Smac/Diablo is a recently identified protein that is released from mitochondria after apoptotic stimuli. It binds IAPs, allowing caspase activation and cell death. In view of its activity it might participate in carcinogenesis. In the present study, we analyzed Smac expression in a panel of cervical cancer patients.We performed semi quantitative RT-PCR on 41 cervical tumor and 6 normal tissue samples. The study included 8 stage I cases; 16 stage II; 17 stage III; and a control group of 6 samples of normal cervical squamous epithelial tissue.Smac mRNA expression was below the detection limit in the normal cervical tissue samples. In contrast, 13 (31.7%) of the 41 cervical cancer biopsies showed detectable levels of this transcript. The samples expressing Smac were distributed equally among the stages  with similar expression levels. We found no correlation between the presence of Smac mRNA and histology, menopause, WHO stage or disease status.de novo in a subset of cervical cancer patients, reflecting a possible heterogeneity in the pathways leading to cervical cancer. There was no correlation with any clinical variable.Smac is expressed  Apoptosis is an evolutionarily conserved biological process that plays a fundamental role in development and tissue homeostasis in metazoans . This tyOne IBM protein is the recently identified Smac/DIABLO ,7. Smac in vitro and in vivo; however, further studies are required prior to clinical testing [IAPs are highly expressed in human tumor cells -17, cont testing -22.Recently, Sekimura and colleagues found that Smac expression was significantly lower in primary lung cancers than in normal tissue ; patient2.Cervical cancer cell lines  were obtained from ATCC and cultured as monolayers in Dulbecco Modified Eagle's Medium (DMEM) containing 10% (V/V) fetal bovine serum  at 37°C in a humidified atmosphere of 5% (V/V) COForty-one cervical cancer samples were obtained from the Instituto Nacional de Cancerologia of Mexico. Written consent was obtained from patients before the samples were collected. Tumors were staged according to the International Gynecology and Obstetric Federation (FIGO) system. The samples comprised 8 at stage IB, 16 at stage IIB and 17 at stage IIIB; and a control group comprising 6 samples of normal cervical squamous epithelial tissue  classification system Table .RNA extraction and RT-PCR analysis were performed as described previously . BrieflyThe PCR cycle parameters for Smac were: 10 min enzyme activation at 95°C followed by 3 cycles of 30 s at 95°C and 2 min at 72°C, then 30 cycles of 30 s at 95°C and 30 s at 68°C, and finally 5 min at 72°C. The corresponding parameters for GAPDH were: 10 min enzyme activation at 95°C followed by 25 cycles of 30 s at 95°C, 30 s at 60°C and 30 s at 72°C. The products were electrophoresed on 1% agarose gels and stained with ethidium bromide. Smac mRNA data were expressed as ratios between the densitometric values (Scion Image software) of Smac gene expression. The PCR products were normalized to the amplified GAPDH, the internal reference gene. Gene expression measurements were repeated at least twice.chi square tests . Kaplan-Meier curves for status were generated and log rank was used to test for differences. The mean follow-up was 14.7 months. The statistical package Intercooled Stata 7.0 was used for analyses and statistical significance was accepted when the p value was less than 0.05.To detect a correlation between pathological tumor parameters and normalized Smac expression we used ANOVA  and To ascertain whether Smac is expressed in cervical cancer we performed semiquantitative RT-PCR analyses on a panel of cervical cancer lines, including HeLa, SiHa, CasKi and CaLo cells. As shown in Figure Next, we measured Smac mRNA levels using the same approach in 41 cervical tumor and 6 normal cervical samples. To ensure accurate determinations and to verify equal RNA input, GAPDH mRNA was amplified simultaneously. Figure Tumors proliferate beyond the constraints that limit growth in normal tissue. Therefore, the resistance of tumor cells to apoptosis is an essential feature of carcinogenesis. This has been confirmed by the finding that deregulated proliferation alone is not sufficient for tumor formation because there is concomitant induction of cell death . Overexpde novo.Smac is a recently identified proapoptotic protein that interacts with and inhibits several IAPs, including survivin ,11. It hUnfortunately, we found no correlation between Smac expression and any clinical variable. This could be attributed to differences in tissue expression of IAPs, which are reported to have different binding affinities for Smac. On the other hand, alternative IAPs such as the recently identified Omi/Htra2  might plCancer treatment by chemotherapy and γ-irradiation kills cells primarily by the induction of apoptosis. However, few tumors are wholly sensitive to these therapies, and the development of resistance to therapy is an important clinical problem. Failure to activate the apoptotic programme represents an important mode of drug resistance in tumor cells . Modulatde novo. This finding contrasts with a previous report for lung cancer [During cervical cancer progression, a subset of tumors express the apoptotic protein Smac g cancer , underliThe author(s) declare that they have no competing interests.JMZ Conceived and coordinated the study.VAML: Conceived and coordinated the study. Statistical AnalysisMEC: Performed RT-PCR assaysDCL: Provided the clinical samples and coordinated patient studyCMLG: Coordinated patient assessment, ethical guidelines.JGGS: Provided clinical assessmentThe pre-publication history for this paper can be accessed here:


To study the effect of retinal/ vitreoretinal surgeries on corneal elevations.Patients who underwent retinal/ vitreoretinal surgeries were divided into 3 groups. Scleral buckling was performed in 11 eyes (Group 1). In 8 (25%) eyes, vitreoretinal surgery was performed along with scleral buckling (Group 2). In 12 eyes, pars plana vitrectomy was performed for vitreous hemorrhage (Group 3). An encircling element was used in all the eyes. The parameters evaluated were best-corrected visual acuity (BCVA), change in axial length, and corneal topographic changes on Orbscan topography system II, preoperative and at 12 weeks following surgery.There was a statistically significant increase in anterior corneal elevation in all the three groups after surgery . The increase in posterior corneal elevation was highly significant in all the three groups after surgery . The increase in the posterior corneal elevation was more than the increase in the anterior elevation and was significant statistically in all the three groups .Retinal/ vitreoretinal surgeries cause a significant increase in the corneal elevations and have a greater effect on the posterior corneal surface. Changes in corneal curvature and axial length have been reported following scleral buckling procedure using keratometer -7. VideoAll these studies have reported a change in the corneal curvature in its anterior surface. However, no study has been performed to evaluate the posterior corneal topographic changes with scanning slit topography system following retinal surgeries. Hence, we made an attempt to study the effect of various retinal surgeries on posterior corneal surface using Orbscan II topography system.A prospective study was performed by enrolling patients admitted in the Retina Services of Rajendra Prasad Centre for Ophthalmic Sciences, New Delhi for retinal/ vitreoretinal surgeries. Thirty one eyes of 31 patients who underwent retinal/ vitreoretinal surgery and came for regular follow-up as per schedule during the period between December 2001 and October 2002 were included in the study. Only those patients were included who had not undergone any previous ocular surgery & did not have any corneal pathology. An informed consent was obtained from all the patients.The patients were evaluated preoperatively on parameters of best-corrected visual acuity (BCVA), axial length (AL) measured by Ultrasound A-scan instrument  and detailed corneal examination on slit-lamp biomicroscope and on Orbscan topography system II . The parameters that were evaluated by Orbscan topography system were anterior elevation, posterior elevation and simulated keratometry.All surgeries were performed by a single surgeon (LV) under local anesthesia by peribulbar injection of 6 ml of 2% xylocaine and 2 ml of 0.5% bupivacaine. Eyes with fresh retinal detachment with clear media and absence of advanced proliferative vitreoretinopathy underwent scleral buckling procedure . In all these eyes, the break/ s were localized, cryotherapy was performed and subretinal fluid was drained. Only circumferential buckle of silicone of style 276  was used and radial buckle or sponge was not used in any eye. The size of the buckle was 90° to 360° depending upon the requirement in individual cases. Encircling element of style 240  was used in all the eyes undergoing scleral buckling.In eyes with associated vitreous hemorrhage or advanced proliferative vitreoretinopathy changes along with retinal detachment, vitreoretinal surgery was performed along with scleral buckling . Pars plana vitrectomy was performed and vitreoretinal membranes were removed either by peeling or by segmentation or delamination. Air fluid exchange was performed followed by Air-Silicone oil exchange. In eyes with only vitreous hemorrhage without the presence of retinal detachment, only pars plana vitrectomy was performed . An encircling element of style 240  was used in all these eyes to counter the anterior traction that could not be fully released by vitrectomy in order to avoid lens damage.The intraoperative details including the nature of surgery, size of the buckle, encircling element, drainage of subretinal fluid, vitrectomy & use of silicone oil or gas, were noted. Postoperative treatment included topical ciprofloxacin 0.3% QID, topical dexamethasone 0.1% QID and topical Homatropine 2% QID.The patients were evaluated at 12 weeks following surgery on similar (preoperative) parameters.The data of all the patients were managed on an excel spreadsheet. All the entries were checked for any possible keyboard error. Preoperative and postoperative measurements in the three retinal surgery groups were summarized by mean and standard deviation. Changes following surgery within each group were assessed using paired 't' test. Preoperative and postoperative values in the three groups were compared using one way analysis of variance (ANOVA), followed by bon ferroni correction for multiple comparison. For the three retinal surgery groups, median was computed for increase in various parameters due to surgery. Kruskal Wallis one way analysis of variance was used to compare median increase in the three groups. STATA 7.0 statistical software was used for data analysis. In this study, p-value smaller than 0.05 was considered as statistically significant.The mean age of the patients was 45.96 ± 15.17 (range: 18–78) years and majority (83.87%) of the patients (N = 31) were males. Of these, right eye was operated in 19 (61.29%) patients.The mean preoperative best-corrected visual acuity (BCVA) was hand motion close to face in 26 eyes; counting finger near to face in 4 eyes and 1/60 on snellen's acuity chart in 1 eye. The mean decimal postoperative BCVA was 0.20 ± 0.12 at 12 weeks follow-up after surgery.The mean preoperative anterior corneal elevation as recorded by Orbscan II topography system in group I was 0.006 ± 0.007 mm, which increased to 0.024 ± 0.013 mm at 12 weeks after surgery (p= 0.003). In group II, it increased from 0.009 ± 0.006 mm preoperatively to 0.021 ± 0.010 mm at 12 weeks (p= 0.008) and in group III, it