluence) were washed twice with pbs, and media were replaced with 0.45 ml of opti - mem i (invitrogen). precomplex of the plasmid dna (0.5 g / well) with fugene (0.5 l / well) in opti - mem was mixed (50 l / well) and incubated at room temperature for 5 min. dna and fugene complexes were added to each well and incubated at 37c with 5% co2. cells were then incubated for 6 h followed by addition of 0.5 ml of fresh media with 34% serum to a final concentration of 17%. after 48 h of transfection, cells were made quiescent by serum deprivation for 24 h, followed by treatment with insulin (100 m) for 0, 5, and 10 min. polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with the following antibodies : anti - irs1 (upstate), antiphospho - irs1 (tyr 941) (santa cruz), anti - akt, antiphospho - akt (thr 308) (cell signaling), and antiactin (sigma). the clinical characteristics of the study participants are shown in table 1. of the genotyped individuals, the available phenotypic data for each phenotype varied from 610 subjects for total cholesterol to 670 subjects for age. in the total sample, 29, 28, and 14% of the subjects had hypertension, t2 dm, and albuminuria, respectively (table 1). however, the gfr data were available for only 453 subjects. of the individuals with gfr data, 29% had t2 dm. clinical characteristics of the genotyped safdgs participants used for the current study irs1 (nm_005544) is composed of two exons, and the full - length protein is encoded by exon-1 (fig. 1). to identify dna polymorphisms in irs1, we sequenced both exons and 2-kb putative promoter region of irs1 in 32 subjects who contributed positively to the linkage of gfr. in total, we identified 11 snps including seven in the coding region, and two each in the 3 utr and putative promoter regions (fig. five are synonymous (asp90asp, gly234gly, his400his, ala804ala, pro893pro) and two are nonsynonymous snps (pro512ala, gly(972)arg). in addition, we chose eight tagging snps (snps-1016, 19) from hapmap database for the analysis to comprehensively cover 59-kb - long region of intron 1 of irs1 (fig. all the 19 snps (11 identified by resequencing and 8 from the public database) shown in fig. 1 (snp-1 to snp-19) were genotyped in the entire data set (n = 670 ; 39 families) by taqman assay. schematic diagram of human irs1 (nm_005544) gene structure on 2q36 and the location of the snps identified and genotyped in safdgs. the exons of the irs1 are represented by solid box and the intron by a thin line. snps with the base change and their reference sequence (rs) numbers are indicated by the vertical arrow. based on the genotypic data, six were excluded from further analysis because one (snp-7) failed to be in hardy - weinberg equilibrium and the minor allele frequencies of five snps (snps-5, 6, 8, 17, and 18) were less than 0.5% (fig. 1). we estimated the pairwise ld (r) between all the 13 snps and found that the pairwise ld ranged from 0 to 1.0 (fig. 2, the relatively high pairwise lds (r > 0.8) were found for the following snps pairs : rs2234931-rs1801278 (r = 1.0), rs2288586-rs10498212 (r = 0.97), rs4675093-rs10205923 (r = 0.92), rs12052364-rs10181778 (r = 0.88), and rs12052364-rs11690232 (r = 0.84). snps are labeled on the y - axis, and the locations (bp) within the gene are shown on the x - axis. pairwise ld is estimated using r values and depicted in the figure by the color intensity of the shaded box, as shown in the legend. the diagonal represents a comparison of each snp against itself (i.e., r = 1.0). (a high - quality color representation of this figure is available in the online issue.) association analysis between genotypic and gfr data were performed using mga. the -coefficients and p values of the covariates used are given in the supplementary data. the location, allele frequencies, and association analyses of 13 snps are summarized in table 2. of the 13 snps examined for association, the g to a substitution at codon 972 changing the amino acids from glycine to arginine (gly(972)arg) with minor allele frequencies of about 4% was found to be significantly associated with gfr - cg (p = 0.0006). given the sample size of 431 for this analysis (table 2 and table 3), genetic effects that account for 2.9% of the total phenotypic variation in gfr - cg could be detected with 80% power with an experiment - wide p value 0.80) (fig. 1 and fig, 911, 13, and 14 resulted in the examination of 256 plausible additive gene action models ; the gly(972)arg variant provided strongest evidence for the observed association with gfr - cg, with estimated posterior probability of > 0.99 (data not shown). such a high posterior probability of effect is suggestive of a direct functional effect for this variant. as revealed by the mga, the mean gfr values by genotypic categories differed significantly (table 3). the carriers of arg972 variant had significantly decreased gfr - cg and gfr - mdrd values. to determine whether gly(972)arg could account for the initial linkage signal, we performed linkage analysis on chromosome 2 conditional on the measured genotype effect. because the sample size available (n = 431) for the gly(972)arg genotypic data was slightly smaller than the sample size (n = 453) used for the original linkage study (5), we repeated the linkage analysis of gfr - cg with genotype - by - diabetes interaction influences. as was done in our original linkage report (5), the lod score obtained from the linkage analysis was adjusted with 2df to 1df, denoted as lodc score. more details on the adjustment of 2df to 1df are provided in the supplementary data. the lodc obtained in the current study was 3.1 (n = 431) compared with the original report of 3.3 (n = 453). by inclusion of the gly(972)arg polymorphism in the model, the lodc score went down from 3.1 to 2.3 ; thus, about 26% of the evidence for linkage of gfr at 2q was explained by this variant (fig. these data suggest that the gly(972)arg contribute to the decrease in gfr and that gly(972)arg may be one of the several potential functional sites regulating gfr or it is in ld with the true functional variant(s) in irs1 or genes flanking irs1. linkage analysis conditional on the gly(972)arg polymorphism on chromosome 2q. to further determine the functional mechanism by which arg972 of variant of irs1 may contribute to variation in gfr, we transfected the human renal mesangial cells with the expression vector pcdna3 carrying wild - type (gly972) and mutant (arg972) irs1 (35) and studied their effect on the insulin - induced tyrosine (941) phosphorylation of irs1 and threonine (308) phosphorylation of akt kinase, a critical downstream effector of pi 3-kinase. transfected cells were treated with 100 nmol / l insulin for 0, 5, and 10 min and lysed. equal amount of total proteins were subjected to sds - page and probed with the following antibodies : anti - irs1, antiphospho - irs1, anti - akt, antiphospho - akt, and actin. in cells expressing arg972 variant of irs1, insulin - stimulated phosphorylation of irs1 was significantly decreased by 57.3% at 5 min and by 65.8% at 10 min of insulin treatment compared with cells expressing gly972 variant of irs1 (fig. 4a). compared with cells expressing gly972 variant of irs, insulin - induced thr308-akt phosphorylation was also significantly reduced in cells expressing arg972 variant of irs1 by 75.9% at 5 min and by 88% at 10 min of insulin treatment (fig. 4b). effect of arg972 on the insulin - induced phosphorylation of irs1 and akt in human mesangial cells. human mesangial cells were transfected with plasmids carrying wild type (wt ; gly972) and mutant (mut ; arg972) irs1 and treated with 100 nm insulin for 0, 5, and 10 min. equal amount of total cellular proteins were separated on sds - page gel and probed with anti - irs1 and antiphospho - irs1. human mesangial cells were transfected with plasmids carrying wild - type (gly972) and mutant (arg972) irs1 and treated with 100 nm insulin for 0, 5, and 10 min. equal amount of total cellular proteins were immunoprecipitated with anti - akt kinase and separated on sds - page gel and probed with anti - akt and antiphospho - akt kinase. association analysis was also extended to available phenotypic data including sbp, diastolic blood pressure (dbp), tgl, hdl - cholesterol (hdl - c), albumin - to - creatinine ratio, and t2 dm. it is noteworthy that the two variants [gly(972)arg and gly(234)gly ] that exhibited significant associations with the gfr measures were also found to be significantly associated with tgl (p = 0.0033) (table 2). in addition, a promoter variant of irs1 (snp-2 ; rs6436635) with minor allele frequency of 10.1% was significantly associated with tgl (p = 0.0328), sbp (p = 0.0007), and dbp (p = 0.0023) (table 2). in addition, a promoter variant (snp-1) with a minor allele frequency of 2.6% and a synonymous variant (snp-3 ; asp90asp) with minor allele frequency of 3.8% also showed statistically significant association with hdl - c (p = 0.0444 and p = 0.0386, respectively). a pair of intronic region snp (rs4675093-rs10205923), which were in strong ld (r = 0.92), also exhibited significant association with sbp (p = 0.0025 and 0.0085, respectively). there is a large interindividual variation in gfr in patients with diabetes and in the general population. it is known that variation in gfr is influenced by environmental and genetic factors as well as their interactive influences (211). in an effort to identify and characterize gfr susceptibility gene(s), we localized a genetic region on chromosome 2q3537 that exhibited linkage to gfr (5). as we report in this study, this locus, which does not confer susceptibility to t2 dm (5), exerts a differential influence on gfr in diabetic and nondiabetic individuals. of the positional candidate genes, irs1 is a strong susceptibility candidate gene to influence gfr since the irs1 protein plays a pivotal role in modulating tissue response to insulin including in the kidney. a structural defect in this protein as a result of genetic variation may alter kidney function. to date, several dna sequence variants have been identified in human irs1. of those, the main focus has been on gly(972)arg variant, since almind. (36) first identified this polymorphism to contribute to t2 dm in a danish cohort. subsequent case control studies in different ethnic groups have examined the association between the gly(972)arg variant of irs1 and t2 dm and related traits. although the association results have been controversial, a meta - analysis combining 27 studies, which included 3,408 cases and 5,419 control subjects, suggested that the carriers of the gly(972)arg variant have a 25% increased risk for developing t2 dm (17). in addition, a marked ethnic difference in the distribution of gly(972)arg variant was also observed. the arg972 variant of irs1 was found to be more common in caucasians (20%) (37) compared with african americans (11%) (38), asians (4%) (39), and mexican americans (4%) (40). in this study, we observed a frequency of about 4% for arg972, which is similar to the one reported in another mexican american population (40). similarly, gly(972)arg failed to show an association with t2 dm as reported (40). of the 13 snps examined for association with gfr, only the gly(972)arg variant of irs1 exhibited significant association with gfr estimated by two formulae. the bqtn analysis using information from eight nonredundant snps also demonstrated that the gly(972)arg variant could influence phenotypic variation in the levels of gfr with estimated posterior probability of > 0.99. this high posterior probability effect reflects the potential functional impact of this variant on gfr. the subsequent conditional linkage / qtn analysis revealed that the gly(972)arg variant explained 26% of the linkage signal for gfr on 2q. taken together, our data demonstrate that the gly(972)arg variant significantly contributes to decrease in gfr. several studies have demonstrated that expression of the 972arg variant of irs1 in cultured adipocytes, muscle, and pancreatic b cells caused a defect not only in tyr-941 phosphorylation of irs1 but also in binding of the p85 regulatory subunit of pi3k to the irs1, resulting in a decrease in irs1-associated pi3k activity and subsequent activation of the ser / thr kinase akt (19,35,4144). 20) generated transgenic mice overexpressing arg972 and confirmed a significant attenuation in insulin signaling pathways downstream of irs1 in the liver, skeletal muscle, and adipose tissue. in the current study, we studied the effect of arg972 on the insulin - stimulated tyrosine phosphorylation of irs1 and threonine phosphorylation of akt in human renal mesangial cells. we show that expression of arg972 in mesangial cells significantly reduces insulin - induced phosphorylation of irs1 and akt kinase compared with wild - type controls. impaired ir / irs1 signaling in mesangial cells provides a potential mechanism by which arg972 variant of irs1 contributes to decreased gfr. alterations in the contractile property of mesangial cells as a result of impaired ir / irs1 signaling by arg972 variant may lead to alterations in glomerular hemodynamics, thereby influencing gfr. because insulin has been shown to be a survival factor for mesangial cells (45), impaired insulin signaling by the arg972 variant may lead to apoptosis of these cells, thereby contributing to structural changes in the glomerulus resulting in a decrease in surface area available for filtration. functional studies are necessary to prove the hypothesis that the arg972 variant of irs1 not only impairs ir signaling to protect mesangial cells against apoptosis but also impairs contractile function of mesangial cells. impaired nitric oxide (no) production in vascular endothelial cells by the arg972 variant may also contribute to the decreased gfr. human umbilical vein endothelial cells isolated from carriers of the arg972 variant of irs1 have impaired enos expression in response to insulin (46). because the impaired ir / irs1 signaling is a potential mechanism for the observed association, we also performed association analysis for the 13 snps using fasting glucose data (n = 541) after excluding t2 dm individuals that are on antidiabetic medications. although the gly(972)arg variant failed to show an association, the promoter variant (rs6436635) was found to be nominally associated (p = 0.035) with glucose levels. the present data also show an association of gly(972)arg variant with tgl in our cohort, and the carriers of arg972 had decreased tgl values. our finding of lower tgl concentrations in carriers of arg972 contradicts previous observations in other populations at high risk to develop atherosclerotic cardiovascular disease (47,48). however, our findings are in agreement with others who reported that patients with t2 dm (49) and patients with insulin resistance (50) have low concentrations of tgl. the functional significance of promoter variants, asp90asp, and a pair of intronic variants (rs4675093 and rs10205923) that exhibit an association with cardiovascular risk factors such as hdl - c, tgl, sbp, and dbp in this study remains to be determined. despite several strengths, our study has potential limitations. to our knowledge, the two estimates of gfr (gfr - cg and gfr - mdrd) have not been validated in the mexican american population. whereas the time - consuming and expensive direct measurement of gfr is difficult to perform (e.g., inulin, iothalamate clearance) in large family studies, the estimated gfr using the abbreviated mdrd and gc formulae has been proposed as the best validated means for transforming serum creatinine measurements into gfr (25,51) and used in several linkage studies (211). another limitation is that because our resequencing effort was based on only 32 individuals, it is possible that we might have missed rare variants specific to this population. however, given the recent emphasis on the role of rare variants in complex disease causation compared with the common variants examined using the genome - wide association studies, it is very interesting to note that the gly(972)arg associated with decline in gfr values has a frequency of 4.0%. we tried to replicate the association between gly(972)arg variant of irs1 and gfr in two independent family studies : the san antonio family heart study (mexican americans, n = 844) and the joslin study on the genetics of type 2 diabetes (caucasians, n = 825)., we provide evidence for the first time that arg972 variant of irs1 is associated with variation in gfr ; carriers of arg972 had significantly decreased gfr values and arg972 variant accounts for 26% of the linkage of gfr on 2q37. it is noteworthy that we show that expression of arg972 in human mesangial cells reduces the insulin - stimulated phosphorylation of irs1 and akt kinase. together, it is possible to speculate that the associated variant is one of multiple potential functional variants influencing variation in gfr in the irs1 gene or is in ld with a potential functional variant(s) in the genes flanking irs1. there is evidence for long - range ld in the human genome (52). however, in the absence of knowledge on such long - range ld of the snp of interest in this study, it is important to note that genetic variants located 500 kb upstream of irs1 were recently shown to be strongly associated with impaired metabolic profiles including insulin resistance and t2 dm. therefore, a thorough screening of the critical region on 2q37 may identify additional variants that could potentially relate to the gfr linkage finding in this study. identification of susceptibility genes that contribute to the decline in gfr may facilitate prediction, prevention, and development of improved treatments of this devastating complication of diabetes. | the objective of this study is to identify and characterize the genetic variants related to the glomerular filtration rate (gfr) linkage on 2q37. of the positional candidate genes, we selected irs1 and resequenced its 2-kb promoter region and exons for sequence variants in 32 subjects. a total of 11 single nucleotide polymorphisms (snps) were identified. to comprehensively cover the 59-kb - long intron-1, eight additional tagging snps were selected from the hapmap. all the 19 snps were genotyped by taqman assay in the entire data set (n = 670 ; 39 families). association analyses between the snps and gfr and type 2 diabetes related traits were performed using the measured genotype approach. of the snps examined for association, only the gly(972)arg variant of irs1 exhibited a significant association with gfr (p = 0.0006) and serum triglycerides levels (p = 0.003), after accounting for trait - specific covariate effects. carriers of arg972 had significantly decreased gfr values. gly(972)arg contributed to 26% of the linkage signal on 2q. expression of irs1 mutant arg972 in human mesangial cells significantly reduced the insulin - stimulated phosphorylation of irs1 and akt kinase. taken together, the data provide the first evidence that genetic variation in irs1 may influence variation in gfr probably through impaired insulin receptor signaling. |