increased from 0.003 ± 0.004 mm preoperatively to 0.012 ± 0.007 mm at 12 weeks follow-up (p = 0.003). On comparative evaluation between the groups, the change in anterior corneal elevation was significant between group I and III (p = 0.04).The mean posterior elevation in group I increased from a preoperative value of 0.016 ± 0.010 mm to 0.043 ± 0.007 mm at 12 weeks after surgery (p= 0.0000). In group II, it increased from 0.014 ± 0.006 mm preoperatively to 0.043 ± 0.007 mm at 12 weeks (p= 0.0001) and in group III it increased from a preoperative value of 0.012 ± 0.005 mm to 0.029 ± 0.006 mm at 12 weeks after surgery (p = 0.0001). A comparative analysis between the groups indicated that the increase in posterior corneal elevation between groups I & III and groups II & III were found to be highly significant .Again, the increase in the posterior corneal elevation was greater than the increase in the anterior elevation in all the 3 groups and on comparative evaluation, the difference in the increase in posterior and anterior elevation was significant statistically in each group .The mean corneal astigmatism in group I increased from 0.89 ± 0.54D preoperatively to 2.50 ± 1.39D at 12 weeks follow-up (p= 0.004). In group II, the average corneal astigmatism increased from 0.87 ± 0.30D to 3.38 ± 2.15D at 12 weeks (p= 0.01) and in group III, the mean preoperative and postoperative corneal astigmatism was 0.85 ± 0.55D and 1.37 ± 0.87D respectively (p= 0.02). A comparative analysis of the change in corneal astigmatism following surgery between groups II & III was significant statistically (p= 0.02).The mean preoperative axial length in group I was 23.27 ± 0.79 mm which increased to 23.98 ± 0.76 mm at 12 weeks after surgery (p= 0.009). The mean preoperative and postoperative (12 weeks follow-up) axial length in group II were 23.92 ± 1.32 mm and 25.94 ± 2.96 mm respectively (p= 0.03). The mean preoperative and postoperative axial length in group III were 22.69 ± 0.87 mm and 22.71 ± 0.83 mm respectively (p= 0.79). Comparative analysis of increase in axial length following surgery between groups I & III and groups II & III were found to be significant statistically .Retinal surgery with or without the use of encircling and buckling elements for external tamponade can alter the shape of the globe. This may cause changes in the refractive status of the eye. Scleral buckling is known to cause a change in the shape of the sclera and can cause induced refractive changes, including astigmatic and nonastigmatic changes -10.We have used Orbscan slit scanning system II to evaluate the corneal topographic changes following retinal/ vitreoretinal surgeries. The data accumulated by Orbscan may be limited by factors such as the accuracy of the system which is ± 20 μm, the measurement noise which leads to both positive and negative difference in the height of the posterior corneal surface and the necessity of aligning the posterior surface before and after surgery which may be a source of error,12. HoweIn the present study, there was a significant increase in both anterior and posterior corneal elevation as detected by scanning slit topography (Orbscan II topography system) following surgery. This increase in the anterior and posterior corneal elevation is probably due to the use of encircling element and/ or buckle in retinal surgeries resulting in corneal steepening. We noted that the change in the posterior elevation was more significant than anterior elevation. A comparative analysis between the three groups indicated that there was no significant difference in the anterior elevation; however the posterior elevation was significantly more in eyes with buckle. It is possible that the buckle and the encircling element have a greater effect on the posterior corneal surface. The increase in the anterior and posterior corneal elevation might be one of the contributing factors for the non improvement of visual acuity in an eye that has undergone retinal/ vitreoretinal surgery. This change might escape detection by routine videokeratography. The anterior protrusion of the corneal back surface induces an increase in the negative power of the corneal surface. Assessing the corneal surface by keratometry or placido disc videokeratography may provide inadequate information regarding refractive change caused by corneal surface alteration that results in retinal/ vitreoretinal surgery.There was an increase in mean corneal astigmatism following surgery in all the groups. This is due to the effect of encircling element or buckle on the corneal surface. Studies have reported that induced astigmatism has been associated with radial scleral buckles,13, circIn retinal/ vitreoretinal surgeries, the encircling band creates a circular indentation of the eye, thereby increasing its anteroposterior axial length; the myopic shift may be upto 3 diopters (D),10,13,16Retinal/ vitreoretinal surgeries result in an increase in the elevation of the corneal surfaces. These changes are more pronounced on posterior corneal surface.None declared.RS designed the study and performed the data collection. NS wrote the manuscript. LKV performed the retinal surgeries. RMP performed the statistical analysis and RBV followed up the patients.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The non-thrombotic effects of thrombin in cardiovascular tissues, as mediated via the protease activated receptors (PARs), and particularly PAR-1, have been the focus of much recent research. The aims of this study were to evaluate the effects of thrombin, a specific PAR-1 activating peptide (PAR1-AP), and a PAR-1 antagonist on human umbilical artery tone in vitro.Human umbilical artery samples were obtained from 17 women at term. Arterial rings were suspended under physiologic conditions for isometric recording. The in vitro effects of thrombin (0.5 units/mL to 3 units/mL), PAR1-AP TFLLR-NH2 [10(-9) to 10(-6) M], and PAR-1 antagonist (N-trans cinnamoyl- p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH2) [10(-9) M to 10(-5) M] on umbilical artery tone were measured.Both thrombin and TFLLR-NH2 exerted a potent cumulative vasodilatory effect on human umbilical artery resistance (P < 0.001). The mean net maximal inhibition (MMI) for thrombin was 53.05%  at tissue bath concentration of 3 units/mL. The MMI with TFLLR-NH2 was 61.50 %  at bath concentration of 10(-6) M. In comparison to vehicle control, the PAR-1 antagonist did not show a significant relaxant or contractile effect (P > 0.05).These findings highlight a potential role for thrombin and PAR-1 receptors in vascular regulation of feto-placental blood flow in normal pregnancy, and in association with the vascular lesions associated with IUGR and pre-eclampsia. In disorders resulting in poor fetal growth, and in pre-eclampsia, thrombotic lesions are frequently observed in the maternal and fetal vascular components of the placenta, -3 and he2 (Thr-Phe-Leu-Leu-Arg-NH2), and the PAR-1 specific antagonist (N-trans cinnamoyl -p-fluoroPhe-p-quanidinoPhe-Leu-Arg-Orn-NH2) on human umbilical artery tone in vitro.There is growing evidence, from vascular tissue studies in several animal models, that non-thrombotic thrombin-mediated signalling events are central to the response to the disease process typical of vascular lesion formation in atherosclerosis . The dirThe study was carried out in the Department of Obstetrics and Gynaecology, University College Hospital Galway, Ireland between May 2002 and April 2003. Sections of human umbilical cord approximately 10 cm in length were excised from the proximal segment of the cord  immediately after elective cesarean delivery. Samples were obtained from 17 women after elective cesarean section at term and from one patient after normal vaginal delivery. All pregnancies were uncomplicated and there was no evidence of hypertensive disease or intrauterine fetal growth restriction. The mean maternal age was 32.47 years (range 25–40 years). The median period of gestation was 38 weeks (range 37–42 weeks). The reasons for cesarean section included previous cesarean section (n = 9), breech presentation (n = 6), previous myomectomy (n = 1) and unstable fetal lie (n = 1). At the time of recruitment 4 women were nulliparous and 13 women were parous.Samples were immediately placed in cold buffered Krebs Henseleit physiological salt solution (pH 7.4) of the following composition: potassium chloride 4.7 mmol/L, sodium chloride 118 mmol/L, magnesium sulphate 1.2 mmol/L, calcium chloride 1.2 mmol/L, potassium phosphate 1.2 mmol/L, sodium bicarbonate 25 mmol/L and glucose 11 mmol/L. Maternal written informed consent was obtained prior to tissue collection, and the tissue collection procedure was approved by the Research Ethics Committee at University College Hospital Galway.2 / 5% CO2) Krebs Henseleit physiological salt solution (PSS) at 37°C and pH 7.4. Rings were allowed to equilibrate for 90 minutes with regular washouts of PSS. During this interval, spontaneous tone developed. After the equilibration period, the vessel rings were challenged with 60 mM potassium chloride (KCl). Three washouts with PSS were carried out once the maximum response had reached a plateau, and a 20 minute recovery period was allowed in order that baseline be attained again. The KCl challenge was performed three times. After the last KCl challenge, 40 minutes recovery was allowed, and contraction was then stimulated by bath exposure of the vessel rings to 5-hydroxytriptamine (5-HT) (10-7 M). Once maximum contractile response to 5-HT was attained, the rings were allowed to remain at plateau for 20 minutes. Concentration- effect experiments were performed by cumulative additions of thrombin, the PAR1-AP, or the PAR-1 antagonist to the tissue bath. Thrombin was added to the tissue bath at an initial concentration of 0.5 units/mL, and this was increased at 20-minute intervals to 1 unit/mL, 2 units/mL and 3 units/mL respectively. The bath concentrations ranges investigated for PAR1-AP were 1 nanomol/L, 10 nanomol/L, 100 nanomol/L and 1 micromol/L (i.e. 10-9 - 10-6M), and for PAR-1 antagonist were 1 nanomol/L, 10 nanomol/L, 100 nanomol/L, 1 micromol/L and 10 micromol/L (i.e. 10-9-10-5M), all at 20-minute intervals.Umbilical arteries were carefully dissected free of Wharton's Jelly and cut in rings 4–5 mm in axial length. Rings were suspended individually on stainless steel hooks inserted into their lumens and mounted under 2 g (30 mN) of isometric tension, in glass-jacketed tissue baths, as previously described ,17. EachThe effects of thrombin, PAR1-AP and PAR-1 antagonist were demonstrated by expressing the mean amplitude calculated during the 20-minute period following addition of each drug concentration, as a percentage of the mean amplitude obtained in the 20 minutes prior to any drug addition. This measurement represents percentage contractility or tone, and subtracted from 100%, provides the percentage relaxation value for each bath concentration of vehicle and study compounds. The net relaxant effect of each compound was calculated by subtracting the percentage contractility value calculated for its respective vehicle , control experiment, at each similar bath concentration. All of the umbilical artery samples used for experimentation for each compound were obtained from different women (i.e. n = 6 for PAR1-AP), for example, was achieved by using umbilical artery samples from 6 different women). The allocation of umbilical artery samples for the different experiments was entirely random.50 value for antagonist inhibition of platelet aggregation stimulated with 1 micromol/L SFLLRN agonist was determined to be 0.1 micromol/L. It was prepared as a 10 millimol/L stock solution in dimethysulphoxide (DMSO) and stored in room temperature protected