for the control of diverse signaling in response to the extracellular milieu, cells develop sophisticated tools to transmit the appropriate signals and thereby orchestrate the responses. the signaling mechanism of an agent involves effector - receptor coupling, production of second messengers, activation of protein kinases, and distribution of these transducers to specific intracellular targets. mitogen - activated protein kinases (mapks) are the serine / threonine kinase family of conserved enzymes that are considered to be the central building blocks in the intracellular signaling networks [15 ]. there are more than a dozen of mapks known in mammals, and these enzymes exist in several isoforms (table 1). mammals express four distinctly regulated groups of mapks, that is, extracellular signal - regulated kinase 1/2 (erk1/2), p38, c - jun n - terminal kinases / stress - activated protein kinases (jnk / sapks), and erk5/big mapk (bmk1) [2, 3, 6 ]. each of these four mapk cascades consist of at least three tiers of protein kinases that are consecutively activated by phosphorylation events : mapk kinase kinase (mapkkk or map3k or mekk) activates mapk kinase (mapkk (mkk or mek) or map2k), which in turn activates mapk. the latter phosphorylates a large array of substrates, including mapk - activating protein kinases (mapkapks) [79 ]. even so, the different tiers are composed of many similar isoforms that can be activated by more than one mapk, increasing the complexity and diversity of mapk signaling. substrates, regulation, and function of different mapks have been discussed in many review articles [1, 2, 4, 5, 911 ], and will not be elaborated upon in great detail here. mapks are important signal transducing enzymes that are involved in transmitting signals from a wide variety of extracellular stimuli including those of growth factors, hormones, cytokines, and neurotransmitters. in fact, mapks are major components of signaling pathways regulating a large array of intracellular events, such as proliferation, differentiation, development, acute signaling in response to hormones, stress response, programmed cell death, gene expression, and steroidogenesis [1, 2, 1214 ]. noteworthy, however, the role of the mapk signaling pathways in steroidogenesis is poorly understood as a consequence of conflicting reports demonstrating stimulation, inhibition, or no effect in different steroidogenic cells [12, 1518 ]. the steroidogenic acute regulatory protein (star) mediates the rate - limiting and regulated step in steroid hormone biosynthesis, that is, the transfer of the substrate for all steroid hormones, cholesterol, from the outer to the inner mitochondrial membrane [1925 ]. as such, this protein plays a crucial role in the regulation of steroid hormones required for life itself, in the case of adrenal steroids, and for maintaining normal reproductive function, in the case of gonadal steroids. star is a rapidly synthesized, labile phosphoprotein, whose expression, activation and extinction are regulated by pka, pkc, and a host of other signaling pathways (reviewed in [23, 2527 ]). the star protein is localized to the mitochondria and consists of several forms of a newly synthesized 30-kda protein, which has a 37 kda precursor form containing an n - terminal mitochondrial targeting sequence [28, 29 ]. star is primarily associated with steroid producing tissues, and studies have demonstrated a tight correlation between the synthesis of star proteins and the synthesis of steroids. the compelling evidence for the critical role star in the regulation of steroidogenesis has been illustrated by the targeted disruption of the star gene and by the study of patients suffering from lipoid congenital adrenal hyperplasia, in which both adrenal and gonadal steroid biosyntheses are severely impaired due to mutations in the star gene [3033 ]. in the adrenal and gonads, camp mediated mechanisms predominantly regulate expression of the star protein and steroid synthesis that involve transcriptional and translational induction. conversely, transcriptional and/or translational inhibition of star expression results in a dramatic decrease in steroid biosynthesis, with the exception of approximately 10%15% of steroid production that appears to occur through star - independent events [3438 ]. it should be noted that phosphorylation of star is required to produce optimal cholesterol transferring ability of the star protein in steroid biosynthesis. two putative pka phosphorylation sites at serine 56/57 and serine194/195 have been identified, in murine and human star respectively, and mutations in these sites demonstrated the functional importance of the latter in the biological activity of star [39, 40 ]. while the camp / pka pathway is unquestionably the major signaling pathway for trophic hormone - stimulated star expression and steroid biosynthesis, it has been well established that the mapk signaling cascades play important roles in regulating these processes. the purpose of this paper is to summarize the findings of a number of laboratories, including our own, that have examined the roles of four distinct mammalian mapk cascades in regulating steroidogenesis, and where evidence exists, on star expression, in steroidogenic cells. the mapk signaling cascade involves activations of several transmembrane signaling molecules, cytoplasmic protein kinases, and a network of interacting proteins, and these, in turn, regulate almost all cellular processes, from gene expression to cell death [1, 3, 7, 14, 41, 42 ]. each of these cascades consists of several tiers of protein kinases that sequentially activate each other upon phosphorylation. activation of each of the mapk pathways is mediated by small gtp - binding proteins (e.g., ras, rap, or other oncoproteins), or by scaffold proteins or by adaptor molecules [41, 43, 44 ]. these events allow the transmission of extracellular signals to appropriate intracellular targets either directly or through three - tiered kinase modules [8, 10, 41 ]. mapks are activated by phosphorylation cascades that contain at least two upstream protein kinases. in all mapk cascades, the kinase immediately upstreaming of the map kinase is a member of the map / erk kinase (mek or mkk) family. these are dual - specificity enzymes that are capable of phosphorylating serine / threonine and tyrosine residues in their map kinase substrates in order to activate the protein 's activity [8, 4547 ]. the meks are also activated by phosphorylation on serine or threonine residues in their activation loops. there are several and diverse mek kinases (mekks) that activate meks [4, 9 ]. in contrast, the activity of mapks is negatively regulated by mapk phosphatases, a group of dual - specificity phosphatases that remove phosphate from serine / threonine or tyrosine residues and thereby inactivate mapks for controlling signals [4850 ]. the mapk signaling pathway has long been implicated in the regulation of cell cycle progression, is used as a biochemical marker in evaluating the mitogenic effects of a variety of stimuli, and is a point of convergence for diverse signaling pathways [12, 15, 16, 51, 52 ]. in mammals, four distinct mapk cascades (erk1/2, p38, jnk / sapk, and erk5/bmk1) are primarily activated by specific mapkks, that is, mek1 and mek2 for erk1/2, mkk3/6 for p38, mkk4/7 for jnk / sapk, and mek5 for erk5/bmk1 [1, 2, 41 ]. nevertheless, each mapkk can be induced by many mapkkks, and presumably each mapkkk confers responsiveness to discrete signals [9, 53 ]. the differential expression, activation, and substrate specificity of these mapks suggest their varying physiological functions in different cellular contexts. a substantial body of evidence indicates that mapk signaling cascades are activated by a large number of extracellular signals and play pivotal roles in regulating the steroidogenic response. as such, a variety of exogenous stimuli (e.g., hormones, growth factors, and cytokines) that influence different mapk cascades exhibit diverse effects on star expression and steroidogenesis in different steroidogenic cell models (figure 1 ; [12, 1517, 54, 55 ]). the erk1/2 signaling cascade is the most widely studied member of the mapks. erk1 (p44 mapk) and erk2 (p42 mapk) are highly conserved throughout eukaryotic cells and bring together transmembrane receptors and a network of various proteins and integrating signals to control many cellular processes, including cell survival, differentiation, tumor progression, and invasion [4, 7, 5658 ]. there are also alternatively spliced forms of erks, namely, erk1b (46 kda), erk1c (42 kda), and erk2b (41 kda). both erk1 and erk2 are activated by numerous extracellular stimuli, including growth factors, cytokines, transforming agents, and ligands that act via g protein - coupled receptors (gpcrs) and tyrosine kinase receptors. the two phosphoacceptor sites, tyrosine and threonine, in the activation loop (tey) are phosphorylated in order to activate the erk1/2 signaling cascade, which occurs exclusively through mek1 and mek2. erks also phosphorylate several substrates (e.g., ribosomal s6 kinase (rsk), the mapk / sapk - activated kinase (msk)) either in the cytosol (e.g., pla2) or in the nucleus (e.g., elk1) [1, 4, 9, 11 ]. however, this activation requires adaptor proteins that are linked to the guanine exchange factors (gefs) of gtp - binding proteins. following stimulation, recruitment of the adaptor protein - gef complex to the plasma membrane results in the induction of ras or related molecules, which then transmit signals to the map3k level of the cascade (raf-1, b - raf, a - raf, tlp-2, and mekk1) [1, 9, 59 ]. generally, the three - tiered system of the erk1/2 signaling cascade is rather complex and involves many mekks that are capable of phosphorylating a number of meks and thereby activating several map kinases and have been discussed elsewhere [1, 2, 4, 11, 41 ]. this signaling pathway is involved in regulating star expression and steroid biosynthesis, but several seeming contradictions have been reported in different steroidogenic tissues [12, 1517, 54, 6062 ]. for example, it has been demonstrated that activation of erk1/2 by hcg increases star expression and testosterone production while inhibition of erk1/2 by u0126 results in decreases in these parameters in hcg - stimulated primary cultures of immature rat leydig cells. in contrast, erk1/2 inhibition enhances star expression as induced by lh, insulin - like growth factor-1 (igf-1), transforming growth factor-, and interleukin-1 (il-1) but decreases steroid levels [2, 12, 52, 62, 63 ]. it has been reported that steroid biosynthesis induced by hcg and fibroblast growth factor-9 (fgf-9) is mediated through the involvement of ras - mapk and pka signaling in mouse leydig cells [64, 65 ]. we have demonstrated that the erk1/2 signaling cascade plays key roles in pka - mediated (activated by (bu)2camp and hcg)- and pkc - mediated (activated by pma) regulation of steroidogenesis [12, 52, 66 ]. all these agents activate erk1/2 ; however, they have diverse effects on star expression and steroid synthesis in conjunction with erk1/2 inhibition by either pd98059 or u0126. in particular, whereas the inhibition of erk1/2 increases camp / hcg - stimulated star expression, it decreases pma - mediated star levels. was not associated with attenuation of the cytochrome p450 side chain cleavage (p450scc) and 3-hydroxysteroid dehydrogenase (3-hsd) enzyme activities, as erk1/2 inhibition had no effect on 22r - hydroxycholesterol - mediated steroid synthesis. noteworthy, the increases in pma, (bu)2camp, and hcg - induced star expression and progesterone levels were inversely correlated with the levels of a negative transcription factor, namely, dosage - sensitive sex reversal, adrenal hypoplasia congenita, critical region on the x - chromosome, gene 1 (dax-1) [12, 52, 66, 67 ]. while u0126 attenuates dax-1 expression, this inhibition can be reversed by pma but not by (bu)2camp / hcg [12, 66 ]. in fact, the differential effects of u0126 on pka- and pkc - mediated steroidogenesis is mediated, at least in part, by alterations in dax-1 expression in mouse leydig cells. furthermore, studies have shown that both (bu)2camp and pma can elevate the levels of scavenger receptor class b type 1 (sr - b1, a high - density lipoprotein (hdl) receptor that binds various lipoprotein particles and facilitates cellular cholesterol influx) and that erk1/2 inhibition decreases sr - b1 expression. the binding of hdl to sr - b1 has been reported to stimulate the erk1/2 pathway following ras activation. sr - bi is involved in importing cholesterol into cells that will eventually be provided to the mitochondria for steroid biosynthesis [52, 69 ]. thus it is plausible that the decrease in steroid synthesis caused by u0126 (during conditions of elevated star) is due to a reduction in cholesterol availability to the mitochondria. recent studies have shown that activation of erk1/2 at the mitochondria plays an important role in leydig cell steroidogenesis [70, 71 ]. specifically, erk1/2 is capable of phosphorylating star at serine 232 in the presence of cholesterol, demonstrating that star is a substrate for erk1/2 and that a mitochondrial kinase complex is essential for maximum cholesterol transferring capacity of the star protein in steroid synthesis. in granulosa cell - derived rlhr-4 and rfshr-17 cell lines, treatments with lh / hcg and fsh, respectively, increase erk1/2 phosphorylation, star expression, and progesterone synthesis [16, 17, 51, 72 ]. conversely, gonadotropin - stimulated star and steroid levels have been shown to be further augmented following inhibition of erk1/2 both by pd98059 and u0126, suggesting that the induction of lh / hcg- and fsh - induced steroidogenesis involves downregulation of the steroidogenic machinery including the erk1/2 cascade. studies have also shown that while pd98059 and u0126 have no effects on lh / hcg, insulin, or igf-1-induced steroidogenesis, they decrease (bu)2camp, cholera toxin and forskolin stimulated progesterone production [54, 60 ]. further, it has been reported that fgf-8 mediated erk1/2 activity is associated with an attenuation of estradiol production in rat granulosa cells. also, erk1/2 activation results in differential effects on lh - induced star and cyp17 expression in bovine theca cells. activation results in an increase in star and a decrease in cyp17 that in turn results in an elevation in progesterone and a reduction in androsterone levels, processes in which dax-1 and steroidogenic factor 1 (sf-1) play pivotal roles. likewise, administration of hcg in vivo or treatment with gonadotropins / camp in vitro is associated with down - regulation of cyp19a1 and upregulation of star and cyp11a1 mrna expression in both mural and cumulus granulosa cells of mouse preovulatory follicles. these events have been shown to be tightly regulated with steroid biosynthesis and involve erk1/2-dependent signaling. recently, the activation of erk1/2, as well as other mapk signaling, by leptin, has been linked to decreases in basal and camp / pka stimulated star expression and progesterone synthesis without affecting the levels of the p450scc and 3-hsd enzymes. additionally, treatment with metformin (a drug that is widely used for treating infertility in women with polycystic ovary syndrome (pcos)) causes an activation of erk1/2 that results in suppression of insulin - stimulated p450 aromatase mrna expression and activity. in theca cells isolated from pcos patients, increases in cyp17 expression and androgen biosynthesis are connected to an attenuation of erk1/2/mek1/2 activity by pd98059, suggesting the involvement of mapk signaling in the pathogenesis of this disease. prostaglandin f2 (pgf2), an agent that influences luteal regression through the induction of the phospholipase c / diacylglycerol / pkc pathway, induces phosphorylation of erk1/2, which, in contrast, decreases basal and lh / hcg - stimulated star expression and progesterone synthesis in human granulosa - luteal cells. these effects of pgf2 in the steroidogenic response can be reversed following inhibition of mek activity by pd98059. moreover, the inhibitory effect of pgf2 on star expression and steroidogenesis, in rat luteal cells, is modulated by the negatively acting transcription factors, dax-1 and ying yang 1 [18, 79 ]. these results demonstrate the multiple effects of the erk1/2 signaling cascade in star expression and steroidogenesis in gonadal cells. in mouse y-1 adrenocortical tumor cells the induction of forskolin - mediated corticosterone synthesis is dependent upon the activation of erk1/2 and is correlated with phosphorylation of sf-1 and increased star gene transcription. accordingly, inhibition of erk1/2, by either pd98059 or u0126, decreases star expression and steroid production without affecting p450scc enzyme activity. increasing evidence demonstrates that acth and angiotensin ii (ang ii) can elevate phosphorylation of p44/p42 mapks and result in increases in star expression and steroid synthesis that involve pkc signaling and ras / raf-1 kinase [8083 ]. the induction of orexins a and b (neuropeptide hormones) mediated star expression and steroid synthesis is mediated by a number of mapk cascades including ekr1/2 activation, where the latter requires multiple g - protein signaling pathways in human h295r adrenocortical cells [55, 84 ]. it has been demonstrated that while proopiomelanocortin (pomc) fragments, 1 - 28-pomc and 1 - 48-pomc, modulate cellular proliferation, they decrease adrenal steroidogenesis by activating erk1/2 signaling. chronic administration of acth induces the phosphorylation of erk1/2 that occurs in parallel with adrenal corticosterone synthesis in adult rats. in both h295r and primary cultures of human adrenocortical cells, treatment with adipokines up - regulates the erk1/2 cascade and results in increases in star expression and aldosterone synthesis, processes that do not require camp / pka signaling [86, 87 ]. taken together, it seems clear that the erk1/2 signaling cascade plays diverse roles in regulating star expression and steroidogenesis. these roles could be a result of the existence of multiple signal transduction pathways that display differences in receptor - effector coupling between tissues and species. the p38 mapk signaling cascade is thought to participate in the response of cells to stress. four members of the p38 mapk family have been cloned and named p38 (mapk14), p38 (p38 - 2), p38 (erk6 or sapk3), and p38 (sapk4) and share approximately 60% homology in their amino acid sequences [10, 8891 ]. also, several alternatively spliced isoforms of p38 mapk include mxi2 (identical to p38) and exip. p38 mapks contain a thr - gly - tyr activation loop sequence (tgy). they are activated by dual phosphorylations on threonine and tyrosine residues in response to numerous stimuli, including cytokines, hormones, gpcrs, heat shock, and other stresses and play important roles in controlling many cellular functions [42, 53, 92, 93 ]. p38 is expressed in most cells ; however, expression of other isoforms is tissue specific. cellular distribution, activation, and substrate specificity of p38 mapks result in diverse biological functions. once activated, p38 mapks either transmit the signals via a three - tier cascade or phosphorylate other regulatory molecules such as pla2, heat shock proteins, and transcriptions factors (c - jun, atf-2, creb, chop, nf - kb, and others) [8, 42, 92, 94 ]. substrates of p38 mapk include mapk - activated protein kinases (mks), that is, mk2, mk3, and mk5 (reviewed in [1, 53, 95, 96 ]). there are also several distinct kinases at the map3k level of the p38 mapk cascade, including mlk2, mlk3, tpl2, dual leucine zipper - bearing kinase, ask1, map three kinase 1, and tak1 [91, 97, 98 ]. for instance, the p38 mapk pathway causes rapid inactivation of the erk1/2 cascade mediated by pp2a. the p38 mapk pathway is involved in tissue homeostasis and several pathologies ranging from inflammation and the immune response to cancer and neurodegenerative diseases [93, 100, 101 ]. it has been demonstrated that il-1 activates p38 mapk and that this event is associated with star expression and testosterone synthesis in immature rat leydig cells [102, 103 ]. the involvement of p38 mapk in steroidogenesis is further assessed by observations in which inhibition of its activity, either by sb203580 or pd169316, results in the coordinate suppression of star and steroid levels. il-1 is also capable of phosphorylating creb and fos / jun through the activation of a p38 substrate which is a rsk family member (rsk - b kinase, also called msk2), suggesting that this process may play a role in the differentiation of immature into adult leydig cells. in addition, it is worth noting that il-1 also activates erk1/2 and inhibition of the latter by u0126 augments expression and phosphorylation of the star protein but decreases androgen synthesis by dissipating the mitochondrial electrochemical potential. these findings suggest that a number of mapk signaling events differentially influence il-1-mediated steroidogenesis in mouse leydig cells. gonadotropins have been shown to activate both p38 and erk1/2 mapks and result in varying effects on star expression and steroidogenesis in ovarian granulosa cells [16, 17, 51, 104 ]. studies have demonstrated that interference of p38 (by sb203580) and erk1/2 (by pd98059 and u0126) activity is associated with increases in lh / hcg / fsh mediated star expression and progesterone synthesis [16, 51, 104 ]. in addition, inhibition of p38 decreases both p450arom and estradiol synthesis, and these events are tightly correlated with liver receptor homolog-1 and dax-1 expression, demonstrating that p38 targets these transcription factors in regulating steroidogenesis. heat shock protein hsp-27 is identified as a downstream phosphorylation target of fsh- and forskolin - mediated p38 mapk activation. in granulosa cell - oocyte cocultures, both bone morphogenetic protein-2 (bmp-2) and bmp-4 exert differential effects on fsh mediated regulation of steroidogenesis through the activation of p38 mapk. indeed, both bmp-2 and -4 increase fsh - mediated p450arom mrna expression and estradiol production but decrease star and progesterone levels. in primary cultures of rat adrenal glomerulosa cells, ang ii activates the p38 and erk1/2 signaling pathways and results in increases in star expression, steroidogenic enzymes, and steroid synthesis [83, 107 ]. concurrently, ang ii inhibits protein synthesis by enhancing p27 expression (a protein known to block the cell cycle in the g1 phase). the effects of ang ii can be reversed through inhibition of p38 activity by sb203580, suggesting that ang ii plays an important role in adrenal physiology. accumulating evidence indicates that activation of the p38 mapk signaling cascade is linked to the aging - induced, oxidative stress - mediated suppression of steroidogenesis in adrenal cells [108110 ]. alternatively, inhibitors of p38 mapks (by either sb203580 or sb202190) and antioxidants (reactive oxygen species (ros) scavengers mntmpyp and n - acetyl cysteine) have been shown to restore corticosterone synthesis in cells from aged rats. these findings indicate that the stress - mediated inhibition of steroid biosynthesis involves the activation of the p38 mapk pathway in the adrenals during the course of aging. also, intense phospho - p38 mapk immunoreactivity has been detected in human brains of postmortem patients afflicted with alzheimer 's disease [111, 112 ], indicating that p38 could be involved in the pathogenesis of this disease. jnk / sapks are also considered as stress - activated mapks ; however, they are different from p38 mapks [1, 113, 114 ]. isolation and subsequent characterization of cdnas encoding these enzymes revealed three genes encoding proteins with 10 or more alternatively spliced forms. three main isoforms, that is, jnk1/sapk (46 kda), jnk2/sapk (54 kda), and jnk3/sapk (52 kda), are approximately 85% identical in their core catalytic domains [115, 116 ]. whereas jnk1/2 mapks are ubiquitously expressed, jnk3 is primarily localized to neuronal tissues, testis and cardiac myocytes. jnk / sapks are activated by cytokines, uv irradiation, growth - factor deprivation, agents that interfere with dna and protein synthesis, as well as other stressors. similar to other map kinases, the activity of jnk / sapks is dependent upon phosphorylation on tyrosine and threonine residues, which are separated by a proline to generate the tpy motif in the activation loop. the resulting signals are then transmitted to three - tier cascades either directly or through map4ks [117, 118 ]. however, two mek family members, mkk4 (sek1, mek4, jnkk1, and skk1) and mkk7 (mek7, jnkk2, and skk4), are predominantly involved in the jnk / sapk cascade for signal integration [117119 ]. several lines of evidence demonstrate the involvement of the jnk / sapk signaling cascade in steroidogenesis. for example, tumor necrosis factor- (tnf) activates jnk / sapk ; however, the latter is associated with decreases in basal and camp - induced steroidogenesis by reducing cyp17 gene expression in ma-10 cells. tnf also decreases the phosphorylation of erk1/2 while simultaneously increasing the abundance of cjun as well as increasing ap-1 binding activity, suggesting the involvement of a number of mapks in tnf signaling. therefore, the activation of jnk / sapk and erk1/2 mapks appears to play a mutually antagonistic role in tnf-mediated steroidogenesis. in rat r2c leydig tumor cells, bisphenol a (bpa), an endocrine disruptor, is capable of activating jnk / sapk and results in an elevation in aromatase activity and an attenuation of testosterone synthesis by targeting both creb and akt. increasing evidence demonstrates that steroidogenesis decreases in aging, a time when the levels of reactive oxygen species (ros) increase. as a consequence, a number of ros, such as superoxide anion (o2) and hydrogen peroxide (h2o2), have been involved in the repression of testicular star expression and steroid synthesis [122124 ]. in k28 mouse leydig cells, the inhibition of ros - mediated star, p450c17 mrna, and steroid levels is mediated, at least in part, through the activation of jnk / sapk mapks and subsequent upregulation of c - jun. these events cause a repression in the trans - activation potential of nur77 on steroidogenic enzyme genes and result in decreases in star expression and steroidogenesis. in human granulosa cells, leptin activates a number of mapks including the jnk / sapk signaling pathway where it decreases camp - induced star protein expression and progesterone synthesis. likewise, bmps can induce phosphorylation of several mapk cascades, exhibit varying actions on steroidogenesis, and play important roles in ovarian follicular growth and maturation [73, 125, 126 ]. also, an oocyte derived factor, fgf-8, has been shown to interact with bmps, activate the jnk / sapk and erk1/2 signaling cascades and subsequently regulate fsh - induced steroidogenesis in a rat granulosa cell - oocyte coculture system. altogether, the jnk / sapk signaling cascade is fundamentally connected with stress - related responses and the activation of jnk / sapk decreases the steroidogenic response in a number of steroidogenic tissues. the erk5/bmk1 is the largest known map kinase (~110 kda) family member. the signaling pathway leading to erk5 activation is poorly understood in comparison with other mapks as a consequence of conflicting findings in the literature [127130 ]. importantly, the c - terminus of erk5 contains 10 consensus map kinase phosphorylation sites which can be autophosphorylated. erk5 is ubiquitously expressed and its activity is regulated by a variety of proliferative (growth factors, phorbol ester, serum, and lysophosphatidic acid) and cell - stressing (h2o2, sorbitol, and uv irradiation) agents [132, 133 ]. the mechanism of upstream activation of the erk5 cascade has not been fully defined. this mechanism may include the action of adaptor proteins (e.g., lad), protein tyrosine kinases, and wnk1 [134136 ]. these components have been shown to induce a number of kinases at the level of map3k, including mekk2/3, tpl2, and mltk [135, 137139 ]. these kinases then phosphorylate mek5 (an upstream component of erk5) on serine and threonine residues. mek5 then activates erk5 on threonine and tyrosine residues in the loop sequence motif (tey) identical to erk1 and erk2. however, erk5 can not be phosphorylated by mek1/2, and mek5 does not phosphorylate erk1/2. another substrate of erk5 is the serum and glucocorticoid - inducible kinase, which may serve as a mapkapk of this cascade, allowing for the possible involvement of a five - tier cascade. erk5 can affect a number of cellular activities (e.g., cellular proliferation, differentiation, and motility) by phosphorylating many transcription factors, including mads box, c - myc, c - fos, myocyte enhancer factors 2a and c, and sap1a [133, 141143 ]. recently, it has been reported that orexins a and b activate erk5 and concomitantly increase expression of the star protein and steroidogenic enzyme genes in h295r cells [55, 84 ]. the effects of orexins on erk5 phosphorylation have been demonstrated to be similar to those of ang ii and are mediated by multiple g - protein signaling pathways. however, orexins simultaneously activate the erk1/2 and p38 mapk signaling cascades, indicating that the regulation of orexin - mediated star expression and steroidogenesis involves several mapks in adrenal cells. information that the activation of the erk5 signaling pathway in gonadal cells is linked to steroidogenesis is currently lacking. erk3, erk4, erk7, nlk, mok, and others are considered as atypical mapks (table 1). regulation, structure, and substrate specificity of these mapks have been described in a recent review. the mapk signaling cascades are the major components of pathways controlling a wide variety of cellular processes, including embryogenesis, gene expression, acute responses to hormones, cell survival, and apoptosis and are of critical importance in the transduction of extracellular signals in cells. noteworthy, the mapk cascade has been implicated in the pathogenesis of a number of human disorders, including alzheimer 's disease, parkinson 's disease, and many cancers. indeed, the multiple effects of mapks could be a result of the involvement of a wide variety of substrates that include protein kinases and phosphatases, transcription factors, cytoskeletal elements, and other signal - regulated molecules. the studies summarized here have emphasized the roles of erk1/2, p38 mapk, jnk / sapk, and erk5 mapks in the regulation of star expression and steroidogenesis in different steroidogenic tissues. the star protein plays an indispensable role in the production of steroid hormones required for bodily homeostasis and normal reproductive development and function. based on the results available at this time, it is obvious that the mapk signaling cascades play diverse roles in controlling star expression and steroid biosynthesis in tissue-, stimulus-, and pathway - specific manners. these processes appear to be dependent on receptor - effector coupling, signaling crosstalk, and/or other factor(s) involved in steroidogenesis. moreover, under specific circumstances, the regulation of mapk - dependent star expression and steroidogenesis involves more than one mapk signaling, and as a consequence different, and even opposing, effects of mapks can be seen in different steroidogenic cells. additionally, different cells express distinct sets of transcription factors, and this diversity may account for the cell - type - dependent specificity of mapk action. given the physiological and pathological roles of the mapk signaling pathways, elucidation of tissue- and disease - specific effects of each of the mapk signaling cascades together with their downstream effectors an abundance of molecular tools including high throughput genomic and proteomic technologies will undoubtedly provide valuable insights into these regulatory mechanisms. | mitogen - activated protein kinases (mapks) comprise a family of serine / threonine kinases that are activated by a large variety of extracellular stimuli and play integral roles in controlling many cellular processes, from the cell surface to the nucleus. the mapk family includes four distinct mapk cascades, that is, extracellular signal - regulated kinase 1/2 (erk1/2), p38 mapk, c - jun n - terminal kinase or stress - activated protein kinase, and erk5. these mapks are essentially operated through three - tiered consecutive phosphorylation events catalyzed by a mapk kinase kinase, a mapk kinase, and a mapk. mapks lie in protein kinase cascades. the mapk signaling pathways have been demonstrated to be associated with events regulating the expression of the steroidogenic acute regulatory protein (star) and steroidogenesis in steroidogenic tissues. however, it has become clear that the regulation of mapk - dependent star expression and steroid synthesis is a complex process and is context dependent. this paper summarizes the current level of understanding concerning the roles of the mapk signaling cascades in the regulation of star expression and steroidogenesis in different steroidogenic cell models. |
the prevalence of obesity and asthma has been increasing throughout the world in recent decades. obesity and increasing adiposity have proven to be a risk factor for incident asthma and make a unique phenotype of the disease. obese patients with asthma demonstrate different asthma phenotype compared with patients of normal weight is associated with decreased glucocorticoid responsiveness, an inability to achieve adequate asthma control, increased symptoms and exacerbations. the aim of this study is, therefore, to evaluate the influence of anthropometric measurements on pulmonary function parameters in asthmatic patients. in this cross - sectional study, subjects were patients who refer to subspecialty clinics of tabriz university of medical sciences from both sexes in june 2012. inclusion criteria were the age 1880 years with a diagnosis of asthma at least 1-year by two pulmonologist according to the global strategy for asthma. then, participants had a history of smoking or other lung disorders and respiratory tract infections, they excluded from the study. demographic characteristics were recorded for each person via a questionnaire created for the study. at the first of this study, this project was approved by the ethics committee of tabriz university of medical sciences. during height and weight measurements patients wore light clothing and were barefoot. body mass index (bmi) was calculated as weight in kilograms divided by the square of the height in meters and was categorized into underweight (18.5), normal weight (18.524.9), overweight (2529.9) and obesity (30) according to the world health organization classification. measuring waist circumference (wc) (halfway between the 10 rib laterally and the most superior part of the anterior superior iliac crest) and hip circumference (hc) (largest circumference between the waist and knees) using a tape with a nearest 0.1 cm was determined by a research assistant. pulmonary function tests (pfts) including forced expiratory volume in 1 s (fev1 [% predicted : % pred ]), peak expiratory flow (pef [% pred ]), forced vital capacity (fvc [% pred ]) and fev1/fvc% were measured by two trained nurses using spirometer (spiroanalyzer st-300 ; fukuda sangyo, tokyo, japan) in the morning. comparison of variables between bmi groups was made using one - way anova and kruskal wallis test for normal and nonnormally distributed data, respectively. in this cross - sectional study, subjects were patients who refer to subspecialty clinics of tabriz university of medical sciences from both sexes in june 2012. inclusion criteria were the age 1880 years with a diagnosis of asthma at least 1-year by two pulmonologist according to the global strategy for asthma. then, participants had a history of smoking or other lung disorders and respiratory tract infections, they excluded from the study. demographic characteristics were recorded for each person via a questionnaire created for the study. at the first of this study, during height and weight measurements patients wore light clothing and were barefoot. body mass index (bmi) was calculated as weight in kilograms divided by the square of the height in meters and was categorized into underweight (18.5), normal weight (18.524.9), overweight (2529.9) and obesity (30) according to the world health organization classification. measuring waist circumference (wc) (halfway between the 10 rib laterally and the most superior part of the anterior superior iliac crest) and hip circumference (hc) (largest circumference between the waist and knees) using a tape with a nearest 0.1 cm was determined by a research assistant. pulmonary function tests (pfts) including forced expiratory volume in 1 s (fev1 [% predicted : % pred ]), peak expiratory flow (pef [% pred ]), forced vital capacity (fvc [% pred ]) and fev1/fvc% were measured by two trained nurses using spirometer (spiroanalyzer st-300 ; fukuda sangyo, tokyo, japan) in the morning. comparison of variables between bmi groups was made using one - way anova and kruskal wallis test for normal and nonnormally distributed data, respectively. in total, 85 asthmatic patients (37 male and 48 female) aged 43.9 10.61 years were participated. demographic characteristics of the study participants (n=85) the mean, minimum and maximum of anthropometric measurements and pulmonary function variables are shown in table 2. hence, according to their bmi, 36.5% normal weight, 37.6% overweight and 23.5% obese were. mean (sd), minimum and maximum of anthropometric and lung function variables for the all participants (n=85) comparing of all anthropometric measurements except height between three groups of bmi indicated statistically significant differences [table 3 ]. comparison of anthropometric and lung function characteristics between bmi categories no significant association between bmi and pfts was observed. in addition, the results showed negative significant correlations between wc with fev1, pef, fvc and fev1/fvc in overweight females. on the other hand furthermore, negative correlation between whr with fev1, pef and fvc in obese females were obtained [table 4 ]. the present study aimed to explore the influence of anthropometric measurements on pulmonary function parameters patients with asthma. in our study, not wc but whr had an inverse significant relationship with lung function tests like many other studies and this appears to be so more in females, unlike other studies. found significant relations of whr with fvc and fev in both men and women. in a recent cross - sectional study in chile, neither bmi nor wc was related to asthma symptoms in 1232 of adults. in return, in a community - based study in sweden, kronander. showed that both bmi and wc were correlated with increased risks for asthma incidence and symptoms, especially in nonatopic. chen. investigated the effect of wc on pfts in three groups of people with normal weight, overweight, and obesity. this supports the hypothesis that an excess of abdominal fat and thoracic region may limit movements of the diaphragm and decrease compliance of the respiratory system. to the effect, obese subjects with asthma who lose large amounts of weight have been shown to experience better conditions for control of their asthma symptoms. adipose tissue is metabolically active and has proinflammatory effects because it secretes a range of substances such as adipose - derived hormones or adipokines. leptin, resistin, and adiponectin are examples of adipokines whose receptors are widely distributed throughout the body, like lungs. leptin and resistin are increased in obesity and have pro - inflammatory effects, including activation of nuclear factor - kb, up - regulation of tumor necrosis factor levels, and enhancement of neutrophilic airway inflammation. in contrast, adiponectin is secreted inversely in obesity, and it is an anti - inflammatory adipokine that inhibits pro - inflammatory cytokines and induces anti - inflammatory cytokines. so, adipose tissue derived hormones may play an important role in managing asthma in obese persons. an important observation of the current study is sex differences, and hence that wc and hc in overweight females and whr in obese females were negatively associated with the lung function variables, whereas there were any significant relationships in males. several cross - sectional studies reported stronger associations between asthma and obesity in women than in men. however, in a systematic review, the effect of wc on pulmonary function parameters was greater among males compared with females. in the california teachers study cohort, obese and overweight women with asthma indicated more severe asthma episodes than normal weight women, as measured by more hospital admissions. these sex differences between asthma and obesity relationship could suggest that estrogen and other sex hormones may play an important role through modulation of th2 cytokine production. moreover, these relationships were found in females are probably associated with a comparable increase in overall muscles of males. on the other hand, the negative correlation between the pfts, whr and hc may be due to the gynoid fat distribution in females versus android fat distribution in males. nevertheless, these findings ensure further investigation and serve to highlight further the differences in the effects of body composition on respiratory function between two genders. therefore, our analysis could not obtain enough statistical power, and a larger population study is needed to confirm these influences. the other shortcoming of this study is if underweight patients were recorded in the study, we could compare their information with other groups and present more comprehensive information. however, bmi has been widely used as the standard index of obesity in a variety of studies, but it seems less efficient. a major limitation of bmi is that it does not distinguish fat mass and muscle mass, while they have different effects on pulmonary function. moreover, another limitation of bmi is that it provides no information on body fat distribution. our study found that pulmonary function was affected by some anthropometric measurements especially in overweight and obese women. these findings particularly reflect that this effect is influenced by the amount and distribution of body fat. | background : obesity is commonly regarded as a risk factor for asthma development, poor asthma control, and poor response to asthma therapy.methods:in a cross - sectional study, 85 asthmatics (37 male and 48 female) participated. pulmonary function tests (pfts) and anthropometric parameters were measured for each patient.results:mean age and median duration were 43.9 10.61 and 6 (314) years, respectively. among anthropometric parameters, only waist - to - hip ratio (whr) indicated significant correlation with pfts in both sex (p < 0.05). there were negative associations between waist circumference, hip circumference and whr with pfts only in overweight and obese women (p < 0.05).conclusions : some anthropometric parameters affected lung function, and it seems that gender differentially contributes to this effect. |