from direct light. Final bath concentrations of DMSO, at the highest concentration of PAR-1 antagonist, did not exceed 1%, for both study and vehicle control strips. Serial dilutions of thrombin, PAR1-AP and the PAR-1 antagonist were made using Krebs Henseleit physiological salt solutions.Fresh Krebs Henseleit physiological salt solution was made and buffered daily. KCl solutions were prepared on the day of experimentation. A stock solution of 5-HT  was made up in de-ionised water and diluted with Krebs solution. Thrombin was purchased from Sigma-Aldrich  and a stock solution of 1100 U/ml prepared in de-ionised water and stored at -20°C. PAR1-AP was purchased from Tocris Cookson Ltd  and a stock solution of 1 millimol/L was prepared using deionized water, with subsequent dilutions in PSS. The PAR-1 antagonist was prepared and assayed by methods previously described . The IC5Comparisons of measurements of amplitude for each bath concentration of thrombin, PAR1-AP and the PAR-1 antagonist, or respective control vehicle, were made using a one-way ANOVA. Post-hoc comparisons were made using the Tukey HSD test. A P value <0.05 was accepted as statistically significant. The statistical package SPSS version 10 was used for statistical calculations.A recording from control experiments  demonstrating umbilical artery tone due to bath exposure of the ring to serotonin, for the entire duration of an experiment, is shown in Figure 2, the PAR1-AP, similarly exerted a cumulative vasodilatory effect on umbilical artery tone. A representative recording of the cumulative effects of PAR1-AP are shown in Figure -6M / 1 micromol/L) was 61.5% .TFLLR-NH2, no relaxation of umbilical artery tone in vitro was observed, in comparison to vehicle only control experiments. Figure For the PAR-1 antagonist, N-trans cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NHThis study demonstrates that thrombin exerts a potent vasodilatory effect on human umbilical artery vasculature in vitro. While it is known that thrombin has an inhibitory effect on vessel tone in other animal and human vascular tissue types -21 this 2. PAR1-APs are recently designed small synthetic peptide ligands which mimic the effects of proteases by binding directly to the activation site of the PAR-1 receptor, bypassing the need for proteolytic cleavage of the receptor. Soluble peptide ligands, as PAR-APs are, can vary greatly in potency as agonists in comparison with proteases [2 is reportedly one of the more selective PAR1-APs and is deemed most preferable for purposes of studying the physiologic role of PAR-1 [We have not elucidated the exact mechanism, or mechanisms, of the thrombin mediated alteration in vascular tone in human umbilical artery. We have however demonstrated that a similar effect is elicited by bath exposure of the arterial rings to the specific PAR1-AP, TFLLR-NHroteases . TFLLR-Nof PAR-1 . In viewof PAR-1 ,14,15 an2, did not alter umbilical artery tone. This peptide is a competitive antagonist i.e., it competes for the agonist binding site and does not appear to have any other activity [2 is inactive towards the PAR-1 receptor. The true role of PAR antagonists, in such experiments, is for the purpose of investigation of the potential effects of an agonist at a PAR subtype. Pre-incubation of the arterial rings with PAR-1 antagonist, for example, should alter the response elicited by PAR1-AP. Such experiments, with selective blockade of the PAR subtypes, in normal and diseased pregnancies, are a subject of further studies.The PAR-1 antagonist, N-trans cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NHactivity . The diractivity . The resThere are some limitations to this study. All umbilical cord artery samples were obtained at elective cesarean section. This was included in the design of the study in order to maintain uniformity in terms of mode of delivery, and, to avoid using cord samples that may have undergone excessive traction in the third stage of labor at vaginal delivery. It is unknown whether this latter point is valid, or not, in studies using in vitro umbilical preparations. We are currently performing comparative studies from samples obtained at vaginal delivery to further evaluate this matter. Secondly, the effects of thrombin and PAR-1 modulation, on smaller vessels in the feto-placenta circulation , and in association with disorders of pregnancy, would compliment our knowledge of the importance of this pathway. Finally, there are limitations in extrapolating from in vitro experiments to the in vivo situation, but the experiments conducted here represent a reliable and valid in vitro model for these vascular preparations.In conclusion, thrombin exerts a potent vasodilatory effect on umbilical artery preparations in vitro. A similar effect is also observed using a specific PAR1-AP. The potential non-thrombotic role of thrombin, and PAR subtype modulation, in regulation of the feto-placental circulation in normal pregnancy, and in pregnancies complicated by hypertensive disease or intrauterine growth restriction, is highlighted by these findings.2) and wrote the manuscript. JJM designed, supervised the study and wrote the manuscript. All authors read and approved the final manuscript.AJO'L performed the experiments and wrote the manuscript. CJO'S and NR performed the experiments. AMF analysed the data and wrote the manuscript. JTE provided the PAR-1 antagonist (N-trans cinnamoyl- p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH


Depending on the species and the lymphoid organ, activation-induced cytidine deaminase (AID) expression triggers diversification of the rearranged immunoglobulin (Ig) genes by pseudo V (ψV) gene- templated gene conversion or somatic hypermutation. To investigate how AID can alternatively induce recombination or hypermutation, ψV gene deletions were introduced into the rearranged light chain locus of the DT40 B-cell line. We show that the stepwise removal of the ψV donors not only reduces and eventually abolishes Ig gene conversion, but also activates AID-dependent Ig hypermutation. This strongly supports a model in which AID induces a common modification in the rearranged V(D)J segment, leading to a conversion tract in the presence of nearby donor sequences and to a point mutation in their absence. Activation induced cytidine deaminase (AID) is required for antibody diversification, a process that varies in different species. By removing one diversification pathway in chickens, AID induces an alternative method of diversification Immunoglobulin (Ig) genes are further diversified after V(D)J rearrangement by gene conversion, hypermutation, or a combination of the two. Surprisingly, even closely related species employ different strategies: mice and humans use exclusively hypermutation , whereasactivation-induced cytidine deaminase (AID) gene. Whereas it was initially proposed that AID is an mRNA editing enzyme  donors activates AID-dependent Ig hypermutation in DT40 cells. This shows that Ig gene conversion and hypermutation are competing pathways derived from the same AID-initiated intermediate. Furthermore we propose ψV knockout DT40 as an ideal model system to approach the molecular mechanism of Ig hypermutation and as a new tool for in situ mutagenesis.guanine phosphoribosyl transferase (gpt) cassette -green fluorescent protein (GFP) transgene, was chosen for the transfection of the ψV knockout constructs. This AID-reconstituted clone, named AIDR, has the advantage that the appearance of deleterious Ig light chain mutations can be easily detected by the loss of sIgM expression, and that GFP-marked AID expression can be shut down after tamoxifen induction of the Cre recombinase transgene inherited from DT40Cre1  or the gpt gene together with the AID-IRES-GFP gene . The disruption of ψV genes in the rearranged light chain locus and the excision of the AID overexpression cassette were confirmed by Southern blot analysis  DT40 variants demonstrated that about a third contained frameshift mutations in the rearranged light chain V segment that were regarded as byproducts of the Ig gene conversion activity . Most interestingly, subclones of the AID-positive ψV knockout clones  rapidly accumulate sIgM(–) populations, whereas subclones of the AID-negative ψV knockout clones  remain sIgM(+)  after 2 weeks' culture for 24 subclones each of the DT40t clones . Analysiactivity . This vi sIgM(+) . This suRψV– clone  of the V(D)J segments, whereas no or few mutations are present in the downstream C region . To inveain gene . Althougain gene B, known RAD51 paralogs induces Ig hypermutation in DT40 cells .It was previously reported that the deletion of 40 cells . To complone see C and to knockout . NeverthRψV– clone, 94 light chain cDNAs from sorted sIgM(–) cells were amplified and sequenced. Although one short insertion and five deletions were detected in this collection  AID dependence, (2) a predominance of single nucleotide substitutions, (3) distribution of the mutations within the 5′ transcribed region, (4) a preference for hotspots, and (5) Ig gene specificity. The only differences between the mutation activity induced by loss of ψV and Ig hypermutation in vivo are the relative lack of mutations in A/T bases and a predominance of transversion mutations in the ψV knockout clones. However, these differences are also seen in hypermutating Epstein Barr virus–transformed B-cell lines  and DT40paralogs , indicatlone see . AlthougRAD51 paralogs, the second stage involves other recombination factors, such as RAD54.The induction of Ig hypermutation by the blockage of Ig gene conversions supports a simple model explaining how hypermutation and recombination are initiated and regulated . InitiatRAD51 paralogs not only decrease Ig gene conversion, but also induce Ig hypermutation  parallel analysis of Ig gene conversion and hypermutation, (2) conditional AID expression, (3) easy genome modifications by gene targeting, (4) normal cell proliferation and repair proficiency, and (5) Ig locus specificity of hypermutation. The ability to induce gene-specific hypermutation in the DT40 cell line might also find applications in biotechnology. One possibility is to replace the chicken antibody coding regions with their human counterparts and then to simulate antibody affinity maturation from a repertoire that continuously evolves by Ig hypermutation.The AIDCre1, which displays increased Ig gene conversion due to a v-myb transgene and contains a tamoxifen-inducible Cre recombinase, has been described previously  and EF6/EF8  primer pairs, respectively. The PCR products of these genes were cloned into the pBluescript plasmid vector (Stratagene) and were sequenced using the M13 reverse primer.To minimize PCR-introduced artificial mutations, PfuUltra hotstart polymerase  was used for amplification prior to sequencing. Long-range PCR, RT-PCR, and Ig light chain sequencing were performed as previously described . The prohttp://www.ncbi.nlm.nih.gov/) accession numbers of the genes discussed in this paper are as follows. AID , RAD54 (GGU92461), RAD52 (U01047), Bu-1 (X92865), and EF1α (NM_204157).The GenBank (