the study was approved by our institutional review board and informed consent was given by the patients. inclusion criteria were skeletal maturity, an isolated fracture involving the proximal one - third of the tibia. pathological fractures, gustilo type iiib, type iiic open fractures,10) tibial fractures accompanied by a soft tissue defect on the anteromedial aspect of the tibia, multiple fractures with spinal injury, and lower limbs with a pre - existing neurological deficit or vascular disease were excluded from this study. between july 2011 and september 2012, 35 patients with an isolated tibial proximal metaphyseal fracture underwent external fixation at our institute. there were 26 men and 9 women with a mean age of 42 years (range, 21 to 62 years). thirteen patients sustained a fracture as a result of a fall, 7 patients sustained a fracture in a motor vehicle accident, and 15 patients sustained a fracture in an automobile - pedestrian accident. the fractures were classified according to the ao foundation and orthopaedic trauma association (ao / ota) guidelines : type 41-a2 in 18 cases and type 41-a3 in 17 cases. there were 10 open fractur11)es, including 3 gustilo type i, 4 gustilo type ii, and 3 gustilo type iiia fractures. the mean time between injury and operation was 3 days (range, 2 to 7 days) in closed fractures. the patient was placed supine on the operating table. under general or spinal anesthesia, the femoral liss plates were provided by many companies, such as synthes (oberdorf, switzerland), smith & nephew (memphis, tn, usa), depuy (warsaw, in, usa), zimmer (warsaw, in, usa), and kanghui (changzhou, china). the ipsilateral femoral liss plate was used, i.e., the right femoral liss plate was used to fix the right proximal tibial fracture and the left femoral liss plate was used to fix the left proximal tibial fracture. the broad end of the plate was placed close to the knee joint since it can allow insertion of more screws for stabilization of the short proximal segment. for closed oblique and spiral fractures, a small incision (3 to 5 cm) was made to expose and clean the fracture gap. a clamp or two k - wires were used to temporarily fix and maintain the anatomic reduction. definitive external fixation was achieved with a femoral liss plate (9 or 11 holes). the plate was placed on the anteromedial aspect of the tibia as close to the skin as possible. a stack of evenly folded towels (1 to 2 cm in thickness) was placed under the plate to keep the plate away from the skin, preventing impingement between the plate and the skin. successive holes were drilled over locking drill - guides through stab incisions where the overlying soft tissue envelope was intact. all screws were bicortical. at each end of the fracture, four to five bicortical locking screws were inserted. the position and orientation of the screws were checked with fluoroscopy. for closed comminuted fractures, under live c - arm fluoroscopy, restoration of length and alignment the steps for placement of screws and plate were the same as those mentioned above. for open fractures, after excisional debridement, external plating was performed during the emergency operation. the steps for placement of screws and plate were the same as those mentioned above. the patients were allowed to walk with partial weight - bearing from the second postoperative day. patients were instructed to visit the clinic for examination every 4 weeks. at each follow - up, radiographs of the tibia were taken and the patients were evaluated using the hospital for special surgery (hss) knee scoring system and american orthopaedic foot & ankle society (aofas) ankle scoring system.12) fracture healing was defined as the presence of cortical bridging on biplanar radiographs. complications were defined by fixation failure, infection, and nonunion. once cortical bridging was observed on biplanar radiographs, the patients were allowed to walk with full weight - bearing for one month before removing the plate in the clinic. the time to fracture healing and postoperative complications the study was approved by our institutional review board and informed consent was given by the patients. inclusion criteria were skeletal maturity, an isolated fracture involving the proximal one - third of the tibia. pathological fractures, gustilo type iiib, type iiic open fractures,10) tibial fractures accompanied by a soft tissue defect on the anteromedial aspect of the tibia, multiple fractures with spinal injury, and lower limbs with a pre - existing neurological deficit or vascular disease were excluded from this study. between july 2011 and september 2012, 35 patients with an isolated tibial proximal metaphyseal fracture underwent external fixation at our institute. there were 26 men and 9 women with a mean age of 42 years (range, 21 to 62 years). thirteen patients sustained a fracture as a result of a fall, 7 patients sustained a fracture in a motor vehicle accident, and 15 patients sustained a fracture in an automobile - pedestrian accident. the fractures were classified according to the ao foundation and orthopaedic trauma association (ao / ota) guidelines : type 41-a2 in 18 cases and type 41-a3 in 17 cases. there were 10 open fractur11)es, including 3 gustilo type i, 4 gustilo type ii, and 3 gustilo type iiia fractures. the mean time between injury and operation was 3 days (range, 2 to 7 days) in closed fractures. the patient was placed supine on the operating table. under general or spinal anesthesia, the femoral liss plates were provided by many companies, such as synthes (oberdorf, switzerland), smith & nephew (memphis, tn, usa), depuy (warsaw, in, usa), zimmer (warsaw, in, usa), and kanghui (changzhou, china). the ipsilateral femoral liss plate was used, i.e., the right femoral liss plate was used to fix the right proximal tibial fracture and the left femoral liss plate was used to fix the left proximal tibial fracture. the broad end of the plate was placed close to the knee joint since it can allow insertion of more screws for stabilization of the short proximal segment. for closed oblique and spiral fractures, a small incision (3 to 5 cm) was made to expose and clean the fracture gap. a clamp or two k - wires were used to temporarily fix and maintain the anatomic reduction. definitive external fixation was achieved with a femoral liss plate (9 or 11 holes). the plate was placed on the anteromedial aspect of the tibia as close to the skin as possible. a stack of evenly folded towels (1 to 2 cm in thickness) was placed under the plate to keep the plate away from the skin, preventing impingement between the plate and the skin. successive holes were drilled over locking drill - guides through stab incisions where the overlying soft tissue envelope was intact. all screws were bicortical. at each end of the fracture, four to five bicortical locking screws were inserted. the position and orientation of the screws c - arm fluoroscopy, restoration of length and alignment was achieved by manual traction and percutaneous manipulation using schanz pins. the steps for placement of screws and plate were the same as those mentioned above. for open fractures, after excisional debridement, external plating was performed during the emergency operation. the steps for placement of screws and plate were the same as those mentioned above. the patients were allowed to walk with partial weight - bearing from the second postoperative day. patients were instructed to visit the clinic for examination every 4 weeks. at each follow - up, radiographs of the tibia were taken and the patients were evaluated using the hospital for special surgery (hss) knee scoring system and american orthopaedic foot & ankle society (aofas) ankle scoring system.12) fracture healing was defined as the presence of cortical bridging on biplanar radiographs. once cortical bridging was observed on biplanar radiographs, the patients were allowed to walk with full weight - bearing for one month before removing the plate in the clinic. all patients were followed up for a mean period of 18 months (range, 13 to 22 months). the mean duration of surgery for closed fractures was 32 minutes (range, 20 to 65 minutes). the mean time to fracture healing was 14 weeks (range, 10 to 20 weeks). after walking with full weight - bearing for one month the average vas score was 3.5 points (range, 1 to 5 points). according to the hss knee scoring system and aofas ankle scoring system, the mean hss score was 91 (range, 85 to 100) and 98 (range, 93 to 100), and the mean aofas score was 94 (range, 90 to 100) and 98 (range, 95 to 100) at 4 weeks postoperatively and final follow - up (table 1). in two patients, effusion from one or two screw sites the other effusion persisted even after one week of cleaning with betadine, and the screws involved in the effusion were removed. there was no case of deep infection, malaligned union, nonunion, and loosening or breakage of plates and screws. the photographs of the representative cases are shown in figs. 1, 2, 3, 4, 5. the early design of an external plate such as the zespol implant coupled with multiple nuts and washers13) may have dissuaded surgeons who may have been otherwise more receptive to this technique.14) with the advent of locking plates with fewer moving parts, this technique was used in the management of open fractures, septic arthritis, and was called the ' supercutaneous plating technique.'15,16,17) however, in most of the previously reported studies, only a small number of patients and open fractures were included, and the tibial fracture was fixed with a tibial locking plate which was thinner than the femoral liss plate. in our study, the femoral liss plate was used as an external fixator to fix both closed and open proximal tibial metaphyseal fractures. the plate was placed on the anteromedial aspect of the tibia, with its broad end close to the knee joint, because we found that the contour of the femoral liss plate matched the anteromedial aspect of the lower leg. the broad end of the femoral liss plate offers greater versatility for inserting the screws as it has 7 holes that allow more precise modulation to obtain the greatest bone purchase in the proximal segment. in contrast to the tibial locking plate, the femoral liss plate is thicker and the diameter of screws in the femoral liss plate is larger than that of the screws in the tibial plate, which can increase the stability of fixation. the anteromedial aspect of the tibia is not covered by bulky muscles and important neurovascular structures, and hence, the surface can be clearly palpated ; thus, facilitating fast and accurate insertion of screws with less risk of neurovascular injuries. in our study, the mean surgical duration was 32 minutes. the plate - bone distance was 20 to 30 mm, which can increase the stability of fixation. according to the report by stoffel.18) on the biomechanics of a locking plate, the number of screws also significantly affected the stability. in our study, a long femoral liss plate with 9 to 11 holes was used. all these features can increase both the axial and torsional stability.19) all fractures healed without any complications in a mean time of 14 weeks. therefore, the motility of muscles was intact, which ensured a good range of motion. because of its low profile the patients could walk wearing normal trousers and there was much less tendency for the locking plate to strike the contralateral lower leg in the swing - through phase of either leg during ambulation.20) in our study, all of the patients were allowed to walk with partial weight - bearing from the second postoperative day. the mean hss score was 91 (range, 85 to 100) and 98 (range, 93 to 100), and the mean aofas score was 94 (range, 90 to 100) and 98 (range, 95 to 100) at 4 weeks postoperatively and final follow - up, respectively. the functional recovery of adjacent joint was satisfactory. in the other report by the authors,21) the tibial liss plates were placed on the lateral aspect of the tibia and staged operations were performed for internal fixation. at the same time, the laterally placed screws passed through the muscles on the anterolateral aspect of the tibia which would compromise the motility of muscles and cause an uncomfortable feeling, pin - site effusion, and even neurovascular injuries. although some techniques have been reported to augment the fixation with nailing, such as a blocking screw,22,23) the placement of a blocking screw is not always easy and would increase the time of surgery. another common complication of nailing is anterior knee pain, regardless of whether a transtendinous or peritendinous procedure is used.24,25) the clinical series with the use of an internal locking plate demonstrated favorable results;26,27,28) however, complications such as malunion and infection are not rare. with external plate fixation, it is not necessary to make two incisions to create a tunnel under the skin, which can provide better protection of the blood supply, decrease the postoperative pain and risk of infection. in contrast, the operation for removal of nails and internal locking plate could be troublesome in some cases. in the report by raja.29) on plate removal, with respect to the rate of infections, it was reported to be approximately 1% in closed tibial shaft fractures, 5% for gustilo type i, 10% for type ii, and > 15% for type iii.30) in our study, all of the 10 open fractures underwent external fixation using the femoral liss plate during the emergency operation. there was no case of deep infection, nonunion, and loosening or breakage of plates and screws. our study has several limitations, including a relatively short follow - up and lack of a control group. the use of the femoral liss plate, which was originally designed for internal fixation of the femur, as an external fixator to fix a tibial fracture is also controversial. in conclusion, for most proximal metaphyseal fractures of the tibia, external fixation using the femoral liss plate the traditional and classic surgical treatment of a closed tibial fracture is internal fixation. in our study, the indication for external plate fixation was expanded to include both closed and open fractures. although the results of external plate fixation are very good, its use is still controversial. | backgroundthe locking plates are often used for internal fixation of closed tibial fractures. the use of a locking plate as an external fixator is still controversial, particularly for closed fractures. the purpose of this study is to evaluate the results of external fixation using the femoral less invasive stabilization system (liss) plate in proximal metaphyseal fractures of the tibia.methodswe prospectively evaluated 35 patients (26 males and 9 females) with a mean age of 42 years (range, 21 to 62 years) who presented with fresh tibial proximal metaphyseal fractures. according to the ao foundation and orthopaedic trauma association (ao / ota) classification, the fractures were identified as type 41-a2 in 18 cases and type 41-a3 in 17 cases, including 25 closed fractures and 10 open fractures. the femoral liss plate was used to fix these fractures, which was placed on the anteromedial aspect of the tibia as an external fixator. the mean follow - up period was 18 months (range, 13 to 22 months).resultsall fractures healed in a mean time of 14 weeks (range, 10 to 20 weeks). there was no case of nonunion, deep infection, and loosening of screws and plates. one month after the appearance of cortical bridging on biplanar radiographs, the locking plate was removed within 3 minutes in the clinic without any difficulty. according to the hospital for special surgery (hss) knee scoring system and american orthopaedic foot & ankle society (aofas) ankle scoring system, the mean hss score was 91 (range, 85 to 100) and 98 (range, 93 to 100), and the mean aofas score was 94 (range, 90 to 100) and 98 (range, 95 to 100) at 4 weeks postoperatively and final follow - up, respectively.conclusionsfor proximal metaphyseal fracture of the tibia, external fixation using the femoral liss plate is a safe and reliable technique with minimal complications and excellent outcomes. its advantages include ease of performing the surgery, use of a less invasive technique, and convenience of plate removal after fracture healing. |
oesophageal stents are used widely to treat benign strictures, oesophageal perforations and fistula and for palliative therapy of oesophageal cancer. oesophageal migration has been reported to occur in 3.6% of cases, and the majority are removed or repositioned endoscopically without complication. this case presentation illustrates a rare and potentially lethal complication of stent migration resulting in mechanical small bowel obstruction. an 81-year - old female with a 24 h history of acute, worsening abdominal pain with associated abdominal distension and bilious vomiting was referred by her gp to the general surgical admissions unit. the patient reported no other gastrointestinal symptoms and there was no prelude to the presentation. she had undergone a billroth i gastrectomy in 1977 for peptic ulcer disease, a splenectomy following blunt trauma in 1989 and further gastric surgery for a cancer in 1999. during a therapeutic endoscopy for a benign stricture this was treated endoscopically with the deployment of a hanaro - antireflux covered stent, covering the defect that was identified at 35 cm. these margins were chosen in order to give a 3 cm cover of the perforation. the extra 2 cm distal cover was because the stent was the only suitable stent available that would be both wide enough to remain in situ and long enough to give adequate cover of the perforation. there were no concerns regarding the apposition of the stent, and the stent was not sutured in place. routine follow - up was arranged for 46 weeks ; however, for an unconfirmed reason, this follow - up never took place. on presentation, blood results on admission, including liver function, urea and electrolytes, crp, amylase, lactate and full blood count, were within normal limits. abdominal x ray showed multiple distended loops of small bowel with a migrated stent inside small bowel fig. 1. abdominal films show multiple dilated loops of small bowel with a visible stent in the abdomen. abdominal x ray taken shortly after presentation. abdominal films show multiple dilated loops of small bowel with a visible stent in the abdomen. following the diagnosis of mechanical small bowel obstruction due to oesphageal stent migration, the patient was booked and consented for a laparotomy. at laparotomy, distended small bowel was found, with the cause of the obstruction being found in the distal ileum. post - operative recovery was unremarkable, and the patient was discharged home with regular follow - up in 68 weeks. in cases of iatrogenic oesophageal perforation, stent placement has been shown to be an effective treatment, and has less associated morbidity and mortality than operative repair. however, the main complication of stent placement is migration, requiring repositioning or replacement. if stent migration is a risk due to large defects, or minimal anatomic barriers to migration, some authors advocate suturing stent into oesophagus, which may avoid this complication. in this case, the patient had unusual anatomy due to her previous surgeries. the stent migration may have been aided by the fact that the patient did not have a pylorus, allowing easy passage into the small bowel. clinicians should consider the risks of stent migration, and make the patient aware of these risks. oesophageal stent migration is a known complication of stent placement, occurring in 3.6% of cases of benign oesophageal conditions requiring stent placement. in a case series of 70 patients where migration has occurred, the authors found that the majority of stents migrate no further than the stomach, exit via the rectum or remain in the body with minimal complications. migration occurred in 11 of the 43 patients (25%) with benign cause of strictures and in 591 of the 845 patients (7.0%) with malignant cause. in the 70 patients, only in 3 was it surgically removed due to intestinal obstruction. in a prospective study of 18 iatrogenic oesophageal perforations treated with stents, all the stents were removed at a mean of 50 20 days. in this case, the stent was placed as a temporary measure to allow closure of the perforation. routine follow - up for removal was arranged, but the patient was lost in follow - up. although complications due to stent migration are rare, perforation, obstruction and haemorrhage all carry significant risks, with high mortality and morbidity. this case illustrates the importance of prompt patient follow - up in order for the stent to be removed. this case also illustrates the importance of making patients aware of the small risk of bowel obstruction and seeking urgent medical attention if the symptoms of bowel obstruction are present. | we report the case of an elderly female patient with a history of a previous billroth i gastrectomy who presented with small bowel obstruction secondary to a migrated oesophageal stent. this patient had an iatrogenic oesophageal perforation following therapeutic endoscopy for a benign stricture 4 months prior to presentation. this was treated endoscopically with a covered stent that was not removed as planned. the stent migrated distally lodging in the terminal ileum, causing small bowel obstruction. oesophageal stent migration is a rare but well - recognized complication of stent placement. endoscopic stenting is an effective treatment for oesophageal perforation with lower morbidity and mortality than operative repair. clinicians should be aware that although patients with a history of previous gastric surgery are at no greater risk of stent migration than others, the altered anatomy can affect the final resting place of the migrated stent and hence the clinical effects and sequalae. |