Tumor-associated antigens recognized by humoral effectors of the immune system are a very attractive target for human cancer diagnostics and therapy. Recent advances in molecular techniques have led to molecular definition of immunogenic tumor proteins based on their reactivity with autologous patient sera (SEREX).Several high complexity phage-displayed cDNA libraries from breast carcinomas, human testis and breast carcinoma cell lines MCF-7, MDA-MB-468 were constructed. The cDNAs were expressed in the libraries as fusion to bacteriophage lambda protein D. Lambda-displayed libraries were efficiently screened with sera from patients with breast cancer.A panel of 21 clones representing 18 different antigens, including eight proteins of unknown function, was identified. Three of these antigens  were found to be overexpressed in tumors as compared to normal breast. A serological analysis of the 21 different antigens revealed a strong cancer-related profile for at least five clones .Preliminary results indicate that patient serum reactivity against five of the antigens is associated with tumor disease. The novel T7-1 antigen, which is overexpressed in breast tumors and recognized specifically by breast cancer patient sera, is potentially useful in cancer diagnosis. A recent development in tumor immunology is based on the idea that the immune system can distinguish between normal and tumor tissues. Various studies suggest that both the cellular and humoral components of the immune system are able to recognize tumors  . The preD gene. The N-terminal display system greatly reduces the selection of artifactual peptides, in comparison with a C-terminal fusion library displayed on lambda  were found to be overexpressed in tumors as compared to normal breast. Recognition by human sera of five of the selected antigens  was associated with cancer diagnosis.Specimens of breast carcinoma and autologous sera from breast cancer patients (B81-B96) were obtained from M. G. Vannini Hospital, Rome. A panel of human sera from breast cancer patients B1-B20, B36-B80 was provided by the Division of Medical Oncology, Federico II University of Naples. All the human biological samples were obtained through informed consent.SpeI, NotI sites at the 5'-end of the D gene in λKM4 vector [SpeI and NotI sites and encodes for a GS linker between the fusion site and gpD, Figure λKM8 was constructed by cloning the oligonucleotide duplex  KM46 5’-CTAGTCTCCTCAGCGGCCGCGGTTCCGGTTCTGGTTCCGGTTCTGGTTCCGGTTCTGGT-3’ and KM47 5’-GGCCACCAGAACCGGAACCAGAACCGGAACCAGAACCGGAACCGCGGCCGCTGAGGAGA-3’ into RsrII and EcoRI restriction enzymes. pKM7 was digested with SpeI and NotI to obtain pKM9, by direct cloning of the oligonucleotide duplex KM48 5’-CTAGCGGTTCCGGTTCTGGTTCCGGTTCTGGTTCCGGTTCTGGCACTAGTCTCCTCAGC-3’ and KM49 5’-GGCCGCTGAGGAGACTAGTGCCAGAACCGGAACCAGAACCGGAACCAGAACCGGAACCG-3’. λKM10 was constructed by cloning pKM9, which was linearized by digestion with XbaI restriction enzyme, into the XbaI site of λDam15imm21nin5 [SpeI, NotI sites at the 3'-end of the D gene and encodes for a flexible GS linker between gpD and the cloned protein fragment, Figure The plasmid pKM7 is a derivative of pKM3 , which wmm21nin5 . The resmRNA from breast carcinoma cell lines MCF-7 and MDA-MB-468 was isolated in a single step by QuickPrep Micro mRNA Purification Kit  according to manufacturer's instructions.Tumor samples from breast carcinoma patients were obtained as surgical specimens and immediately frozen in liquid nitrogen. Total RNA was prepared by Total RNA Isolation System  and purified to Poly A+ RNA using PolyATract mRNA Isolation Systems (Promega).Total RNA from normal testis was purchased from Genpak, UK (# 061023). Total RNA from normal breast (pool of 3) was purchased from Stratagene, La Jolla, CA (# 735044).+ RNA from cell lines or human tissues were used to synthesize cDNA by random priming, using TimeSaver cDNA Synthesis Kit . RNasin Ribonuclease Inhibitor (Promega) was added to first-strand synthesis reaction.From 1 to 5 μg of the purified poly(A)GCGGCCGCTGGNNNNNNNNN-3'), K79 (5'-GCGGCCGCTGGCNNNNNNNNN-3'), and K81 (5'-GCGGCCGCTGGCANNNNNNNNN-3') was used for priming. They all carry a NotI site (underlined) at their 5' end, and a random sequence of nine nucleotides at their 3' end, positioned in the three possible reading frames. The second strand was synthesized by nick translation according to the manufacturer's instructions.A mixture of the following oligonucleotides (130 pmol): K64 (5'-One hundred ng of ds cDNA were randomly primed with 25 pmol of oligonucleotide K56 (5'-GGCCGGCCAACNNNNNNNNN-3'), constituted by a constant sequence at the 5' end, and a random 3'sequence. The reaction mixture was purified by QIAgen QIAquick columns.ACTAGTGGCCGGCCAAC-3'), K60 (bio-5'-GCACTAGTCGGCCGGCCAAC-3'), K61 (bio-5'-GCACTAGTCGGGCCGGCCAAC-3') and K65 (bio-5'-GGAGGCTCGAGCGGCCGCTGG-3'). K59, K60 and K61 carry the same constant sequence of K56 positioned in the three possible frames with respect to a SpeI site (underlined) allowing directional cloning. K65 carries a NotI site (underlined), that anneals to the 5' end of the reverse strand of cDNA.Approximately 0,2 ng of the above randomly primed ds cDNA was amplified by PCR with biotinylated primers: K59 , filtered by Microcon-100 columns  to reduce the number of small fragments and additionally fractionated by 6% PAGE. DNA smear, corresponding to 300–1000 base-pair fragments, was cut and eluted from gel according to standard procedure .SpeI and NotI enzymes, in order to remove the biotinylated extremities and uncut fragments, a 20-minute incubation with streptavidin M-280 Dynabeads  was performed. After additional filtration on Microcon-100 the insert was cloned in λKM8 or λKM10 vectors.After digestion with SpeI, NotI enzymes and dephosphorylated. For each library 5 ligation mixtures, each one containing 0.5 μg of vector and about 3 ng of insert, were performed. After overnight incubation at 4°C the ligation mixtures were packaged in vitro by lambda packaging extract . BB4 cells were infected by lambda and plated in top-agar on 100 (15 cm) NZY plates. After overnight incubation phages were eluted from the plates with SM buffer, purified, PEG/NaCl precipitated [The vector was digested with ipitated  and stor4, 1% Triton) for 30 minutes at 37°C under gentle agitation. 1010 pfu of lambda library were then added to the preincubated mixture for a further incubation of 1 hr. Magnetic beads (100 μl), linked to Protein A  were washed twice with the blocking solution. Mixture of library with serum was incubated with the beads for 10 min at RT under agitation. The beads were washed 10 times with 1 ml of washing solution . The bound phages were recovered by infection of 600 μl BB4 cells added directly to the beads. After a 20-minute incubation 10 ml of molten NZY-top agar (48°C) was added to the mixture of beads with infected cells and immediately poured onto NZY plates (15 cm). Next day the phage particles were harvested by incubation of the plates under agitation with 15 ml of SM buffer for 4 hours at 4°C. The phage particles were purified by PEG/NaCl precipitation and stored in 1/10 of initial volume of SM with 0.05% NaN3 at 4°C.Two μl of human serum were preincubated with 10 μl of BB4 bacterial extract and 10 μl of UV-killed lambda phage in 1 ml of blocking buffer . For maximum sensitivity specific primers for the different genes were designed to amplify sequences located near the 3' end of gene's transcript. Twenty-five cycles of PCR were performed from 1 μl of each cDNA template, normalized through PCR amplification of the β-actin gene.Five hundred ng of poly(A)Lambda libraries were constructed by directional cloning of randomly primed cDNA from human breast carcinoma cell lines MCF-7 and MDA-MB-468, from human breast carcinomas or from human testis into the phage display vector λKM8 to generate fusions with the N-terminus of gpD . The other antigens either reacted with a low percentage of cancer sera, or the total panel of the tested sera was too small to offer any clear conclusion.Phage lysates were prepared from all the selected clones as previously described  and testTwenty-one positive clones were found to encode fragments from 18 different gene products, such as 4 clones  showing homology to different regions of the same reverse transcriptase gene . Only 4 clones with specific tumor-related reactivity were isolated from the T6 library. However, C-terminal fusion might allow efficient display and selection for some antigenic C-terminal protein domains. In fact, the C-terminal fragment of fucosyltransferase (clone T6-7) was isolated from the T6 library.The panel of selected TAAs in Table 2 see  containsOther selected antigens with known or predicted intracellular location are alpha-6-fucosyltransferase (clone T6-7) and zinc finger protein 258 . Alpha-fucosyltransferase catalyzes the transfer of GDP-fucose to oligosaccharide chains linked to proteins, lipids and sugars  and resiThe human myc oncogene is transcribed from four alternative promoters giving rise to mRNAs for Myc1, Myc2, MYCHEX1 and 5'ORF . Clone TAmong isolated antigens, there are 4 clones  having between 55–91% sequence identity with that of a reverse transcriptase homolog . The four underlined gene products from the list in parenthesis were analyzed for mRNA expression in tumors and normal breast tissue. The mRNA expression levels were analyzed by PCR from SMART-cDNA template in 7–10 breast cancer specimens, one metastasized lymph node, normal breast, testis and peripheral lymphocytes from healthy donors. Two of these 4 unknown antigens and T11-3 were found to be frequently overexpressed in breast cancer. In particular clone T7-1, which was classified as encoding for an unknown protein since it has 100% identity only with KIAA1288 from EST database, was found to be overexpressed in breast carcinomas. This finding, together with the good reactivity of T7-1 protein with sera from tumor patients, identifies this antigen among the most promising targets for diagnosis of the disease.Eight proteins in the tumor antigen panel are unknown, or hypothetical proteins with unknown functions . MGCIn spite of the fact that several promising antigens were identified from cDNA library constructed from testis mRNA, none of the antigens derived from testis or other libraries could be classified as specific testis/cancer (CT) antigen, because of their low expression in testis  or expression in other tissues as well.In this work we analyzed the frequency of the immune response to the 21 identified antigens by using a panel of sera from tumor patients and healthy donors. In general, we observed a low frequency of serum reactivity with the antigens, which was expected and is similar to that of the vast majority of SEREX-identified clones . A signiTaken together, these results lead us to believe that analysis of a complex panel of serologically-defined TAAs, with very large panels of sera from patients classified according to clinical parameters, i.e., age of patient, stage, extent and outcome of disease, etc. could lead to a much clearer understanding of the role, specificity and significance of the immune response versus disease in cancer patients.We demonstrated that a lambda display-based approach permits the efficient identification of tumor antigens, potential immunological targets in breast cancer. The list of 21 antigens identified in this work contains eight proteins of still unknown function. Three of the genes  were found to be overexpressed in tumors as compared to normal breast. Five of the selected antigens  were recognized specifically by breast cancer patient sera.aa, amino acids; Ag, antigen; EST, expressed sequence tags; ds cDNA, double-stranded cDNA; SEREX, serological identification of antigens by recombinant expression cloning; PAGE, polyacrylamide gel electrophoresis; PEG, polyethylene glycol; pfu, plaque-forming units; Sos1, son of sevenless homolog 1; TAA, tumor-associated antigen.EP, PV, AP, GM, EB, FF and OM are salaried by an organization holding two patents relating to the content of the manuscript.EP and PV contributed equally to the work. EP, PV and AP carried out cDNA library experiments, recombinant protein production, immunoassays and database search. GM and EB performed selection experiments. SB, MLD and MC contributed to immunological analysis of tumor antigens. ADPC and AL performed clinical studies. EC coordinated all medical aspects of the work. OM planned and performed molecular biology experiments, and teamed with FF in design and coordination of the entire project. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Table 2. List of identified TAAs. The File is given in Microsoft Word format. The table contains information about selected antigens.Click here for file