for the past half century, myopia has emerged as an extremely important health issue and the related literature has increased exponentially [1, 2 ]. and epidemiological data suggested that the prevalence and incidence of myopia were increasingly high, causing serious social and economic burdens. so both scientists and ophthalmologists showed great interest and applied significant effort to elucidate the mechanism of myopia development. as an important component in the field of myopia research, animal model has greatly expanded our knowledge on the visual regulation of refractive development [4, 5 ]. these animal models such as monkeys, chicks, tree shrews, fish, and guinea pigs each have its advantages and disadvantages [68 ]. recently, the establishment of mouse model of myopia has contributed to a recent surge in interest and revealed several essential findings [911 ]. the mouse model offers a highly efficient model in which to study the genetic and environmental basis of the growth of the eye, as well as gene - environment interactions [1214 ]. nevertheless, available techniques for device attachment in mice are limited. in methods using sutures, glue, or a combination of techniques, the goggle or defocusing lens may be easily scratched off and lead to poor ocular health [11, 15, 16 ]. other researchers introduced an elizabethan collar to avoid troublesome scratches, but the collar may seriously impact the animal 's activity and systemic health [11, 17 ]. the head - mounted goggle apparatus was reported to be highly stable and efficient. however, the components of goggle apparatus were not convenient fabrication and the surgical procedures were complicated. then, the purpose of this study is to introduce a simple head fixation device for myopia induction in mice that (1) can be attached with simplified procedures, (2) should keep stable for a certain period of time, (3) effectively induce experimental myopia, and (4) is reliably secured to the eye and whole health. the head fixation device is composed of two parts, the nylon connector and the metal spectacle frame. the nylon connector, including m3 8 nylon screws, gaskets, and caps, was designed and produced (shanghai zhisheng plastic co., ltd.). the spectacle frames were first designed using rhinoceros software (version 5.0, robert mcneel & associates ; figure 1) and then were made of 304 stainless steel by laser cutting (versolsolar hangzhou co., ltd.). a single spectacle frame has four functional components including the (1) kidney - shaped slotted hole for position control, (2) rounded lens clip for experimental lens fixation, (3) nose pad for strengthening fixation, and (4) connecting rod for integration. the spectacle frames underwent manual shaping to be attached to the mouse head ; the physical dimensions and a computer representation of the 3d shape for the frame are shown in figures 1 and 2. the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (10 mg / kg, figure 3(a)).the dorsal cranial fur was shaven ; the surgical area was cleaned with betadine (figure 3(b)).a middle line incision (810 mm) was made to expose the dorsal cranial surface of the skull.the exposed fascia and periosteum were removed from the coronal suture to the sagittal suture, and the surface was cleaned and dried (figure 3(c)).the screw was sutured to the bilateral temporal muscle, cervical trapezius muscle, and frontal skin with 6 - 0 nylon sutures (figure 3(d)).the incision was sutured with 6 - 0 nylon suture, and antibiotic ointments were applied (figure 3(e)).the gasket and screw were fixed to supply a platform supporting the spectacle frame (figure 3(f)).the spectacle frame was placed on the head, and the cap was screwed in and adjusted to position the lens clip (figure 3(g)).the fixation was strengthened by suturing the nose pad to the skin, and the experimental lenses were fixed (figure 3(h)). the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (10 mg / kg, figure 3(a)). the dorsal cranial fur was shaven ; the surgical area was cleaned with betadine (figure 3(b)). a middle line incision (810 mm) was made to expose the dorsal cranial surface of the skull. the exposed fascia and periosteum were removed from the coronal suture to the sagittal suture, and the surface was cleaned and dried (figure 3(c)). the screw was sutured to the bilateral temporal muscle, cervical trapezius muscle, and frontal skin with 6 - 0 nylon sutures (figure 3(d)). the incision was sutured with 6 - 0 nylon suture, and antibiotic ointments were applied (figure 3(e)). the gasket and screw were fixed to supply a platform supporting the spectacle frame (figure 3(f)). the spectacle frame was placed on the head, and the cap was screwed in and adjusted to position the lens clip (figure 3(g)). the fixation was strengthened by suturing the nose pad to the skin, and the experimental lenses were fixed (figure 3(h)). male c57bl6j mice aged postnatal 28 days (p28) underwent baseline retinoscopy refraction and axial length measurement using a custom - built optical coherence tomography (oct), which has been detailed in [19, 20 ] (figure 4). two groups were set in the current study. in the experimental group (n = 25), myopia was monocularly induced with the head - mounted spectacle frame over the right eye using a 15.0 diopter (d) lens (hangzhou boston optics co., ltd.) ; no defocusing lens or plano lens was fixed to the left lens clip. in the control group the experimental mice were housed in isolation, and the control mice were housed in groups of five. the body weight was measured weekly in experimental group from the time of purchase (p21) to the sacrifice date (p56). all mice were exposed to a 12 h light : 12 h dark cycle and checked daily for 28 days to assess the defocusing lens position stability. then mice aged p56 were refracted and measured again to assess the effectiveness of myopia induction. in addition, a coronal segment of the tissue corresponding to the locations of the underlying screws was serially sectioned and h&e stained. this study was approved by the institutional animal care and use committee (iacuc) at zhejiang university (permit number : zju201306 - 1 - 01 - 066). all experimental procedures were conducted in accordance with the guidelines for the care and use of laboratory animals of zhejiang university. all mice were sacrificed by an anesthetic overdose, and all efforts were made to minimize suffering. there were no intraoperative complications, and none of the animals experienced postoperative incision infection. the mice wearing head - mounted spectacle frames appeared to have normal activity and were well groomed and freely feeding (see video in supplementary material available online at http://dx.doi.org/10.1155/2016/8497278). mice weights were monitored weekly, and weight gain of mice in experimental group was appropriate when compared with the reference values from the jax lab (figure 5). there were two situations to be noted in the assessment of device stability, a spectacle frame lost and an entire apparatus lost. in these cases, the defocusing lens could flip away from the eye and allowed for some normal visual input, which required timely intervention. the lost of spectacle frame was always the result of a loosened screw cap or nose pad and could be retightened in minutes. an entire lost apparatus was usually caused by frequent scratching, tissue reaction, or accidental clamping with the cage, and the nylon connector and spectacle frame should be refixated under general anesthesia. in this study (table 1), 2 cases of spectacle frame loss but no entire apparatus loss were found in the first week (p29p35), 8 cases of spectacle frame loss and 2 cases of entire apparatus loss occurred in the second week (p36p42), 12 cases of spectacle frame loss and 3 cases of entire apparatus loss occurred in the third week (p43p49), and 13 cases of spectacle frame loss and 3 cases of entire apparatus loss occurred in the fourth week (p50p56). with this technique, 9/30 mice lost the spectacle frame or entire apparatus once, 14/30 mice lost the spectacle frame or entire apparatus twice, and 2/30 lost the spectacle frame or entire apparatus three times during the 28 days induction period. in the first two weeks, no mice lost the spectacle frame or entire apparatus three or more times. our apparatus stability was relatively superior to the way that directly glued to the fur and was essentially the same as the head - mounted goggling apparatus reported by faulkner. refractive error data in table 3 demonstrated no significant difference between the right and left eyes in the experimental group at p28 (0.08 1.26 d ; paired t - test p = 0.75) or in the controls (0.70 1.34 d ; paired t - test p = 0.13). 28 days later, the right eye of mice in experimental group showed a significant myopic shift compared to the left eye (4.90 2.17 d ; paired t - test p = 1.69 10), but not found in the control group (0.90 1.37 d ; paired t - test p = 0.07). the axial length measurements using oct (table 3) showed no significant difference between the right and left eyes in both experimental (0.004 0.034 mm ; paired t - test p = 0.58) and control group (0.004 0.016 mm ; paired t - test p = 0.48) at p28. after the induction period, the right eyes of mice in experimental group were significantly longer than the contralateral eyes (0.025 0.037 mm ; paired t - test p = 0.002), while no significant difference was found in the controls (0.013 0.031 mm ; paired t - test p = 0.22). postmortem evaluation showed general reactive hyperplasia of the skin and no obvious alteration of the skull underlying the screws. histological examination revealed chronic inflammation of the hypoderm due to the foreign body reaction in the h&e stained sections when compared with the normal controls, and the skull at the screw locations appeared healthy with no bony destruction (figure 6). it is generally known that form deprivation and lens defocusing are the two classical methods for myopia induction in animal, and they were also reported to be used in the study of mouse myopia [9, 21 ]. no matter which method is chosen, existing techniques for device attachment can be shared. in this study, we used the lens - induced myopia model for the reason of renewed interest that myopia progression in humans and animals can be altered through optical intervention [5, 22 ]. a literature survey showed that tejedor and de la villa made the first attempts by lid - suturing to induce deprivation myopia in mouse ; however, it was not suitable for defocusing lens attachment. later, schaeffel. introduced the method that goggles directly glued to the fur around the eye, and it was improved by barathi. to be used in lens - induced myopia [16, 17 ]. the method using glue was technically simple, but the device stability was not reliable enough and may lead to poor ocular health. thus, tkatchenko. reported a further refined technique, using a combination of glue and sutures. although the application of this method may not be restrained by mouse age and the effectiveness of myopia induction was reported to be good, no compliance data was shown. and then some researchers used plastic collar to improve stability ; however, it would inevitably affect the physical activity and whole health [11, 17 ]. faulkner. developed a noncontact head - mounted goggle apparatus for the study of murine myopia. however, the surgical procedures for the delicate device were quite complicated and time consuming and may lead to skull or brain injury. in addition, it was not suitable to be used in younger mouse with fragile skull, which may also make restriction on its popularity. thus, it is still necessary to invent an easily operated, safe, and effective device for the myopia induction in mice. thus we developed the head - mounted spectacle frame and introduced a relatively easy - to - learn fixation method. firstly, this device is lightweight (the nylon connector, spectacle frame, and defocusing lens amount to 0.500.60 grams) and could enhance the compliance of spectacle frame wearing. comparing with the head - mounted goggle apparatus, our research results demonstrated at least evenly matched position stability. in our study, there were still situations that indicated that the defocusing lens was not permanently fixed, and the ability to maintain device stability tended to decrease with extended induction time. therefore, it was of great importance to tighten the screws when operating and to check the fitting situation carefully during the experimental period. the body weight gradually increased during the experimental period, with no obvious difference with the reference values. with sutures fixation but not skull implantation, there were only chronic subdermal inflammations found at the screw locations, but no pathological change in the skull or adjacent brain. thirdly, and perhaps most importantly, our device and method induced myopic shift and axial elongation in the experimental eyes. literatures have shown that a 10 d lens fitted over the mouse eye could induce a refractive shift of 13.03 d in 46 days (p10p56) under a 12 : 12 h light : dark cycle, and a 25 lens induced a 14.6 d myopic shift in 21 days (p24p45) under constant light. in this study, the myopic shift of 4.90 d in 28 days (p29p56) is relatively small and is approximately equal to that reported by faulkner. using a head - mounted goggling apparatus (5.66 d myopic shift in 14 days of form deprivation). many explanations may contribute to the reduced effectiveness, such as species variability of the mice, a later induction initiation, or others. in any case, the head - mounted spectacle frame apparatus is reliable and practicable. in addition, our device is economical and the surgical procedures are easy to learn, for only requiring commonly used microscopic suture materials but not professional equipment. this is also a beneficial aspect of our device and method in consideration of the popularization. first, the physical dimensions of the one - piece frame were fixed in this study, which may not be compatible with mouse younger than p28. thus frames with optimized physical dimensions adapting to younger mice during the susceptible period for myopia induction could substantially increase a future use of this device. second, the introduced method is a rather invasive procedure, which requires recovery and infection and pain management. third, although other reported methods and procedures were not easy to duplicate, it was still of significance to make comparison between these methods and our method at the same time, including effectiveness, security. and experiments along these lines are currently in progress in our laboratory. in summary, we described a novel apparatus named the head - mounted spectacle frame apparatus and represented a simplification of the currently employed head fixation techniques for study of myopia in mouse. our results demonstrated several advantages, including the improved compliance, effective myopia induction, and reliable security. all of these features would likely prove useful in small animal models among investigators studying experimental myopia trials. | the mouse model has been widely employed to explore the mysteries of myopia. for now, existing techniques for induction of experimental myopia in mice can be classified into three types : (1) devices directly glued to the fur ; (2) devices attached using a combination of glue and sutures ; (3) devices attached using a skull - mounted apparatus. these techniques each have its advantages, disadvantages when considering the devices stability, safety, complexity, effectiveness, and so forth. thus, techniques for myopia induction in mice have yet to be further refined to popularize the applications. in this pilot study, we introduce a new head fixation device named the head - mounted spectacle frame apparatus for the study of mouse lens - induced myopia. surgical procedures for device attachment were relatively simple and easy to learn in our study. effective myopia induction was validated by retinoscopy refraction and axial length measurement using optical coherence tomography. in addition, it showed improved compliance and reliable safety when compared to the published methods. the head - mounted spectacle frame apparatus provides a new choice for the study of lens - induced myopia in mouse. it also allows for the use of form deprivation, making it attractive for future experimental mouse myopia trials. |
haemodialysis is the most effective modality in treatment of end stage renal disease (esrd) (1, 2). patients undergoing the haemodialysis treatment are at a greater risk of acquiring systemic infections (3). standard haemodialysis treatment sessions last 46 hours and patients are exposed to 15,00020,000 litres of dialysis fluid, annually. in haemodialysis procedure, dialysis water is directly administered in the form of dialysate and infusate up to 3,4006,800 litres through two or three dialysis water monitor ultrafilters (4). the trend of using online haemodialysis procedures increased dialysate contamination which in turn may lead to the infections caused by many antibiotic resistant bacteria (5). inefficient systems in treating water (filtration and reverse osmosis) and ineffective methods of disinfection are responsible for the majority of bacteraemia and sepsis among patients in haemodialysis (6). therefore water systems in haemodialysis should be under constant microbiological monitoring (6, 7). on the other hand, haemodialysis services in hospitals have the same water distribution networks. occasionally, these networks encounter problems such as low flow of water to meet the required tap water for the procedure. therefore, haemodialysis machines are frequently connected to a storage water tank to ensure adequate volume and pressure of water especially in times of peak demand (8). however, with any water distribution network, those in the hospitals are subjected to biofilm formation (9). a number of pathogens, such as legionella, pseudomonas, and mycobacteria grow well in biofilms and may be more resistant to disinfectant utilized in the haemodialysis water than their planktonic forms (9). are thin, gram - negative, obligate aerobic and non - spore forming rods with complexed nutritional requirements which contaminate the water distribution networks particularly dialysis water and can be transmitted from water to the air via hospital water supply. inhalation of legionella contaminated aerosols is a common source of human infections (10). in addition, some species of legionella such as legionella pneumophila are strongly associated with asymptomatic infections (legionnaires disease) or produce mild cough, sore throat and pontiac fever (11). several comprehensive epidemiological studies have linked the exposure of individuals to the contaminated water distribution systems of hospitals to the acquisition of nosocomial legionnaires disease (12, 13). however, few studies have been conducted on detection of legionella infection through haemodialysis water systems. the objective of the present study was to investigate the prevalence of legionella spp. and other bacterial infections in air, water and dialysate utilized in a haemodialysis centre in an educational hospital in iran. one separated machine was especially assigned for haemodialysis treatment of patients infected with hepatitis b virus. the centre performs approximately 1100 haemodialysis sessions monthly in three shifts a day for 95 patients suffering from esrd and approximately 45 sessions of haemodialysis treatment for acute cases of renal failure. water treatment system is a built - in unit which includes tap water pre - treated with filter system, a water softener, and an activated carbon filter followed by a final purification with reverse osmosis (ro) process. treated water is stored in a reservoir tank where it will be distributed to the haemodialysis unit and dialysis machines. after each session of haemodialysis, the machines are rinsed and disinfected according to the protocols enforced by the ministry of health. a total of 50 water and dialysate samples were weekly collected from november 2013 to january 2014. the water samples (500 ml) were collected according to the water sampling guidelines (14) from 5 sites as follows : distribution loop (n=6), raw water (n=4), reverse osmosis (ro) (n=11), water storage tank (n=11) and dialysate effluent (n=18). the total and faecal coliforms were determined by utilizing the most probable number (mpn) method (15). moreover, samples were plated on macconkey (mc) agar (conda, spain) to determine the presence of faecal coliforms. after incubation at 37 c for 24 h, the grown colonies were identified by standard biochemical tests. the tubes showing gas formation and the presence of escherichia coli were tested for determination of mpn for faecal coliforms. the tubes that only showed turbidity were also plated on mc agar plates to be tested for non - fermentative bacteria. isolation of legionella spp. was done as follows : sterile water samples were used as negative control whereas sterile water samples inoculated with legionella atcc33152 (1010 cfu / ml) were used as positive controls. water samples of 1000 ml volume were passed through the cellulose nitrate membrane filters (sartorius ag, germany) with a pore size of 0.45 m. membranes were fragmented into small pieces while they were poured into 100 ml sterile plastic beaker with 50 ml original filtered water. this beaker was placed on a shaker at 37 c for 30 min for releasing bacteria from the filter into the water. one ml was taken from each container and heat - treated at 50 c for 30 min to inactivate the microorganisms other than legionella spp. afterwards, 100 l of each water sample was inoculated on a bcye medium (difco, usa) supplemented with glycine, vancomycin, cycloheximide and polymyxin b (gvpc). the plates were incubated under microaerophillic condition at 35 c (90% humidity, 3% co2) for 7 days (16). conditions of pcr and size of the amplified fragments were as described by hosseini. (18). airborne bacteria were sampled using a single stage andersen air sampler (model quick take 30 