A paucity of information about male reproductive health and a perceived interest in involvement among local men provided the impetus for carrying out a village based male reproductive health camp. The aim was to investigate men's willingness to participate in such camps, and to describe reproductive health problems in men.Structured interviews were carried out with 120 men attending a reproductive health check-up in a village in rural West Bengal, India. General information, details of family planning methods used and data on reproductive health complaints were collected. Clinical examinations were also carried out. Socio-demographic characteristics were compared for men with and without reproductive health and urinary complaints.Three quarters of the married men were using contraception, but the majority stated that their wives were responsible for it. The most common reproductive health complaint was urinary problems; 28% had burning on urination, and 22% reported frequent and/or difficult urination. There were few social or demographic differences between men with and without problems. Seventeen percent of the men had clinically diagnosed reproductive health problems, the most common being urethral discharge. None of the men with diagnosed problems were using condoms.This study highlights the interest of men in their reproductive health, but also highlights the high proportion of men with problems. In addition, a number of men with clinically diagnosed problems had not reported them in the interviews, illustrating either the reticence to report or the lack of knowledge about symptoms of reproductive health problems. Recommendations for future programmes and research in this field are given. Reproductive health is a major world priority, with particular problems in developing countries. However, as Ndong  states, The documentation of the prevalence of male reproductive health problems has been identified as a research priority by the World Health Organization (WHO) . Until nInformation about male reproductive health problems may help in the planning of future services, the needs for screening, prevention and treatment. In order to collect this information, a village based reproductive health camp for men was carried out. Health camps are used to provide services in places where the perceived need and demand is high but access to treatment is poor due to lack of information about available services, distance from health facilities, or low economic status of the population. It is one of the preferred methods of service provision in rural villages. The usual model is a temporary clinic set up by a team of health personnel at a community-owned location  to provide services and medicines usually free of cost based on the health needs of the community. The aim of this study was to investigate men's willingness to participate in such camps, and to describe reproductive health problems in this group of men. This paper reports on the findings from this health camp.Men aged 15–60 years, both married and unmarried and living in one rural village were invited to attend. The village is relatively small (approximately 4000 residents), mostly consisting of low-income, Muslim population, and had been highlighted by local health workers as having particularly poor knowledge about reproductive health issues.The camp was held in a village school on one day in February 2000. The community was consulted about the camp through a variety of routes, including men's groups, Panchayats , schoolteachers and specially convened public meetings; all of these groups agreed to the holding of the camp in the village. The camp was publicised through peer educators, health workers and group meetings, and was described as a reproductive health check-up, with free treatment if necessary. A male team was used as feedback from health workers and peer educators in the village indicated that men would be more willing to attend if male doctors carried out the examinations.Health workers carried out a confidential interview on arrival to obtain demographic information , information about personal hygiene, details about family planning issues and about general health complaints  by using a structured interview schedule. Male doctors then asked about reproductive health complaints  and current sexual activity (sex outside marriage or with a prostitute), and carried out clinical examinations . Blood samples were taken for Venereal Disease Research Laboratory (VDRL) test from all attendees.Those men detected as having sexually transmitted infections and urinary problems were given the appropriate medication, provided with condoms and referred to hospital when necessary. Advice and instructions for condom use were given and patients were encouraged to bring their partners for treatment.The Child in Need Institute (CINI) is a non-governmental organisation (NGO) working in West Bengal, and is one of the four national NGOs in India. The organisation has, for the last 30 years, focused on the health and nutrition of women and children, with the additional involvement of adolescents and men. CINI started working on reproductive health with men in rural areas in 1997. To date, this involvement has entailed orientation and education with men's groups, intensive training of voluntary peer educators and facilitators and individual interaction with health workers. There are at present no specific clinical services for men provided by CINI: men who are identified as having a problem are given individual information and counselling and are referred to other clinics for treatment.The information collected during the interviews, clinical examination and pathology reports was entered into the Epi-Info data entry and analysis package . StatistOne hundred and twenty men attended the camp; all men responded freely to the interview, and all but two agreed to a clinical examination. This sample represents about 10% of the local male population aged 15–60 years.The mean age in this group of men was 29.6 years (range 16–60). Fifty-nine percent of the men were married (n = 71); the mean age at marriage was 22.6 years (range 14–34). Fifty-two percent of the attendees had children (comprising 90% of the married men); among the men with children, the mean number was 2.8 (range 1–7). The literacy rate was 71% among the attendees, although more than half of these had only studied up to class V or less. Eighty four percent of the attendees  had a monthly income of less than Rs. 2000 (~$45); 40% (n = 48) were daily wage earners, and a further 30% (n = 36) were in service , 15% (n = 18) were either unemployed or students. One third of the men worked away from home for at least one night each month, half of these for 1–5 days, and half for more than 5 days per month. Only 11 attendees indicated that they consumed alcohol.Twenty percent (n = 24) reported good personal hygiene , however 28% (n = 33) had very poor hygiene practices .Four fifths of men (82%) reported at least one problem with their general health, the most commonly reported problems were coughs and colds , and weakness , other problems included indigestion, backache and headache. Two men were identified as having hypertension.Forty-three percent of all men (n = 51) reported that they or their wives were using some kind of family planning, this represented 73% of the married men; only one unmarried man was using contraception (condoms). Over half of the men stated that their wives are responsible for family planning, they are using the oral contraceptive pill, have an intrauterine device or had sterilisation. Eight men were using condoms, and one man had had a vasectomy. The main reason given for not using family planning was being unmarried. Two men reported a lack of knowledge of family planning, two men had religious objections and five men said that they wanted children.Six married men admitted to having extramarital sex, three of them with prostitutes. Seven unmarried men (14%) reported having sexual relations; three of them with prostitutes. None of the men visiting prostitutes reported using condoms, however one married man having extra marital sex reported using condoms. It did not appear that the men visiting prostitutes or having extra-marital sex were any different from the other men in terms of monthly income, education or number of nights working away from home.Half of the attendees reported at least one reproductive health complaint (n = 60) table . Only thOn clinical examination, 20 men (17%) had findings of reproductive health problems, table , fourteeThere was a trend that men having extramarital sex and men having sex with prostitutes were more likely to have positive clinical findings than men not having extramarital sex . None of the eight men using condoms had positive clinical findings compared with 18% (n = 20) of the men not using condoms.This study indicates that there is a high level of reproductive health problems among those men attending a reproductive health camp in a rural area of India. More than one in ten men had urethral discharge and over one third reported urinary problems. The high number of men reporting urinary symptoms is similar to that of an unpublished study in Uttar Pradesh, India . HoweverThe enthusiasm from men for involvement in reproductive health programmes has been reported elsewhere in Southern Asia ,15. FactWang  identifiThe high proportion of men reporting urinary problems has also highlighted the need testing of urinary samples. One study in Bangladesh indicated that among 47 men reporting pain passing urine, only one was infected with Chlamydia trachomatis ; we are A significant number of men did not complain of reproductive health problems  during the interview, but had positive clinical findings table . This inThere are some limitations to this study. First of all the numbers were relatively small and a larger sample size might have given further insight. Information obtained from urethral smears of all attendees may have provided important information. However, this was not possible in our setting due to technical problems. Further, not all areas of relevance to reproductive health were addressed in this study. Psychosexual problems have been commonly reported elsewhere , but werThis study provides important information about male reproductive health problems in a sample of men in rural West Bengal, India. The response of the men and the fact that nearly all attenders were willing to undergo a clinical examination is encouraging, and emphasises the possibilities for future research. The high level of reproductive health problems identified on clinical examination, but not reported in interviews, plus the low levels of condom use illustrate the need for reproductive health interventions with such groups. Future research could build on the findings of this exploratory study.CINI = Child in Need InstituteVDRL = Venereal Disease Research LaboratoryThe author(s) declare that they have no competing interests.KMD participated in the design of the study, performed the statistical analysis and drafted the manuscript. SD conceived the study, participated in its design, co-ordination, data entry and analysis and contributed to the manuscript. RD conceived the study, and participated in the design, co-ordination and analysis. All authors read and approved the final manuscript.


Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target protein in an unmodified form or by applying modifications using expressivity and solubility tags.Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed for the production of protein preparations bound for downstream purification and processing.  Escherichia coli are outstanding factories for recombinant expression of proteins. An expression system for the production of recombinant proteins in E. coli usually involves a combination of a plasmid and a strain of E. coli [Microorganisms like the enterobacterium  E. coli . The mai E. coli .Under normal cellular conditions a subset of cytoplasmic proteins are able to fold spontaneously  while agE. coli cytoplasm. Refolding from inclusion bodies is in many cases considered undesireable, but is however sometimes the method of choice. The major obstacles are the poor recovery yields, the requirement for optimization of refolding conditions for each target protein and the possibility that the resolubilization procedures could affect the integrity of refolded proteins. In addition, the purification of highly expressed soluble protein is less expensive and time consuming than refolding and purification from inclusion bodies. Maximizing the production of recombinant proteins in a soluble form is therefore an attractive alternative to in vitro refolding procedures. The methods used to mediate soluble expression can be divided into procedures where target modification is avoided and procedures where the target sequence is engineered , which are withdrawn from the host metabolism for maintenance and expression of the foreign DNA . The forin vivo aggregation of recombinant proteins consists of cultivation at reduced temperatures [A well known technique to limit the eratures . This steratures  and refeE. coli chaperones are increased at temperatures around 30°C [The aggregation reaction is in general favored at higher temperatures due to the strong temperature dependence of hydrophobic interactions that determine the aggregation reaction . A direcund 30°C ,12. The However, a sudden decrease in cultivation temperature inhibits replication, transcription and translation . TraditiDifferent strategies aimed at optimizing the expression of recombinant proteins at low temperature are as follows.cspA promoter was developed for the expression of proteins at low temperature [cspA promoter is highly induced at low temperature and is well repressed at and above 37°C. A sequence encoding the TolAI-β-lactamase fusion protein which is toxic to E. coli and rapidly degraded at 37°C was placed under the control of the cspA promoter. Temperature downshift to 15 or 23°C abolished degradation of the fusion protein and the toxic phenotype associated with expression at 37°C was suppressed. It was suggested that this system is a valuable tool for the production of proteins containing membrane-spanning domains or otherwise unstable gene products in E. coli.A system based on the perature . The cspOleispira antarctica RB8T) allow E. coli to grow at high rates at 4°C [O. antarctica RB8T was co-expressed with Cpn60 and Cpn10 in E. coli at 4°C. This procedure increased the specific activity of the purified esterase 180 fold as compared to enzyme prepared from cultivations at 37°C. It was concluded that the low temperature was beneficial to folding and the system was suggested as a tool for expression and correct folding of recombinant proteins in the cytoplasm of E. coli.A principle that allows for protein expression and folding at 4°C was presented recently . This prs at 4°C . An esteNumerous specialized host strains have been developed to overcome the metabolic burden related to high level protein expression.E. coli mutant strains have contributed significantly to the soluble expression of difficult recombinant proteins. C41(DE3) and C43(DE3) are mutants that allow over-expression of some globular and membrane proteins unable to be expressed at high-levels in the parent strain BL21(DE3) [1Fo ATP synthase subunit b membrane protein in these strains, in particular C43(DE3), is accompanied by the proliferation of intracellular membranes and inclusion bodies are absent [ and a high number of reports on their use in expression of difficult proteins have been published [Two L21(DE3) . Expresse absent . These sublished -23. A reublished .E. coli cytoplasm are actively kept reduced by pathways involving thioredoxin reductase and glutaredoxin. The disulfide bond dependent folding of heterologous proteins is improved in the Origami strains from Novagen. Disruption of the trxB and gor genes encoding the two reductases, allow the formation of disulfide bonds in the E. coli cytoplasm. The trxB (Novagen AD494) and trxB/gor (Novagen Origami) negative strains of E. coli have been selected in several expression situations [trxB) [Cysteines in the tuations -27. Folds [trxB) . OverexpThe simplest way to produce a recombinant protein is by batch cultivation. Here all nutrients required for growth are supplied from the beginning and there is a limited control of the growth during the process. This limitation often leads to changes in the growth medium such as changes in pH and concentration of dissolved oxygen as well as substrate depletion. Furthermore inhibitory products of various metabolic pathways accumulate. Cell densities and production levels are only moderate in batch cultivations.In fed batch cultivations, the concentration of energy sources can be adjusted according to the rate of consumption. Several other factors can also be regulated in order to obtain the maximal production level in terms of target protein per biomass. The formation of inclusion bodies can be followed in fed batch cultivations by monitoring changes in intrinsic light scattering by flow cytometry . This alE. coli recombinants were cultivated in the presence of 0.1 mM Mg2+ [Folding of some proteins require the existence of a specific cofactor. Addition of such cofactors or binding partners to the cultivation media may increase the yield of soluble protein dramatically. This was demonstrated for a recombinant mutant of hemoglobin for which the accumulation of soluble product was improved when heme was in excess . Similar mM Mg2+ . An impoE. coli encode chaperones, some of which drive folding attempts, whereas others prevent protein aggregation [E. coli ribosome they associate with the trigger factor chaperone [A possible strategy for the prevention of inclusion body formation is the co-overexpression of molecular chaperones. This strategy is attractive but there is no guarantee that chaperones improve recombinant protein solubility. regation ,11,33. Ahaperone . Exposedhaperone . GroEL  required the co-expression of gene product 31 (phage co-chaperonin gp31) [E. coli.Soluble expression in in gp31) . ExpressAnother study reports the purification of a heterodimeric complex by expression of each subunit (pheromaxein A and C) as a fusion to thioredoxin . Each suin vivo solubility of target proteins. New systems for co-expression of multiple proteins involved in complex structures enable such strategies [Conclusively, interaction partners potentially favour rategies .Target proteins are not always expressed in a soluble form by the strategies described above. The last part of this review discusses how misfolded proteins can be engineered or pushed to evolve and selected to gain soluble expression.in vivo [The use of affinity tags in recombinant protein purification has a long tradition. Not only have they been exploited for the development of generic purification strategies. Affinity tags have been observed to improve protein yield, to prevent proteolysis and to increase solubility in vivo ,45.E. coli maltose binding protein (MBP) and the E. coli N-utilizing substance A (NusA). MBP (40 kDa) and NusA (54.8 kDa) act as solubility enhancing partners and are especially suited for the expression of proteins prone to form inclusion bodies. Although many proteins are highly soluble, they are not all effective as solubility enhancers. E. coli MBP proved to be a much more effective solubility partner than the highly soluble GST and thioredoxin proteins in a comparison of solubility enhancing properties [E. coli MBP [Among the most potent solubility enhancing proteins characterized to date are the operties . Solubilcoli MBP . A precicoli MBP ,49. The coli MBP .E. coli cytoplasm [. The Wilkinson-Harrison model along with experimental data identified NusA as a highly favorable solubility partner [E. coli [Wilkinson and Harrison proposed a model for the theoretical calculation of solubility percentages of recombinant proteins expressed in the ytoplasm . A webse partner . The maj[E. coli ,53. Nume[E. coli -57.cis trans isomerases (PPIases) [Natural molecular chaperones that have been used as solubility enhancers include prolyl PPIases) , thioredPPIases)  and dsbAPPIases) .in vivo and in vitro when fused to the C-terminus of the light chain variable domain of an IgG [Fusion partners such as MBP and NusA are relatively large proteins. We recently suggested the use of a highly soluble N-terminal fragment of translation initiation factor IF2 (17.4 kDa) as a solubility partner . The usef an IgG .In a recent study it was shown that a 17 residue C-terminal extension of Pfg27 resulted in several fold enhancement of soluble expression . SeveralScreening strategies have been employed to select for favorable fusion partners in a high throughput manner. In such a system more than 80% of the proteins tested showed high levels of expression of soluble products with at least one of eight fusion partners including NusA, intein, thioredoxin, His-tag, MBP, calmodulin binding protein and glutathione-S-transferase . These rStructural and functional genomics and proteomics are important elements in the evaluation of gene function. The expression and purification of properly folded proteins in a high throughput manner are key elements in these studies. A number of different approaches to the high throughput screening of soluble expression products have been described recently.The intrinsic folding yield, stability and solubility of target proteins can be improved by engineering the target protein. When structural information is available, the solubility of the expressed protein has been improved by rational site directed mutagenesis . A more E. coli cells expressing target genes fused to the GFP-gene is related to the solubility of the target gene expressed alone [E. coli can be improved by directed evolution approaches for a certain target protein by screening for fluorescing mutants. This approach evolved three insoluble proteins including Pyrobaculum aerophilum methyl transferase, tartrate dehydratase β-subunit and nucleoside diphosphate kinase to be 50%, 95% and 90% soluble respectively [Fluorescence of ed alone . Hence, ectively . The GFPectively .A similar approach is the use of selective pressure. By fusing target proteins with chloramphenicol acetyl transferase (CAT) more soluble fusion protein mutants were selected on media containing progressively higher levels of chloramphenicol . Furtherin trans. Active β-galactosidase can be detected when the α peptide becomes soluble and restore enzyme activity by binding to lacZΩ [Another fusion reporter method use the β-galactosidase α peptide as fusion partner in a screen for lacZα complementation in a system where inactive lacZΩ is supplied to lacZΩ .E. coli promoters that are up-regulated when misfolded proteins are expressed. It was found that the promoter for the small heat shock protein ibpA could be fused to lacZ and used as a reporter for misfolded protein [An innate host cell response is induced when recombinantly expressed proteins are misfolded. This response can be monitored by the transcription from  protein . This re protein .Soluble fusion proteins are not necessarily biologically active and properly folded. Several reports have demonstrated that soluble preparations of fusion proteins have low biological activity as compared to the non-fused protein . It was E. coli cytoplasm.A few strategies that are radically different from the conventional fusion partner and selection approaches have been developed for the potential rescuing of recombinant proteins from misfolding in the A system based on artificial oil bodies was developed and illustrated by a fusion protein composed of oleosin and GFP . The expin vivo rescuing system based on the E. coli ribosome was recently presented [in vivo aggregation by fusing them to ribosomal protein L23. The fusion protein is expressed in a strain of E. coli deficient in the essential L23 ribosomal protein. This allows for the covalent coupling of target proteins to the highly soluble ribosomal particles. Ribosomes with coupled target protein can subsequently be isolated by centrifugation methods and the target protein released in a highly enriched form by site specific protease cleavage.An resented . Target E. coli recombinants. A subset of the methods focus on relieving the cellular stress that is a response to the extreme metabolic situation experienced by the host cell during the process of hyperexpression of a single or a few proteins. A second subset of methods focus on improving the solubility and structural stability of the expressed protein, by the combination of the target protein with specific peptide tags. A common trait in modern expression strategies is the skillful combination of the utensils in the genetic toolbox, but also a constant reconsideration of the accepted paradigms in trade of protein expression.We have reviewed the most recent improvements in obtaining soluble and functional protein preparations from