skc, scientific co) which impacted air at a rate of 28.3 l / min for 10 min / day (30 min for legionella) through a narrow slit. the air sampler was located approximately 100 cm away from the patient s bed (at the height of 91 cm). after each sampling, the culture plates were immediately transferred to the laboratory and incubated at 37 c for 2448 h. trypticase soy agar (conda, spain) plates and cellulose nitrate membrane filters with a pore size of 0.45 m were used to isolate the other bacteria and legionella spp. after sampling, the number of grown colonies on each plate was recorded and the concentration of the airborne bacteria and legionella spp. finally, the mean levels of airborne bacteria in the haemodialysis centre were compared with the european union good manufacturing practices guidelines (1 cfu / m in class a rooms and 100 cfu / m in class c rooms) (19). one separated machine was especially assigned for haemodialysis treatment of patients infected with hepatitis b virus. the centre performs approximately 1100 haemodialysis sessions monthly in three shifts a day for 95 patients suffering from esrd and approximately 45 sessions of haemodialysis treatment for acute cases of renal failure. water treatment system is a built - in unit which includes tap water pre - treated with filter system, a water softener, and an activated carbon filter followed by a final purification with reverse osmosis (ro) process. treated water is stored in a reservoir tank where it will be distributed to the haemodialysis unit and dialysis machines. after each session of haemodialysis, the machines are rinsed and disinfected according to the protocols enforced by the ministry of health. a total of 50 water and dialysate samples were weekly collected from november 2013 to january 2014. the water samples (500 ml) were collected according to the water sampling guidelines (14) from 5 sites as follows : distribution loop (n=6), raw water (n=4), reverse osmosis (ro) (n=11), water storage tank (n=11) and dialysate effluent (n=18). the total and faecal coliforms were determined by utilizing the most probable number (mpn) method (15). moreover, samples were plated on macconkey (mc) agar (conda, spain) to determine the presence of faecal coliforms. after incubation at 37 c for 24 h, the grown colonies were identified by standard biochemical tests. the tubes showing gas formation and the presence of escherichia coli were tested for determination of mpn for faecal coliforms. the tubes that only showed turbidity were also plated on mc agar plates to be tested for non - fermentative bacteria. isolation of legionella spp. was done as follows : sterile water samples were used as negative control whereas sterile water samples inoculated with legionella atcc33152 (1010 cfu / ml) were used as positive controls. water samples of 1000 ml volume were passed through the cellulose nitrate membrane filters (sartorius ag, germany) with a pore size of 0.45 m. membranes were fragmented into small pieces while they were poured into 100 ml sterile plastic beaker with 50 ml original filtered water. this beaker was placed on a shaker at 37 c for 30 min for releasing bacteria from the filter into the water. one ml was taken from each container and heat - treated at 50 c for 30 min to inactivate the microorganisms other than legionella spp. afterwards, 100 l of each water sample was inoculated on a bcye medium (difco, usa) supplemented with glycine, vancomycin, cycloheximide and polymyxin b (gvpc). the plates were incubated under microaerophillic condition at 35 c (90% humidity, 3% co2) for 7 days (16). conditions of pcr and size of the amplified fragments were as described by hosseini. (18). airborne bacteria were sampled using a single stage andersen air sampler (model quick take 30 skc, scientific co) which impacted air at a rate of 28.3 l / min for 10 min / day (30 min for legionella) through a narrow slit. the air sampler was located approximately 100 cm away from the patient s bed (at the height of 91 cm). after each sampling, the culture plates were immediately transferred to the laboratory and incubated at 37 c for 2448 h. trypticase soy agar (conda, spain) plates and cellulose nitrate membrane filters with a pore size of 0.45 m were used to isolate the other bacteria and legionella spp. after sampling, the number of grown colonies on each plate was recorded and the concentration of the airborne bacteria and legionella spp. finally, the mean levels of airborne bacteria in the haemodialysis centre were compared with the european union good manufacturing practices guidelines (1 cfu / m in class a rooms and 100 cfu / m in class c rooms) (19). a total of 50 water samples were taken from dialysis systems among which 24 samples (48%) showed bacterial contamination. the highest rate of legionella contamination was observed in the storage tank (67 cfu / ml) whereas they were not isolated from the dialysate effluents. the most total bacterial count was related to the dialysate effluent (785 185.3 mpn / ml, pv 0.05) and the maximum total count of coliforms was related to the reverse osmosis (48 31.7 mpn/100ml, pv 0.05). in contrast, the minimum number of total bacteria and total coliforms was observed in the distribution loop (353 107.9 and 0, respectively, pv 0.05) (table 1). mean value of detected bacteria in the haemodialysis water system (p 0.05) table 2 illustrated other bacteria isolated from water samples of dialysis systems. a great number of isolated bacteria were gram - negative bacilli (mostly pseudomonas spp.) which accounted for 48.3% of culture positive samples. frequency of gram - positive cocci (mostly micrococcus spp.) and gram - positive bacilli (mostly bacillus spp.) was 24.1 and 16.7%, respectively. the maximum and minimum bacterial counts were reported from dialysate effluents as well as distribution loops. were isolated from 4 cases (8%) using bcye medium from reverse osmosis outlets (two cases), distribution loop outlet (one case) and storage tank (one case). tracking of 16s rrna gene via pcr revealed five positive cases were the source of contamination which was confirmed by culture method as well. overall, 20 air samples were taken from the haemodialysis centre (table 3). in these systems, the mean (sd) of airborne bacterial colony count was 169.52 (29.09). a majority of the isolated bacteria were gram - positive cocci which were mostly non - pathogenic. staphylococcus aureus and pseudomonas aeruginosa were the most important pathogenic bacteria isolated from the air samples. the mean (sd) of airborne fungal colony count among the investigated samples was 30.7 (9.8) which were saprophytic molds mostly belonging to the aspergillus genus. the hospitalized patients undergoing haemodialysis treatment are exposed to large volumes of water in each haemodialysis session. innovative procedures are being implemented to improve efficacy of haemodialysis treatment for patients suffer from end stage renal disease. however, they are prone to infections caused by various pathogenic agents (20). in the present study, most of the gram - negative bacteria isolated from haemodialysis systems were non - fermentative bacteria. these bacteria can easily grow in sterile distilled water and dialysate which may be due to the presence of bicarbonate and glucose in these samples (21). two cases (8.3%) of burkholderia cepacia were isolated from the dialysate effluents and the storage tanks. burkholderia is an opportunistic respiratory bacterium with high rate of antibiotic resistance (23). the coliforms isolated from the dialysis system were not from a faecal origin which was probably due to the environmental contamination of the salt and resins used in the dialysis water system. diverse forms of bacteria isolated from dialysis water system in different studies may be related to different times of sampling, different methods of sterilization, inadequate disinfection of dialysis machines, burnout of dialysis water system and availability of various types of dialysis machines (21, 23, 24). in the present investigation on dialysis water system, 4 legionella isolates were grown on plates but 5 isolates were detected by pcr among which the fifth pcr - detected isolate was originated from storage tank. when the dialysis machine is operating, the temperature increases up to 6070 c. according to the studies, most cases of legionella have been isolated at lower temperatures (4560 c) (25). in the present study, a case of legionella was also isolated from raw water. given that the dialysis water system is connected to the hospital pipelines it is likely that legionella strains enter into the haemodialysis water system through the hospital pipelines and colonize in different areas and form biofilms. it is believed that some legionella strains are able to tolerate continuous treatments of disinfection. this tolerance is related to the biofilm production by legionella in the dialysis water systems. according to the european union good manufacturing practices guidelines, haemodialysis centres are placed in the class c of air surveillance standards (100 cfu / m) (19). in the present study, the total mean (sd) of airborne bacterial colony count in the haemodialysis centre was more than the standard value which indicates lack of suitable air conditions in the haemodialysis centre. the presence of s. aureus and p. aeruginosa in the air and dialysis water systems raises the possibility of bacterial recirculation in the physical milieu of the haemodialysis centre. the results of the total count of bacteria in the air and the pattern of isolated bacteria were consistent with other studies conducted in health centres and other wards such as intensive care units (2628). current conventional techniques applied in water treatment process in civic areas can not considerably reduce the bacterial endotoxin. although urban public water supplies are chlorinated, they may possibly contain small amounts of gram - negative bacteria. water containing gram - negative bacteria with the dialysate together can enhance overgrowth of bacteria in the absence of chlorine. therefore, the reverse osmosis (ro) is being used for complete treatment of water used for dialysis. however, small amounts of gram - negative bacteria and non - tuberculosis mycobacteria in the water may pass through the filters or will colonize in the reverse osmosis unit (8). the high viable agents detected in this study indicated that the microbiological quality of the haemodialysis water was lower than the limits recommended by the aami (29). after each period of disinfection, increased contamination was observed which was probably due to the bacterial biofilms generated in the water pipes. the worrisome finding in the present study was the presence of legionella isolates along with non - fermentative bacteria in the haemodialysis water system which are capable of forming biofilms in the haemodialysis water system pipes. the presence of biofilm in the pipes makes bacteria to grow recurrently few hours after each disinfection period. it is recommended that the storage tank and the reverse osmosis membranes should be weekly disinfected rather than once a month to remove biofilms adequately. the accumulation of water in the reservoir holding tank is a good source for growth of bacteria. dialysis machine is an important source of contamination such as staphylococcus, pseudomonas and legionella. therefore an efficient prevention program is needed to eliminate bacterial contamination from the dialysis water system. periodic surveillance programs for microbiological qualification in haemodialysis centres can also lead to a better planning for disinfection of haemodialysis water acknowledgment. this study was supported by shahroud university of medical sciences (grant no : 9012). | background and objectives : the microbiological monitoring of the water used for haemodialysis is important especially for legionella and non - fermentative bacteria since patients with end stage renal disease (esrd) are suffering from deteriorated function of immune system.materials and methods : a total 50 water and dialysate samples were weekly collected over a period of 10 weeks from 5 sites. total and faecal coliforms were determined by utilizing the most probable number (mpn) method. for isolation of legionella, water samples were inoculated on a bcye medium. dna extraction was performed and was used to amplify 16s rrna gene of legionella species. airborne bacteria were sampled using a single stage andersen air sampler.results:out of total 50 water samples, 24 samples had bacterial contamination. the highest rate of legionella contamination was observed in the storage tank (67 cfu / ml). legionella was not isolated from the dialysate effluent samples. the highest rate of total bacterial count was related to the dialysate effluent and the maximum total count of coliforms was related to the reverse osmosis. the isolated bacteria were gram - negative bacilli (mostly pseudomonas isolates), gram - positive cocci (mostly micrococcus spp.) and gram - positive bacilli (mostly bacillus spp.). six samples were contaminated with coliforms. no faecal coliform was isolated from the samples.conclusion:these results indicated that dialysis machine is an important source of contaminations such as staphylococcus, pseudomonas and legionella. therefore an efficient prevention program is needed to eliminate bacterial contamination of dialysis water system. moreover, in haemodialysis centres, periodic surveillance programs for microbiological qualification can lead to a better planning for disinfection of haemodialysis water systems. |
any reduction in the normal carrying angle of the elbow produces cubitus varus deformity. the most common cause of this deformity is late complication of supracondylar fracture of the humerus.1, 2 it is a poorly tolerated cosmetic deformity with no practically functional impairment, although there is some difficulty in carrying heavy loads. the deformity following humeral supracondylar fracture is non - progressive, while progressive deformity could be due to physeal injury of medial condyle, malunited fracture of lateral condyle and congenital cubitus varus deformity. non - progressive deformity is usually treated by one surgery while progressive cubitus varus deformity may require repeated surgeries. various corrective osteotomies have been described for the cubitus varus such as lateral closing wedge osteotomy, medial opening wedge osteotomy, dome osteotomy, step - cut osteotomy and pentalateral osteotomy.3, 4, 5, 6, 7 we reported a case of progressive cubitus varus deformity secondary to medial humeral condyle physeal arrest, managed by lateral closing wedge osteotomy of lateral pillar of distal humerus, which was a novel surgical technique. a 5 years old boy presented with complaint of cubitus varus deformity of the right upper limb. according to his father, the patient had the history of falling on outstretched hand three years ago, and afterwards he was not able to move his right elbow and was taken to local hospital. after removal of cast, the parents noticed deformity of the right elbow which was kept on progressing over the next three years. at presentation, the patient brought no old records, so the type of fracture and the initial follow - up was not known. in clinical examination, the child had the cubitus varus deformity of 30 as compared to carrying angle of 6 on the left upper extremity. there was flexion arc of 100 on the affected side as compared to 120 on the opposite side. the patient underwent x - ray examination of both the upper limbs, with elbow extended and forearm supinated. the plain radiograph showed the shortening of medial column of the elbow with deformed distal humeral anatomy. humeroulnar angle was 25 on the affected side as compared to 5 on the left side. mri of the affected elbow joint was done, indicating physeal scarring of the medial condyle of the humerus (fig. 1). as the deformity was progressive and grotesque, his father was told about the requirement of operative intervention and informed consent was obtained for surgery. a line diagram of the radiograph with elbow extended and forearm supinated was made on the tracing paper, humeroulnar angle was calculated and the wedge of 1.5 cm was removed from the lateral pillar, without breaching the medial cortex of the distal humerus. a 5 cm incision was made over the lateral aspect of the distal humerus. the site of osteotomy was marked distally, at the level of olecranon fossa and another point 15 mm proximal. osteotomy was done and 15 mm wedge of bone was removed from the lateral column. the condyle of the humerus was rotated proximally and laterally and fixed with two k - wires. he was given splintage for another 4 weeks, and then active elbow range of motion was started. after 12 months of follow - up, he had cubitus rectus at the elbow, with flexion arc of 120 and full supination and pronation (fig. 2). cubitus varus deformity is one of the most common complications seen in paediatric patients (age group : 58 years) following trauma.1, 2, 3 the cubitus varus can increase the risk of lateral condylar fracture, pain, tardy posterolateral rotatory instability, internal rotational malalignment and poor cosmesis. bellemore reported supracondylar osteotomies in 16 patients using a modified french method. this technique, originally described in 1959 with a lateral closing wedge through a posterior approach, used an intact periosteal hinge medially and two screws with a wire loop laterally to control the distal fragment. voss described lateral closing wedge osteotomies through lateral approaches in 36 patients. hui used a medial approach with a lateral closing wedge in 14 cases, with one case of transient ulnar nerve paresis with residual varus. derosa and graziano applied a step - cut technique of distal humerus valgus osteotomy using one cortical screw for fixation in 11 patients. all the described techniques were for cubitus varus deformity following malunited fracture of supracondylar humerus. in the present case, the deformity was progressive and it was caused by medial condyle physeal injury with altered condyle anatomy. so in such scenario, usual lateral closing wedge and medial opening wedge osteotomy could not correct the deformity. thus the novel approach of lateral column resection was done with good postoperative outcome. in conclusion, cubitus varus deformity is common entity, physeal injury is one of the cause and should not be missed. the non - progressive deformity can be easily managed by single surgery while progressive deformity may require multiple surgeries. in case of progressive deformity, different osteotomy pattern can be devised for distorted distal humerus anatomy with good clinical outcome. | the outward angulation of elbow with supinated forearm is cubitus varus deformity. this deformity is often seen as sequelae of malunited supracondylar fracture of humerus in paediatric age group of 58 years. the deformity is usually non - progressive, but in cases of physeal injury or congenital bony bar formation in the medial condyle of humerus, the deformity is progressive and can be grotesque in appearance. various types of osteotomies are defined for standard non - progressive cubitus varus deformity, while multiple surgeries are required for progressive deformity until skeletal maturity. in this study we described a novel surgical approach and osteotomy of distal humerus in a 5 years old boy having grotesque progressive cubitus varus deformity, achieving good surgical outcome. |
the endemic carnivorous plant venus flytrap (dionaea muscipula ellis) produces a rosette of leaves, each divided into two parts : the lower part called the lamina and the upper part called the trap. the trap catches prey by very rapid movement of its bilobed halves that shut when the trigger hairs protruding from the upper leaf epidermis are stimulated by touch. at room temperature, two touches activate the trap, which snaps shut in a fraction of second (juniper., 1989). at higher temperature (3540 c) only one stimulus is required for trap closure (brown and sharp, 1910). the stimulation of trigger hairs activates mechanosensitive ion channels and generates a receptor potential, which induces an action potential. electrical signals are the immediate cause of the trap movements irrespective of the way in which the signal is triggered ; for example by mechanical stimulation or by electrostimulation (volkov., 2007, 2008a, b, c, 2009a, b). in animals, the ionic mechanism of the action potential of axons depends on inward - flowing na (depolarization) and outward - flowing k ions (repolarization), whereas the excitation of plant cells depends on ca, cl, and k ions (fromm and lautner, 2007). the action potentials in dionaea have been extensively studied (e.g. burdon - sanderson, 1873 ; affolter and olivo, 1975 ; hodick and sievers, 1986, 1988, 1989 ; sibaoka, 1991 ; trebacz and sievers, 1998 ; krol., 2006 ; volkov., 2007, 2008a, b, c, 2009a, b). they propagate from mechanosensitive trigger hairs of the lobe to the trap midrib, more rapidly across the lower (abaxial) surface than across the upper one, while they are not recorded in adjacent lamina (burdon - sanderson, 1873 ; burdon - sanderson and page, 1876 ; williams and pickard, 1980 ; volkov., 2007). (2007) found that the generated action potential had a duration of 1.5 ms and a velocity of 10 m s. trigger hair - induced generation of action potentials is not associated only with trap closure. the struggling of the entrapped prey in the closed trap results in generation of further action potentials which cease to occur when the prey stops moving. over 100 action potentials were recorded in the trap with prey in the first 2 h and the mechanical stimulation triggered secretion of digestive fluid (affolter and olivo, 1975 ; lichtner and williams, 1977). in a previous study it was shown that repeated irritation of trigger hairs temporarily reduced the rate of photosynthesis (an) and the effective photochemical quantum yield of photosystem ii (psii), and stimulated the rate of respiration (rd) in the traps but not in the adjacent laminae (pavlovi., 2010). these findings are not surprising because the inhibitory effect of electrical signals on an and psii has also been well documented in non - carnivorous plants (koziolek., 2003 ; lautner., 2005 ; bulychev and kamzolkina, 2006a, b ; hlavkov., 2006 ; kaiser and grams, 2006 ; fromm and lautner, 2007 ; krupenina and bulychev, 2007, 2008 ; grams., 2009). however, the exact mechanism underlying the photosynthetic limitation caused by electrical signals is not yet known. it is difficult to conclude whether the changes in psii, which measures the proportion of light absorbed by chlorophyll associated with psii that is used in photochemistry (for definition, see genty., 1989 ; maxwell and johnson, 2000), are the reason for or just a consequence of decreased carboxylation efficiency. in fact, reduced carboxylation efficiency decreases psii, which prevents overexcitation of psii and protects it against photoinhibition (for a review, see kramer., 2004). it has been proposed that subcellular alternations in ion fluxes (e.g. ca) and ph may be involved in the photosynthetic responses, which modify the enzymatic activities in the cytoplasm or chloroplast (lautner., 2005 ; bulychev and kamzolkina, 2006a, b ; krupenina and bulychev, 2007). bulychev and kamzolkina (2006a, b) found that the depression of electron transport after action potentials in cells of chara was largely due to non - photochemical quenching (npq) in psii. (2003) suggest that transient knockout of photosynthesis mediated by electrical signals in mimosa pudica is too fast to be a result of zeaxanthin - dependent npq or chemical signals, as was later proposed by hlavkov. (2006), and propose that the rapid decline of psii might result from direct interference with electron transport chains in chloroplasts through direct impact of electrical signals. in addition, a direct effect of the electrical field on charge separation and recombination in the reaction centre of psii can not be excluded (meiburg., 1983 ; dau and sauer, 1991, 1992 ; bulychev and vredenberg, 1999 ; vredenberg and bulychev, 2002, 2003 ; vredenberg., 2009). with the present state of knowledge it is still difficult to conclude what is the site effect of electrical signal - induced inhibition of photosynthesis. here a detailed analysis of chlorophyll a fluorescence kinetics simultaneously with gas exchange measurements is provided during irritation of trigger hairs, which induce the generation of action potentials in d. muscipula. first, the relationship between electrical signals and chlorophyll fluorescence in the light at atmospheric co2 concentration was examined. in the second experiment, the calvin cycle reactions were inhibited by lowering the co2 concentration to zero, while electrons still move on alternative electron acceptors, allowing determination of whether electrical signals have a direct impact on the electron transport chain. in the dark, the maximum quantum yield of psii (fv / fm) together with fast chlorophyll fluorescence induction kinetics (o - j - i - p), reflecting the filling up of the psii electron acceptor plastoquinone pools qa and qb, were measured in the presence of electrical signals. are the primary targets of electrical signal - induced inhibition dark or light reactions of photosynthesis ? twenty 3- or 4-year - old d. muscipula j. ellis plants were grown in a growth chamber at an irradiance of 150 mol m s photosynthetic active radiation (par) and a 14/10 h light / dark period, in well - drained peat moss in plastic pots irrigated with distilled water. the trap was closed by mechanical stimulation and the leaf was cut near the base. the trap and thin wire (0.1 mm diameter) placed in the closed trap were sealed into a leaf cuvette (plc6, pp - systems, hitchin, uk), which monitors co2 and h2o exchange. the base of the lamina protruding outside the cuvette was submerged in distilled water in an eppendorf tube to prevent it drying out. the trigger hairs in the closed trap in a hermetically closed cuvette were repeatedly stimulated for 15 s by moving the thin wire protruding outside. movements of the wire in the empty closed cuvette had no effect on co2 and h2o exchange, confirming that the movements of the wire had no effect on the gas - tight seal. measurements of chlorophyll a fluorescence were performed with a fluorcam fc-1000 lc (photon systems instruments, brno, czech republic) attached to the plc6 cuvette connected with a ciras-2 infrared gas analyser (pp - system, hitchin, uk). before each measurement one leaf was cut and placed inside a plc6 cuvette. because the diameter of the cuvette window was 18 mm after 2 min, the minimal fluorescence (f0) at a light intensity < 0.1 mol m s par was measured. thereafter, the maximal fluorescence (fm) was measured using a saturation pulse (light intensity 3000 mol m s par, duration 800 ms). then an actinic light was switched on (80 mol m s par) and, after stabilization of the net photosynthetic rate (an), three saturation pulses were given every 60 s (3000 mol m s par, 800 ms duration) for determination of the maximal fluorescence in the light - adapted state (f'm) afterwards the trigger hairs of the trap were mechanically stimulated by manipulation of the wire protruding outside the cuvette for 15 s. after 17 s a fourth saturation pulse was given followed by the remaining five pulses at regular 60 s intervals. in some experiments, after 17 s the actinic light was switched off and f'0 was recorded for estimation of the excitation pressure (1qp). photosynthetic parameters (fv / fm, psii, npq, and 1qp) were calculated according to maxwell and johnson (2000). for definition of the parameters, simultaneously, the infrared gas analyser monitored co2 and h2o exchange every 2 s at a leaf temperature of 221 c, ambient co2 concentration of 380 l l, a relative air humidity of 6070%, and a light intensity of 80 mol m s par (red - emitting leds, =620 nm). in the second experiment the measurements were done in exactly the same way, but without co2 (1 l finally, the whole experiment was performed in the dark at ambient co2 concentration of 380 l l. changes in the co2 concentration in the measuring chamber were recorded with a constant delay of 9 s (the time taken for gas to pass from the cuvette to the infrared gas analyser). this delay was taken into account in figures presented here, and the bold lines on the x - axis were moved 9 s to the left. after measurements, the leaves were dried at 70 c for 5 d, weighed, and the an and rd were calculated in nmol co2 g dw s. data shown are representative of a total of five independent measurements. at high excitation irradiance, dark - adapted leaves show characteristic polyphasic fluorescence kinetics with four distinct steps named o - j - i - p (for reviews, see strasser., 2004 ; lazr, 2006). because vredenberg and bulychev (2002) hypothesized that the i - p phase may be under photoelectrochemical control, the polyphasic increase in chlorophyll a fluorescence in the d. muscipula trap was measured in control (non - irritated) and irritated traps using a fluorpen fp 100max (photon systems instruments, brno, czech republic). the fast increase in chlorophyll a fluorescence was measured over a time span of 10 s to 1 s. before the measurements, the trap was closed and dark adapted for 30 min. then the fast chlorophyll a fluorescence induction kinetics were measured in non - irritated traps. after 30 min in the dark the same trap was stimulated by the thin wire protruding outside the trap for 15 s. after 17 s the saturation pulse was given (2000 mol m s par, duration 1 s). the measurements were repeated three times on the same trap in the following order : control irritated control irritated control irritated with a 30 min dark interval between measurements to ensure that the previous saturation pulse had no effect on the shape of the curves and the differences are caused by trigger hair irritation. two - tailed paired t - test was used to find significant differences between the o, j, i, and p phases of the increase in chlorophyll a fluorescence (statgraphics, centurion xv). each cut leaf was fixed inside a small measuring chamber so that the lamina was dipped in a mild saline solution (0.1 mm cacl2, 0.5 mm kcl), while the electrodes were placed on the abaxial surface of the closed trap. a glass micropipette containing an ag / agcl wire and filled with 3 m kcl was mounted with a half cell holder and connected to the headstage of the probe. an identical electrode was placed in the measuring chamber to serve as a reference electrode. the electrodes were connected to the amplifier and the signal was recorded continuously during trap stimulation at a 1 khz rate of sampling frequency with home - made lab - view software. the action potentials were measured in the light (80 mol m s par) at ambient co2 concentration, in an atmosphere without co2 (1 l five traps from different plants were selected for each treatment ; 10 measurements were performed. the statistical differences between treatments (amplitude and number of action potentials) were evaluated by student t - test (statgraphics, centurion xv). twenty 3- or 4-year - old d. muscipula j. ellis plants were grown in a growth chamber at an irradiance of 150 mol m s photosynthetic active radiation (par) and a 14/10 h light / dark period, in well - drained peat moss in plastic pots irrigated with distilled water. the trap was closed by mechanical stimulation and the leaf was cut near the base. the trap and thin wire (0.1 mm diameter) placed in the closed trap were sealed into a leaf cuvette (plc6, pp - systems, hitchin, uk), which monitors co2 and h2o exchange. the base of the lamina protruding outside the cuvette was submerged in distilled water in an eppendorf tube to prevent it drying out. the trigger hairs in the closed trap in a hermetically closed cuvette were repeatedly stimulated for 15 s by moving the thin wire protruding outside. movements of the wire in the empty closed cuvette had no effect on co2 and h2o exchange, confirming that the movements of the wire had no effect on the gas - tight seal. measurements of chlorophyll a fluorescence were performed with a fluorcam fc-1000 lc (photon systems instruments, brno, czech republic) attached to the plc6 cuvette connected with a ciras-2 infrared gas analyser (pp - system, hitchin, uk). before each measurement one leaf was cut and placed inside a plc6 cuvette. because the diameter of the cuvette window was 18 mm after 2 min, the minimal fluorescence (f0) at a light intensity < 0.1 mol m s par was measured. thereafter, the maximal fluorescence (fm) was measured using a saturation pulse (light intensity 3000 mol m s par, duration 800 ms). then an actinic light was switched on (80 mol m s par) and, after stabilization of the net photosynthetic rate (an), three saturation pulses were given every 60 s (3000 mol m s par, 800 ms duration) for determination of the maximal fluorescence in the light - adapted state (f'm) afterwards the trigger hairs of the trap were mechanically stimulated by manipulation of the wire protruding outside the cuvette for 15 s. after 17 s a fourth saturation pulse was given followed by the remaining five pulses at regular 60 s intervals. in some experiments, after 17 s the actinic light was switched off and f'0 was recorded for estimation of the excitation pressure (1qp). photosynthetic parameters (fv / fm, psii, npq, and 1qp) were calculated according to maxwell and johnson (2000). for definition of the parameters, simultaneously, the infrared gas analyser monitored co2 and h2o exchange every 2 s at a leaf temperature of 221 c, ambient co2 concentration of 380 l l, a relative air humidity of 6070%, and a light intensity of 80 mol m s par (red - emitting leds, =620 nm). in the second experiment the measurements were done in exactly the same way, but without co2 (1 l finally, the whole experiment was performed in the dark at ambient co2 concentration of 380 l l. changes in the co2 concentration in the measuring chamber were recorded with a constant delay of 9 s (the time taken for gas to pass from the cuvette to the infrared gas analyser). this delay was taken into account in figures presented here, and the bold lines on the x - axis were moved 9 s to the left. after measurements, the leaves were dried at 70 c for 5 d, weighed, and the an and rd were calculated in nmol co2 g dw s. data shown are representative of a total of five independent measurements. at high excitation irradiance, dark - adapted leaves show characteristic polyphasic fluorescence kinetics with four distinct steps named o - j - i - p (for reviews, see strasser., 2004 ; lazr, 2006). because vredenberg and bulychev (2002) hypothesized that the i - p phase may be under photoelectrochemical control, the polyphasic increase in chlorophyll a fluorescence in the d. muscipula trap was measured in control (non - irritated) and irritated traps using a fluorpen fp 100max (photon systems instruments, brno, czech republic). the fast increase in chlorophyll a fluorescence was measured over a time span of 10 s to 1 s. before the measurements, the trap was closed and dark adapted for 30 min. then the fast chlorophyll a fluorescence induction kinetics were measured in non - irritated traps. after 30 min in the dark the same trap was stimulated by the thin wire protruding outside the trap for 15 s. after 17 s the saturation pulse was given (2000 mol m s par, duration 1 s). the measurements were repeated three times on the same trap in the following order : control irritated control irritated control irritated with a 30 min dark interval between measurements to ensure that the previous saturation pulse had no effect on the shape of the curves and the differences are caused by trigger hair irritation. two - tailed paired t - test was used to find significant differences between the o, j, i, and p phases of the increase in chlorophyll a fluorescence (statgraphics, centurion xv). each cut leaf was fixed inside a small measuring chamber so that the lamina was dipped in a mild saline solution (0.1 mm cacl2, 0.5 mm kcl), while the electrodes were placed on the abaxial surface of the closed trap. a glass micropipette containing an ag / agcl wire and filled with 3 m kcl was mounted with a half cell holder and connected to the headstage of the probe. an identical electrode was placed in the measuring chamber to serve as a reference electrode. the electrodes were connected to the amplifier and the signal was recorded continuously during trap stimulation at a 1 khz rate of sampling frequency with home - made lab - view software. the action potentials were measured in the light (80 mol m s par) at ambient co2 concentration, in an atmosphere without co2 (1 l five traps from different plants were selected for each treatment ; 10 measurements were performed. the statistical differences between treatments (amplitude and number of action potentials) were evaluated by student t - test (statgraphics, centurion xv). repeated 15 s irritation of trigger hairs in a closed trap decreased the effective photochemical quantum yield of psii (psii), indicating that linear electron transport was inhibited. after stopping the mechanical irritation, the inhibition of psii was confined mainly to the digestive zone of the trap (fig. this rapid inhibition resulted in a transient increase in the intercellular co2 concentration (ci ; fig. 2b), while the stomatal conductance (gs) was not affected (data not shown ; see pavlovi., 2010). during 15 s of irritation of trigger hairs in a closed trap, 3.70.7 (1 se) (maximum 7) action potentials with an average amplitude of 40.13.5 mv (maximum 80 mv) were recorded (fig. 2a, inset). the detailed analysis of chlorophyll a fluorescence kinetics in the digestive zone of the trap revealed that the decrease of psii is caused at first by the increase in the steady - state fluorescence in the light (ft), whereas maximal fluorescence in the light - adapted state (f'm) was not affected immediately after irritation (fig. the fluorescence increase upon reduction of plastoquinone is due to a decrease in the rate of radical pair formation (forward electron transfer) and an increase in the rate of radical pair recombination (backward electron transfer). the reduced plastoquinone pool results in increased excitation pressure at the psii reaction centre, promoting photoinhibition (increased 1qp ; fig. 2d) which is prevented by a series of down - regulatory processes known as npq. within 1 min after irritation, ft is quenched by npq as indicated by the large drop in f'm (fig. f'm was rapidly reversed in darkness, indicating that it represents the fast relaxing energy state quenching (qe) rather than photoinhibitory quenching (qi ; data not shown). spatiotemporal changes of effective photochemical quantum yield of psii (psii) in a d. muscipula closed trap assessed by chlorophyll fluorescence imaging. the trap was irritated by a thin wire between 162 s and 177 s. typical responses to trigger irritation in the venus flytrap (d. muscipula) at a light intensity of 80 mol m s par and an atmospheric co2 concentration of 380 l l at a leaf chamber temperature at 22 c. net photosynthetic rate (an ; a), electrical signals (a ; inset), intercellular co2 concentration (ci ; b), chlorophyll fluorescence kinetics (c), and chlorophyll fluorescence parameters, means 1 se (d). psii, the effective photochemical quantum yield of psii, open circles ; 1qp, the excitation pressure, open triangles ; npq, the non - photochemical quenching, open squares. the duration of trigger hair irritation is denoted as a bold line on the x - axis. data shown are representative of a total of five measurements. because light and dark reactions of photosynthesis are coupled together by the production and consumption of atp and nadph, from the above - mentioned results it is difficult to conclude whether the decreased psii is a reason for or just a consequence of reduced an. transiently increased 1qp before induction of npq indicates that a traffic jam of electrons in the electron transport chain occurred. this is probably due to a decreased concentration of the oxidized form of nadp (an electron acceptor from psi) determined by a decreased activity of the calvin cycle. therefore, the concentration of co2 was decreased, to inhibit the dark reactions of photosynthesis, allowing the electrons to move on alternative electron acceptors (e.g. o2, activation of cyclic electron flow and photorespiration, n metabolism) to observe the direct impact of action potentials on psii. the experiment in a co2-free atmosphere (1 l l) showed the reverse effect of trigger hair irritation on psii, despite the same efflux of co2 (fig. trigger hair irritation decreased ft and increased f'm, and thus psii slightly increased (fig. the absence of co2 had not significant effect on action potentials, as the amplitude and number of pulses were comparable with those in the previous experiment (number 3.20.5, p=0.545 ; amplitude 37.23.2 mv, p=0.485, fig. typical responses to trigger hair irritation in the venus flytrap (dionaea muscipula) at a light intensity of 80 mol m s par and an atmospheric co2 concentration of 1l l at a leaf chamber temperature of 22 c. net photosynthetic rate (an ; a), electrical signals (a ; inset), intercellular co2 concentration (ci ; b), chlorophyll fluorescence kinetics (c), and chlorophyll fluorescence parameters, means 1 se (d). psii, the effective photochemical quantum yield of psii, open circles ; 1qp, the excitation pressure, open triangles ; npq, the non - photochemical quenching, open squares. the duration of trigger hair irritation is denoted as a bold line on the x - axis. data shown are representative of a total of five measurements. in the dark - adapted leaf the plastoquinone pool is oxidized that is, the reaction centres are open and calvin cycle enzymes are inactivated, allowing estimatation of the maximal photochemical activity of psii (fv / fm). trigger hair irritation resulted in transient efflux of co2 from the trap, indicating that the increased respiration rate (rd) is the major contributor to the decreased an [an is a function of rd and gross photosynthesis (ag) (fig. trigger hair irritation had the opposite effect on fluorescence in dark - adapted and light - adapted traps at ambient co2 concentration. this indicates that the fv / fm, which is proportional to the quantum yield of o2 evolution from psii, was slightly higher after irritation (fig. however, the effect of action potentials on the fluorescence in