Prediction of the transmembrane strands and topology of β-barrel outer membrane proteins is of interest in current bioinformatics research. Several methods have been applied so far for this task, utilizing different algorithmic techniques and a number of freely available predictors exist. The methods can be grossly divided to those based on Hidden Markov Models (HMMs), on Neural Networks (NNs) and on Support Vector Machines (SVMs). In this work, we compare the different available methods for topology prediction of β-barrel outer membrane proteins. We evaluate their performance on a non-redundant dataset of 20 β-barrel outer membrane proteins of gram-negative bacteria, with structures known at atomic resolution. Also, we describe, for the first time, an effective way to combine the individual predictors, at will, to a single consensus prediction method.We assess the statistical significance of the performance of each prediction scheme and conclude that Hidden Markov Model based methods, HMM-B2TMR, ProfTMB and PRED-TMBB, are currently the best predictors, according to either the per-residue accuracy, the segments overlap measure (SOV) or the total number of proteins with correctly predicted topologies in the test set. Furthermore, we show that the available predictors perform better when only transmembrane β-barrel domains are used for prediction, rather than the precursor full-length sequences, even though the HMM-based predictors are not influenced significantly. The consensus prediction method performs significantly better than each individual available predictor, since it increases the accuracy up to 4% regarding SOV and up to 15% in correctly predicted topologies..The consensus prediction method described in this work, optimizes the predicted topology with a dynamic programming algorithm and is implemented in a web-based application freely available to non-commercial users at  Transmembrane proteins are divided to date into two structural classes, the α-helical membrane proteins and the β-barrel membrane proteins. Proteins of the α-helical membrane class have their membrane spanning regions formed by hydrophobic helices which consist of 15–35 residues ,3. TheseIn this work, we evaluate the performance of the available prediction methods to date. Using a non-redundant dataset of 20 outer membrane β-barrel proteins, with structures known at atomic resolution, we compare each predictor in terms of the per-residue accuracy  and thaThe results obtained from each individual algorithm, on the test set of the 20 proteins are summarized in Table -4). This means, that there is truly a difference in the vector of the five measured attributes across the different methods and the type of sequence that we use as input. By including in the model the interaction term between the two factors, we get a marginally insignificant p-value (p = 0.0619), indicating that some of the methods behave differently with input sequences of different type. Examining each one of the attributes independently (Table -4), correctly predicted topologies (p = 0.0193) and correct barrel size (p = 0.0001). In all cases, the type of the method is a highly significant factor (p < 10-4), reflecting the fact that there are true differences in the performance of the methods. The interaction term in the ANOVA is significant only for the SOV measure (p = 0.0272), and marginally significant for the correctly predicted residues (p = 0.402). However, these results do not provide us with a clue as to which method performs better (or worse) than the others; it states that one or more methods depart significantly from the mean. The ranking of the methods has to be concluded by observing Table The MANOVA test Table  yields aly Table , we obse p = 0.069, indica-4) and the interaction term (p = 0.0025), giving us a clear indication that the visually observed superiority of the HMM-based methods has a statistically significant justification. The statistically significant interaction of the 2 factors, furthermore suggests that the decrease in some of the measured attributes when submitting full-length sequences, is smaller (if anything) for HMM-based methods than for the NN and SVM-based ones. In fact, considering the three top-scoring HMM methods, we observe that the per-segment measures are not influenced from the type of the input sequence whereas the per-residue measures are significantly increased with full-length sequences as input, reflecting the fact that more non-transmembrane residues are correctly predicted, as noticed already in [In order to discover the statistically significant differences between the methods, we proceeded by grouping the methods according to the type of the algorithm they utilize. This way, we grouped together the HMM-based methods  and the NN and SVM-based methods . Thus, instead of having a factor with 8 levels describing the methods, we now have a factor with 2 levels (HMM and not HMM). The MANOVA test Table  once agaready in . ConsideThese findings suggest, that the HMM-based predictors perform better, on average, than the NN and SVM-based methods, in almost all of the measured attributes. We should mention here, that the difference between HMM and NN/SVM methods is larger for the measures of per-segment accuracy than for per-residue accuracy. Even the simplest and less accurate HMM-based method, BETA-TM, that uses single sequence information compares favorably to the refined NN/SVM methods that use profiles derived from multiple alignments. As a matter of fact, only B2TMPRED, which uses a dynamic programming algorithm to refine the prediction, predicts more accurately than BETA-TM the correct topology and/or the barrel size of the proteins, but still cannot reach the accuracy of the other HMM-based methods. Furthermore, the HMM-based methods are not influenced significantly whether full-length sequences or just the β-barrel domains are submitted for prediction. Interestingly, the NN/SVM methods, often falsely predict the signal peptide sequences as transmembrane strands in the precursors whereas HMMs do not. This observation is consistent with the theory regarding the nature of HMM and NN-based methods. Thus, it is consistent with the fact that the regular grammar of the HMMs can capture more effectively the temporal variability of the protein sequence and map successfully the proteins' modular nature to a mathematical sound model. Therefore, it is not surprising that also for α-helical membrane proteins' topology prediction the best available predictors are those based on HMMs . On the These observations, will have a significant impact not only on isolated predictions for one or few proteins, but also on predictions for sequences arising from genome projects where one expects to have the precursor sequences. Thus, predictions on such sequences will be more reliable, when obtained from HMM-predictors rather than NN and SVM-based ones. However, the performance of even the best currently available predictors are not as good as the predictions obtained for α-helical membrane proteins . This isOne issue that was not possible to investigate statistically is that of the use of evolutionary information in the form of profiles derived from alignments. It is well known, that the inclusion of information arising from alignments, increases significantly the performance of secondary structure prediction algorithms . This waComparing the performance of individual methods, one has to keep in mind several important aspects of the comparison. From the one hand, the limited number of β-barrel membrane proteins known at atomic resolution, resulted in having a test set, that includes some  of the proteins used for training each individual method or a close homologue. This does not imply that the comparison of the methods is biased (regarding the ranking), but that the absolute values of the measures of accuracy may be influenced. Thus, when it comes to newly solved structures, we may expect somewhat lower rates in the measures of accuracy for all methods examined. On the other hand, when comparing the results of the individual methods, as they appear in the original publications, we observe some discrepancies. These arise, mainly due to the fact, that when reporting results of a prediction method, the authors usually report the measures of accuracy obtained in the jackknife test . Furthermore, the authors of the individual methods report the measures of accuracy obtained using as input different types of sequences, and comparing using as observed different annotations for the transmembrane strands. For instance, other authors report measures of accuracy obtained from the β-barrel domain of the proteins, others from the sequences deposited in PDB, and others report also the results from precursor sequences. As for the observed transmembrane strands used for comparisons, most of the authors used the annotations for the strands found in PDB, and only PRED-TMBB used manually annotated segments that resemble better the part of the strand inserted into the lipid bilayer. The last observation, partly explains the better prediction accuracy obtained by PRED-TMBB, mainly in the measures of per-residue accuracy (correctly predicted residues and correlation coefficient).One important result of this study is the development of the consensus prediction method, for predicting the transmembrane strands of β-barrel membrane proteins. Even though consensus prediction has been proved to be a valuable strategy for improving the prediction of α-helical membrane proteins ,29,38, nHowever, in the web-based implementation of the consensus prediction method, we allow the user to choose at will the methods that will be used for the final prediction. This was decided for several reasons: Firstly, for a newly found protein, we might have larger variations on the predictions, and we could not be sure if the choice of different algorithms will give better results or not. Secondly, the different predictors are not sharing the same functionality and availability. For instance, some predictors respond by e-mail , most of the others by http , and others may be downloaded and run locally , whereas one of the top-scoring methods (HMM-B2TMR) is available as a commercial demo only, requiring a registration procedure. These facts, forced us not to have a fully automated server (but instead we require the user to cut 'n paste the predictions) but also to allow flexibility on the chosen methods, and let the user decide alone which methods he will use. For this reason, we also give to the users the opportunity to provide, if they wish, custom predictions. This way, a user may choose to use another method, that will come up in the future, or, alternatively, to use manually edited predictions., where the user may choose which of the individual predictors will include, in order to obtain the final prediction.We have evaluated the currently available methods, for predicting the topology of β-barrel outer membrane proteins, using a non-redundant dataset of 20 proteins with structures known at atomic resolution. By using multivariate and univariate analysis of variance, we conclude that the HMM-based methods HMM-B2TMR, ProfTMB and PRED-TMBB perform significantly better than the other (mostly NN-based) methods, in both terms of per-residue and per-segment measures of accuracy. We also found, a significant decrease in the performance of the methods when full-length sequences are submitted for prediction, instead of just the β-barrel domain. However, the HMM-based methods are more robust as they were found largely unaffected by the type of the input sequence. This is an important finding that has to be taken in account, not only in the cases of single proteins' predictions, but mostly in cases of predictions performed on precursor sequences arising from genome projects. Finally, we have combined the individual predictors, in a consensus prediction method, that performs significantly better even than the top-scoring individual predictor. A consensus prediction method is for the first time been applied for the prediction of the transmembrane strands, of β-barrel outer membrane proteins. The consensus method, is freely available for non-commercial users at N. meningitidis [The test set that we used has been compiled mainly with consideration of the SCOP database classification . In partngitidis  was alsongitidis  was usedngitidis . The remS. aureus is active as a transmembrane heptamer, where the transmembrane domain is a 14-strand antiparallel β-barrel, in which two strands are contributed by each monomer. Both structures are not included in the fold "transmembrane beta-barrels" of the SCOP database. In summary, the test set , which is considered to be the most reliable measure for evaluating the performance of secondary structure prediction methods [TOP), i.e. when both the strands' localization and the loops' orientation have been predicted correctly, and the correctly predicted barrel size (BS), i.e the same with the correctly predicted topologies, but allowing for one strand mismatch [βQ) in a two-state model (transmembrane versus non-transmembrane), and the well known Matthews Correlation Coefficient (βC) [For assessing the accuracy of the prediction algorithms several measures were used. For the transmembrane strand predictions we report the well-known  methods . We alsomismatch . As measent (Cβ) .TYPE) that could be either the full precursor sequence or the transmembrane barrel domain only, a factor with two categories, and the individual predictive method (METHOD), which has 11 categories. Furthermore we tried to group the methods to those based on a Hidden Markov Model and those that were not. This factor (HMM) was evaluated later, in order to assess the impact of the type of the prediction method. The formal way to assess the overall statistical significance is to perform a two-way multivariate analysis of variance (MANOVA) [TYPE, METHOD), will imply that the vector of the measured attributes varies significantly across the levels of these factors. We also included into the models, the interaction term between the two factors (TYPE*METHOD or TYPE*HMM). This was necessary in order to investigate, the potential differences of the dependent variables in the various combinations of the independent variables. For instance, a significant interaction of TYPE with HMM, will indicate that the effect of the input sequence will be different on the two types of methods.The measures of accuracy mentioned earlier are the dependent variables that we wish to compare. We treat each prediction on each protein as an observation, and as independent variables we use the type of the submitted sequences ((MANOVA) . For theHaving obtained a significant result from the MANOVA test, we could use a standard 2-way analysis of variance (ANOVA) for each of the dependent variables, in order to be able to confirm which one of the measured attributes, varies significantly across the two factors. In the ANOVA models, we also included the interaction terms. In all cases, statistically significant results were declared those with a p-value less than 0.05. We report for the ANOVA and MANOVA models, the test statistic and the corresponding p-value, for the fitted models (including the interaction term).In order to produce a combined prediction, we have two alternatives: One is to use some kind of ensemble Neural Network, or, alternatively, to summarize the individual predictions using a consensus method. Ensemble Networks show a number of significant advantages over the consensus methods ,66, but L, denoted by:Suppose we have an amino acid sequence of a protein with length x = x1, x2,..., xL,i we have the prediction of the jth predictor and for each residue where, Si by averaging over the independent contributions of each predictor:Thus, we can define a per-residue score This way, we can obtain a consensus prediction score for the whole sequence,This score is capable of yielding inconsistent predictions, such as a strand with 3 residues for example. For this reason it is then submitted to a dynamic programming algorithm, to locate precisely the transmembrane strands. The algorithm is essentially the same used by , with thPGB conceived of the study, performed the collection and analysis of the data and drafted the manuscript, TDL participated in data collection, implemented the consensus algorithm and designed the web interface and SJH supervised and coordinated the whole project. All authors have read and accepted the final manuscript.