dark - adapted traps was much less obvious (changes in f0 up to 4%) than in light - adapted traps (changes in ft up to 60%, and of in f'm up to 35% ; compare figs 2c and 4c) ; therefore, the changes in fm were omitted in the calculation of npq. the amplitude and number of action potentials were comparable with our previous experiments, indicating that darkness had no effect on electrical signalling (number 3.80.3, p=0.837 ; amplitude 45.95.1 mv, p=0.332 ; fig. data from chlorophyll a fluorescence transient (o - j - i - p) values are the means 1 se from 18 measurements. statistical differences were evaluated by two - tailed paired student t - test ; significant differences before and after irritation of the same trap (paired data) are in bold. typical responses to trigger hair irritation in the venus flytrap (dionaea muscipula) in darkness and an atmospheric co2 concentration of 380 l l at a leaf chamber temperature of 22 c. net photosynthetic rate (an ; a), electrical signals (a ; inset), intercellular co2 concentration (ci ; b), chlorophyll fluorescence kinetics (c), and chlorophyll fluorescence parameter, means 1 se (d). fv / fm, the maximal quantum yield of psii photochemistry, is shown by open circles. the duration of trigger hair irritation is denoted as a bold line on the x - axis. measurements of the polyphasic increase in chlorophyll a fluorescence (o - j - i - p) in dark - adapted leaves have advantages over a single parameter such as the well known fv / fm. the decrease in the fluorescence rise in the 20200 ms time range (o - j - i rise) was compensated by an increase in the rise in the 20200 ms time range (i - p rise ; fig. the values of four distinct phases of the increase in chlorophyll a fluorescence (o - j - i - p) are summarized in table 1. significant differences were found in o, j, and p phases of the increase in chlorophyll a fluorescence. chlorophyll a fluorescence transients in non - irritated (black line) and irritated (grey line) d. muscipula traps given on a logarithmic time scale. the results reported here and in a previous study (pavlovi., 2010) confirmed that the irritation of the trigger hairs and the subsequent generation of action potentials in the digestive zone of the closed trap of d. muscipula resulted in a transient decrease in psii and an (figs 1, 2). the generation of action potentials and their negative effect on photosynthesis were confined to the trap and were not recorded in the adjacent lamina (volkov. convincing evidence on the role of the electrical signals in the regulation of photosynthesis has been described by numerous authors (herde., 1999 ;, 2003 ; lautner., 2005 ; bulychev and kamzolkina, 2006a, b ; hlavkov., 2006 ; kaiser and grams, 2006 ; fromm and lautner, 2007 ; krupenina and bulychev, 2007, 2008 ; grams., 2009). in accordance with krupenina and bulychev (2007), the inhibition of psii and the rapid efflux of co2 are longer than the duration of the action potential itself. these authors called it the long - lived state effect of action potentials on photosynthesis. (2009) that if the electrical signals have an impact on cytosolic ph, changes in enzyme activity might play a role in photosynthetic limitation (e.g. carbonic anhydrase, a ph - dependent enzyme important in the regulation of mesophyll conductance). bulychev and kamzolkina (2006a, b) proposed that action potentials suppress the calvin cycle reactions by increasing [ca ] in chloroplast stroma. (2009) found in isolated chloroplasts that the increase of [ca ] had no effect on the psii. (2005) suggested direct involvement of increased [ca ] in o2 formation of psii, and koziolek. (2003) proposed that the rapid decline in psii might result from an interference of the electron transport chains in chloroplasts through the direct impact of electrical signals. (2006) suggested that an increased level of jasmonic acid and abscisic acid had a direct inhibitory effect on the photosynthetic apparatus and stomata closure, respectively, in response to electrical signals evoked by local burning in tobacco. however, chemical signals are too slow to account for the photosynthetic response in sensitive plants (e.g. mimosa or dionaea), as was concluded by koziolek. however no changes in gs were found during irritation of the trap in this and a previous study, and the stomatal limitation of photosynthesis in carnivorous d. muscipula can be excluded (pavlovi., 2010). in this study evidence is provided that the decrease in psii is a consequence of reduced activity of enzymes involved in the dark reaction of photosynthesis, due to a feedback mechanism of co2 assimilation on electron transport rather than a direct effect of electrical signals on the light reaction, which seems not to be affected substantially. this assumption is supported by the observation that during the first seconds after trigger hair irritation in the light, the electron transfer chain became over - reduced (increased 1qp) before the lumen could be sufficiently acidified to initiate npq (fig. 2d). rapid relaxation of npq in the dark indicates that energy state quenching (qe) is the major contributor to npq (data not shown). the release of npq by nigericine and the rapid reversal of action potential - triggered npq in darkness in chara cells also indicates npq 's relationship to qe (bulychev and kamzolkina, 2006a, b ; krupenina and bulychev, 2007). a correlation between npq and zeaxanthin accumulation after 5 h in response to current application in solanum lycopersicum zeaxanthin dissipates excess excitation energy as heat and prevents photoinhibition of psii (pospil, 1997). absorption of sunlight that exceeds a plant 's capacity for co2 fixation results in a build up of the thylakoid ph that is generated by photosynthetic electron transport. the lumen acidification and subsequent activation of violaxanthin de - epoxidase, which catalyses the conversion of violaxanthin first to antheraxanthin and then to zeaxanthin and is connected to qe, might be explained by the decreasing atp consumption in the calvin benson cycle. cyclic electron flow around psi may also contribute to lumen acidification, because a role in down - regulation of psii via production of ph and subsequent activation of qe has been proposed (mller., 2001 ; kramer., 2004 ; niyogi., 2005). however, a zeaxanthin - independent npq mechanism localized in the psii core complex or the role of lutein can not be excluded ; both are also activated by generation of ph and are rapidly relaxed in darkness. these types of quenching form rapidly and may precede zeaxanthin - dependent quenching (ruban and horton, 1999 ; finazzi. it seems that electron transport is not directly inhibited by electrical signals. in the absence of co2, when calvin cycle reactions are inhibited by unavailability of co2 substrate, 3d). even a slight decrease in ft and increase in f'm in the light resulted in higher psii (fig. it is tempting to assume that a transient increase of ci after irritation, as a result of transiently increased rd, decreased 1qp and npq and slightly and transiently increased psii by the stimulation of the calvin cycle which consumes nadph and restores the oxidized form of nadp, an electron acceptor from psi (fig. however, the possibility of a direct impact of the electrical signals on the charge separation recombination reaction in psii and subsequent increased fluorescence yield also can not be excluded, as discussed below. the changes in chlorophyll a fluorescence in dark - adapted traps and fv / fm are not so obvious as the changes in the light - adapted state (with or without co2) and are rather minor (fig. the polyphasic increase in chlorophyll a fluorescence has advantage over a single parameter such as the well known fv / fm and takes into account all the steps of sequential fluorescence increase upon sudden illumination. quantitative models enable calculation of the energy cascade from psii light absorption to electron transport using o - j - i - p curves (for a review, see strasser., 2004). it has been proposed that the o step is the fluorescence signal coming from excited chlorophylls of light - harvesting antenna before the excitations reach the reaction centre of psii, the j step reflects light - driven accumulation of qa, and steps i and p reflect light - driven accumulation of qb and qb, respectively ; however, several other explanations have been proposed (for reviews, see lazr, 2006, 2009). at first glance, it seems that trigger hair irritation and subsequent generation of action potentials in d. muscipula resulted in an increase in fv / fm and thus increased photochemical efficiency of psii (table 1). however, care must be taken in the interpretation of the results, because the models relating variable psii fluorescence and energy trapping are based on the assumption that the energetic state of psii reaction centres is determined and quantified by the redox state of qa (two - state trapping model). a three - state trapping model, proposed by vredenberg (2000, 2004), suggests that the saturation of photochemistry does not necessarily result in saturation of the changes in fluorescence yield, as pheophytin (pheo) and oxidized secondary donor tyrosine (yz) may also act as efficient fluorescence quenchers of psii. therefore, any calculations of energy fluxes in psii according to the two - state trapping model (strasser., 2004) were avoided and only the differences at four distinct steps of the increase in chlorophyll a fluorescence were quantified (table 1). the decrease in the o - j - i rise and increase in the i - p rise in an irritated trap is in accordance with electrochemical stimulation of the fluorescence yield supplementary to photochemical quenching (fig. 5, pospil and dau, 2002 ; vredenberg and bulychev, 2002, 2003 ; vredenberg, 2004 ; vredenberg., 2009). it was proposed that an electric field in the vicinity of the reaction centre could influence the chlorophyll fluorescence (meiburg., 1983 ; dau and sauer, 1991, 1992 ; bulychev and vredenberg, 1999 ; vredenberg and bulychev, 2002 ; vredenberg, 2004 ; vredenberg., apart from the influence of qa oxidation, the electrical field may exerts its effect on recombination of charges in psii by decreasing the gibbs free energy difference (g0) between the excited states in the reaction centre of psii and the charge - separated state (p680 pheo). as far as is known, this is the first time that the generation of action potentials has impact on yield of chlorophyll a fluorescence during the o - j - i - p transient, and provides convincing evidence that the fluorescent rise is under electrochemical control. (2006) found no changes in the fluorescence induction in response to variation potentials generated by tobacco leaf in response to local burning. it seems that the donor side inhibition of photosynthesis (electrons from water) was not significantly affected as the k step in the increase in chlorophyll a fluorescence has not appeared. (1997) concluded that a typical k step in the increase in chlorophyll fluorescence is due to the decrease in the continuous supply of electrons to the reaction centre of psii from water. because the effect of electrical signals on the fluorescence yield of psii in a dark - adapted trap of d. muscipula is relatively small, it is suggested that the main site effect of electrical signals on inhibition of photosynthesis is in dark reactions. for better understanding, 6 summarizes the hypothesis about the target of electrical signals on photosynthesis in d. muscipula. hypothesis about the effect of action potentials on photosynthesis in dionaea muscipula upon trigger hair irritation. in accordance with the present results, it is supposed that the main targets of action potential - induced inhibition of photosynthesis are dark reactions of photosynthesis (1). suppression of calvin cycle reactions or co2 availability by inhibition of carbonic anhydrase (an important part of the regulation of mesophyll conductance) may decrease atp and nadph consumption. unavailability of adp and the oxidized form of nadp inhibits atp synthesis and linear electron transport (2). this results in increased excitation pressure at psii (3) due to the accumulation of the reduced plastoquinone pool and increased emission of fluorescence (4). inhibition of atp synthesis and probably also enhanced cyclic electron flow around psi (5) decrease the ph in the thylakoid lumen (6). the decrease in ph within excess excitation energy is dissipated as heat, and chlorophyll fluorescence is quenched (7). the result also support the hypothesis about the direct effect of electrical signals on charge separation recombination reactions in psii (8), although the effect seems to be small rather than substantial. the results of the experiment performed in the dark indicate that rapid efflux of co2 originates not only from inhibition of photosynthesis but also from stimulation of respiration (fig. a transient rise in rd after generation of action potentials was also documented in the liverwort conocephalum conicum (dziubinska., 1989). the results suggest that at least some of the energy connected with the rise of rd is utilized for the restoration of the state of the ionic balance (i.e. restores the resting state). jaffe (1973) and williams and bennet (1982) found that during trap closure in d. muscipula, 29% of atp is lost. subsequent availability of an increased concentration of adp may stimulate enzymes in early steps of the respiration pathway (for an overview, see taiz and zeiger, 2002). however, the role of atp is not only in rapid closure of the trap, but also in generation of action potentials, as suggested by dziubinska. (1989), because repeated mechanical irritation in a closed trap resulted in transient stimulation of rd (fig. the action potentials generated by trigger hair irritation in the carnivorous plant d. muscipula have an impact on both light and dark reactions of photosynthesis as chlorophyll a fluorescence measurements indicate. however, the changes in the yield of chlorophyll a fluorescence in dark - adapted traps are small in comparison with the changes in the light - adapted state. it is concluded that the main target of action potential - induced inhibition of photosynthesis is in the dark reaction, whereas the decreased electron transport (expressed as psii) is only a consequence of impaired co2 assimilation, preventing photooxidative damage of psii by dissipation of excitation energy via npq. recombination reactions in psii ; in this case the effect is small rather than substantial but provides important and convincing evidence about the electrochemical component of chlorophyll a fluorescence in vivo. | mechanical stimulation of trigger hairs on the adaxial surface of the trap of dionaea muscipula leads to the generation of action potentials and to rapid leaf movement. after rapid closure secures the prey, the struggle against the trigger hairs results in generation of further action potentials which inhibit photosynthesis. a detailed analysis of chlorophyll a fluorescence kinetics and gas exchange measurements in response to generation of action potentials in irritated d. muscipula traps was used to determine the site effect of the electrical signal - induced inhibition of photosynthesis. irritation of trigger hairs and subsequent generation of action potentials resulted in a decrease in the effective photochemical quantum yield of photosystem ii (psii) and the rate of net photosynthesis (an). during the first seconds of irritation, increased excitation pressure in photosystem ii (psii) was the major contributor to the decreased psii. within 1 min, non - photochemical quenching (npq) released the excitation pressure at psii. measurements of the fast chlorophyll a fluorescence transient (o - j - i - p) revealed a direct impact of action potentials on the charge separation recombination reactions in psii, although the effect seems to be small rather than substantial. all the data presented here indicate that the main primary target of the electrical signal - induced inhibition of photosynthesis is the dark reaction, whereas the inhibition of electron transport is only a consequence of reduced carboxylation efficiency. in addition, the study also provides valuable data confirming the hypothesis that chlorophyll a fluorescence is under electrochemical control. |
amyand 's hernia is a rare presentation of acute appendicitis, in which an incarcerated or perforated appendix is found in the right inguinal canal. only a few cases have been diagnosed either by computed tomography (ct) or ultrasonography before operation. we report a 24-day - old neonate who presented atypically with tender swelling in the right scrotum. ultrasonography findings suggested amyand 's hernia preoperatively, which was confirmed by histopathological examination following surgery. a 24-day - old male neonate presented at the department of pediatric surgery of the first hospital of jilin university in changchun, china with right scrotal swelling for 4 days. local examination revealed a firm, tender swelling of 3.0 2.0 2.0 cm in the right inguinal region extending into the scrotum. ultrasonography detected a colon - type echo in the right inguinal canal [figure 1 ] measuring 30.0 5.0 mm. the wall of the colon - type structure measured 3.5 mm in diameter and was thickened. the right testis measured 9.9 5.2 mm with an anechoic region of 7.0 12.0 mm anterior to the right testis [figure 2 ]. an ultrasonic diagnosis of right inguinal hernia and right encapsulated hydrocele of the tunica virginals was considered. longitudinal ultrasonography view of the incarcerated appendix (arrows) ultrasonogram demonstrates the right testis (arrows) and hydrocele of the right testis since the patient 's presentation was not typical of a hernia, a primary clinical diagnosis of testicular tumor or epididymitis was considered. because the neonate was considered to have epididymitis, he was started on antibiotics for 2 days. repeat ultrasonography showed similar features as before, including right inguinal hernia and encysted hydrocele in the right scrotum. in addition, the tunica vaginalis, right testis and epididymis were found to be hyperemic and swollen with discharge of purulent material. the tunica vaginalis also contained a colon - type structure adhering to the swollen and inflamed right testis and epididymis. this colon - type structure extended into the right inguinal region and was identified as the appendix. an appendectomy was performed through the inguinal incision alone, and the right hernia sac was ligated. histopathological examination of the appendix specimen revealed fibrous tissue hyperplasia in the interstitium, with acute and chronic inflammatory cell infiltration, vasodilatation, and congestion. an ultrasonography scan 1 week later revealed a normal scrotum and testes on both sides, and the patient was discharged from the hospital. there have been fewer than 200 cases of amyand 's hernia in the literature ; however, only two cases have presented as scrotal swelling. although acute scrotum is a common clinical presentation of torsion of the testis or its appendages and epididymo - orchitis, incarcerated hernia and scrotal and/or inguinal abscess should also be considered in the differential diagnosis. clinical presentations of torsion caused by undescended testes may be quite similar to those observed in amyand 's hernia. ultrasonography is useful in distinguishing torsion of undescended testes from amyand 's hernia because of its high efficiency, low exposure, and real - time properties. ultrasonography can detect hernias with sacculations and moving content of the colon. in this case, ultrasonography detected a soft tissue structure similar to a thick wall of the colon without sacculations, and the hernia contained an isolated soft tissue with a thick wall and moving contents in the inguinal canal. second, ultrasonography, due to its real - time monitoring, can identify the moving content in the intestine. testicular tumors and epididymitis can be reasonably excluded even though these conditions have similar symptoms. in the present case, ultrasonography identified an inflamed appendix and encapsulated fluid around the right testis that was confirmed by surgery as transudate due to appendicitis. in this case, a neonate presented with right scrotal swelling without any signs of peritonitis. this was most likely because inflammatory transudate surrounded only the testis, and thus irritated the testis and its surrounding tissues. since the appendix, rather than the colon, was incarcerated in the inguinal canal, symptoms of intestinal rhythm alteration were not present. specifically, ultrasonography revealed the presence of an echogenic intestinal canal in the right inguinal canal and encapsulated hydrocele of the tunica vaginalis. in conclusion, we would like to highlight that ultrasonography might be a valuable tool in the preoperative diagnosis of amyand 's hernia. | acute appendicitis secondary to hernia incarceration presenting as scrotal swelling is exceptionally rare in neonates. we report a neonate who presented with tender swelling in the right scrotum. ultrasonography detected features of a rare amyand 's hernia. surgical exploration and histopathological examination confirmed the diagnosis. |
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