Loa in a human volunteer (the author of this paper) by means of 1. Transplanting adult simian Loa worms from a wild drill (Mandrillus leucophaeus) to man; and 2. Infecting the same volunteer by sub-cutaneous inoculation with infective larvae of simian Loa from a laboratory-bred, experimentally infected Chrysops silacea.This paper describes unsuccessful attempts to induce a nocturnally-periodic infection with simian  Loa loa and the highly similar Loa parasites which were found in several species of local monkey, especially in the drill (Mandrillus leucophaeus) but also, less frequently, in several long-tailed monkey species, Cercopithecus nictitans martini, C. mona mona and C. preussi.In the mid 1950s, one of the main lines of research followed by the Helminthiasis Research Unit (HRU), at Kumba in the then British Cameroons was to establish the relationship between human infections with Loa infection after a quarantine period of 6–8 months in screened cages), which were experimentally infected by transplantation of live adult simian Loa worms extracted from wild drills shot in the local forests, revealed that the parasites exhibited a nocturnal microfilarial periodicity with microfilariae (mf) that were significantly longer than those of human L. loa.Observations on 109 young drills  were of shorter length than the normal wild monkey parasites [By contrast, drills which had been infected by inoculation of infective larvae from the day-biting Chrysops species revealed that in nature the diurnally periodic human Loa loa was transmitted among humans by the day-biting species C. silacea and C. dimidiata, whereas the nocturnally periodic simian parasite was being transmitted among monkeys by two forest-canopy-dwelling species, C. langi and C. centurionis, both of which bite in the forest canopy at night and are presumed to feed mainly on sleeping monkeys [Further studies on the biting habits of local  monkeys .Loa were quite capable, when transplanted together under experimental conditions into uninfected drills, of inter-breeding and producing microfilariae of intermediate dimensions and periodicity [Loa are never transmitted to monkeys, but such events, if they do occur, appear to be rare).No naturally-acquired diurnally periodic microfilarial infection was seen in 21 wild drills, the periodicities of whose adult worms were examined by transplantation into uninfected animals; despite the fact that male and female worms of human and simian iodicity . . They also noted that out of 547 patients from this area, whose blood films were examined both by day and night, 197 showed Loa mf only by day, 322 showed them by day and by night; and 16 showed mf only at night. Although the latter cases, with a complete reversal of the normal periodicity, showed only light microfilarial loads , among those persons who showed Loa mf by day and by night, 62 showed microfilarial concentrations that were nearly as high by night as by day and 10 showed more mf by night than by day  [Loa responsible for them may be closely related to the simian parasite.Later, Belgian workers in the Mayumbe District in the south-western part of the Democratic Republic of the Congo (DRC), commented on the local human Loa-encephalopathy have been reported following mass treatment with ivermectin by the African Programme for Onchocerciasis Control (APOC) in areas where loiasis is co-endemic with onchocerciasis [Loa-encephalopathy has had a deleterious effect on the popularity of the APOC campaign in that area [personal communication).Recently in the Republic of Cameroon, cases of erciasis -11. A rehat area . FurtherLoa-encephalopathy following treatment with DEC or with ivermectin, mainly seen in patients heavily infected with Loa microfilariae, are not well understood, and co-factors may exist that account for the fact that some patients do not develop SAEs despite having high Loa microfilaraemia. Experimental work by Dr Samuel Wanji using an animal model is currently trying to reproduce heavy microfilaraemic infections of human L. loa in experimentally infected monkeys (mainly Mandrillus spp) and to investigate the biochemical and pathological changes that accompany the development of any Loa-encephalopathy following ivermectin treatment.The patho-biological reasons for the occurrence of Loa parasite or with the diurnally-periodic human parasite, it was obviously far more difficult to determine whether the nocturnally-periodic simian parasite could be transferred to man. Nevertheless, at that time, before the discovery of the potentially deadly viruses such as Ebola, Marburg and HIV that are believed to originate from monkeys, attempts to infect a human (the author) experimentally with a simian strain of the Loa parasite were undertaken. Today, such experiments would not only be viewed as unethical but also as potentially life-threatening.In the original work at the HRU, Kumba, where it was relatively easy to infect young drills experimentally (either by inoculation of infective larvae or by transplantation of adult worms) with either the nocturnally-periodic simian Loa-encephalopathy in some individuals following treatment with ivermectin. These attempts at experimental infection of a human being with simian Loa are as follows:In 1954 and 1955, the author  daily as a prophylactic for malaria), took part in two such experiments, which have not been previously published but are relevant in the light of the localised occurrence of Loa worms, all alive, undamaged and motile, were collected from the subcutaneous and intermuscular tissues. The worms were placed in sterile normal saline solution, along with a small quantity of merthiolate, in the same manner that had been used previously to transplant adult simian Loa worms successfully into other monkeys. Two and a half hours later, 12 of these live, motile, adult female worms and five males were inserted, under local anaesthesia, into the upper, anterior part of the right thigh of the author by the Medical Officer-in-Charge of the Kumba Medical Field Unit. After making a 3-inch, longitudinal incision through the skin and the superficial fascia, the simian Loa worms were inserted, some into the sub-cutaneous tissue and others under the deep fascia of the rectus femoris muscle. The fascial layers were then sewn up, the skin closed with nylon sutures, and intramuscular penicillin was given to counteract infection.In July 1954, a large male drill, which had been shot in the forest near Kumba some 3–4 hours previously, was brought into the laboratory by the Unit's hunter. It was immediately dissected and a total of seven male and fifteen female mature simian Loa worm(s) appeared under the skin or crossing the eye, nor did any Loa mf appear in the peripheral blood over the next 46 years. .Over the ensuing week the transplant area became considerably swollen and painful over an area of approximately 6–8 × 5–6 inches and over the following month it itched frequently. Day and night blood films (50 cu. mm) were taken once a week over a period of six months, and thereafter fortnightly for the next six months, but none of them detected any microfilariae. No Loa, which had been dissected out in normal saline from a laboratory-bred female Chrysops silacea that had taken a blood-meal 10 days previously from a captive drill infected with the nocturnally-periodic simian strain of Loa. Previous experimental work with monkeys had shown that this method of infection was more effective than trying to induce a Chrysops containing infective larvae of Loa to feed on, and thus transmit infective Loa larvae to, an uninfected person. There was a slight reddening and itching of the skin in the area around the injection site over the following week but otherwise no papular eruption or other reaction developed. No Loa microfilariae were detected in the peripheral blood by day or by night over the ensuing eight years; nor, over the same period was any skin reaction seen that could have been attributed to the death of L3 or subsequent stages of L. loa dying in or under the skin, as were reported subsequently when infective larvae of L. loa from the bite of an experimentally-infected Chrysops silacea were subsequently killed by dosage with diethylcarbamazine citrate (DEC) used as a chemoprophylactic [Eighteen months after the transplant, the author also injected himself subcutaneously in the left thigh with 35 live, motile, infective larvae of simian hylactic .Loa. Obviously the failure to infect a single individual must be interpreted with caution. Nevertheless these observations are worth recording especially now that research is in progress to try and ascertain:Both these attempts failed to infect a healthy human volunteer with the nocturnally-periodic simian strain of Loa-encephalopathy in certain areas of central Africa, where ivermectin is now being used currently for the control of onchocerciasis, or where in the past it has occurred following treatment with diethylcarbamazine; and1. The factors leading to the localised occurrence of Loa-encephalopathy if it can be induced in drills heavily infected with Loa mf when treated with ivermectin.2. The biochemical and histological changes that may be associated with a None declared.Brian Duke was sole author