In the Western world, endometrial cancers are the most common gynaecological neoplastic disorders among women. Initial symptoms are often vague and may be confused with several other conditions or disorders. Thus, there is a need for an easy and reliable diagnostic tool. The objective of this work was to identify a gene expression signature specific for endometrial adenocarcinomas to be used for testing potential endometrial biomarkers.Gpx3, Bgn and Tgfb3). An independent analysis of differential expression, revealed a total of 354 cDNA clones with significant changes in expression. Among the 10 best ranked clones, Gpx3, Bgn and Tgfb3 were found.Changes in expression between endometrial adenocarcinomas and non-/pre-malignant endometrium from the BDII EAC rat model were compared in cDNA microarray assays. By employing classification analysis (Weka) on the expression data from approximately 5600 cDNA clones and TDT analysis on genotype data, we identified a three-gene signature (Taken together, we present a unique data set of genes with different expression patterns between EACs and non-/pre-malignant endometrium, and specifically we found three genes that were confirmed in two independent analyses. These three genes are candidates for an EAC signature and further evaluations of their involvement in EAC tumorigenesis will be undertaken. Endometrial carcinomas (ECs) are the most frequently occurring malignancies in the genital tract among women in the Western world. As in most other cancer diseases, neoplastic progression to EC is very complex, and involves high penetrance genes as well as intricate interactions of multiple low penetrance genes . ECs canDue to the genetic heterogeneity among the human population and the complexity of tumor etiology, it is attractive to use inbred animal model systems in studies of carcinogenesis. The BDII/Han inbred rat model, first described in 1987, is genetically well characterized and has since then become a useful model for studies of human endometrial adenocarcinoma. More than 90% of the female BDII/Han virgins spontaneously develop EC during their life time, where the majority of the neoplasms are EACs. The endometrial carcinogenesis in the females of the BDII rat strain is hormone dependent and represents an outstanding model for spontaneous hormonal carcinogenesis. -6Genomic approaches such as gene expression profiling by DNA microarrays, provide unprecedented tools to handle the complexity of cancer at the transcriptional level. In the present study, we have applied different statistical and classification approaches on global expression data to identify potential classifiers, i.e. sets of marker genes whose expression profiles can be used to differentiate between EAC tumors and non/pre-malignant endometrial lesions. Furthermore, we have used genotype data from the female progenies of BDII crosses ,8 to invp-value adjustment. The 50 most differentially expressed genes were subjected to hierarchical clustering and their expressions are shown in Figure The significance analysis of the microarrays demonstrated 890 dysregulated genes. However this number was considerably reduced to 354 when applying FDR Tgfb2 is involved in 12 different processes commonly aberrant in tumors and Thy1 in 10.Out of the top 50 genes, 31 genes were found to be involved in cellular processes commonly implicated in human tumorigenesis Figure . As illuThe overall classification results obtained with the models derived by the machine learning algorithms in Weka are summarized in Supplementary Table Gpx3  and Bgn .For many classification algorithms the output from Weka also includes the genes that the classifier is based on, and for some algorithms it is possible to determine the gene that had the most influence on the classification results. Table Tgfb3) with significant association to a susceptibility region  in the BN background. In this region, all animals from crosses with the BN background that developed tumors were homozygous for the BDII alleles. We chose to include genes in the proximity (within 10 Mbp or a 20 cM region), which means that there is at least 80% probability that the loci are inherited together.The significance analysis revealed 354 genes to be differentially expressed between non/pre-malignant and endometrial samples. We choose to investigate the locations in the genome of the 50 genes with the highest differential expression. By means of TDT analysis on samples on the backcross progeny (n = 39), we could only identify one gene . However, in some cases, no malignant cells were detected in the removed cell mass when pathologically characterized. These tissue samples represent normal or pre-malignant endometrium, and hence provide new valuable information concerning the developmental stages during carcinogenesis. In a previous study, we re-classified the samples predicated on the expression patterns in combination with observed cell morphology/physiology. In the re-classification the expression of genes regulated by the TGF-β signaling pathway as well as global expression data were used, and two major groups with distinct differences in the expression patterns were recognized . One groAs expected, many genes showed differential expression between non-/pre-malignant endometrium and endometrial adenocarcinomas. In total, we found 354 genes that were significantly differentially expressed between the groups. In Figure We also examined the gene functions of the top 50 genes using DAVID , were 31Tgfb,3 that was located in a susceptibility region identified in progenies from backcrosses with the BN background. Independent of tumor grade , we found a significant chi-square value from the TDT analysis (p < 0.05). Moreover, Tgfb3 was also found among the top 10 genes with differential expression between endometrial tumors and non-/pre-malignant endometrium. This result indicates that Tgfb3 is a susceptibility gene candidate and thus that the BDII strain might harbor an SNP involved in the initiation of EAC which will be further investigated. However, the expression data only correlated with grade of tumor as the gene was down-regulated in EAC and up-regulated in non-/pre-malignant cell lines regardless of the genetic background. An explanation for the differences might be that Tgfb3 expression is correlated to different genetic changes occurring during tumor development/progression. Since TGFβ3 is involved in multiple cellular processes the effects of genetic variation on mRNA expression may vary during tumor progression due to interactions with other affected genes. Thus, if the hypothesized genetic change in Tgfb3 is inherited, the effect on the expression level might be different in early and late stages of EAC.When we performed the TDT analysis on genotyping data from the genome-wide screen with microsatellite markers, we could pin-point one gene, transforming growth factor beta 3, Gpx3 (nine classifiers) and Bgn (seven classifiers). Based on the content of these classifiers, Gpx3 and Bgn are the best individual marker genes and should be investigated for their potential value in diagnosis of human endometrial adenocarcinoma. The value of Gpx3 and Bgn as signature genes is strengthened by the fact that both of them are within the 10 most significant genes in the microarray differential expression analysis described above.To investigate whether more specific signature genes that differentiate between normal/pre-malignant endometrium and endometrial adenocarcinomas could be created, we used the classifier tool Weka, which contains a number of different machine learning algorithms. A majority of the algorithms applied performed very well in deriving classifiers that could classify the samples classes, with an average accuracy of 97.1%, and several algorithms performed at an accuracy of 100%. Depending on the type of classifier derived, it may or may not be possible to identify which individual gene was the most important for the classification result. For example, tree-based classifiers select a small subset of genes and place the most important of these at the root of the tree, whereas multi-layer perceptrons use all genes as input, and consequently the importance of each gene cannot be readily determined. In our results, 16 algorithms derived classifiers where the most important gene could be determined, and the two genes that were most frequently identified as top genes in the classifiers were Gpx3, exhibits a critical role in detoxifying reactive oxidative species and maintaining the genetic integrity of mammalian cells. Gpx3 has been found to be either deleted or highly methylated in exon 1 in prostate cancer cell lines [Gpx3 is thought to be associated with its ability to repress the expression of Met. Expression data of Met in the present work implies that samples exhibiting an up-regulation of Gpx3 also show a low expression of Met. However, additional experimenting is essential to further evaluate the implications of loss of expression of Gpx3 in EAC carcinogenesis.Plasma glutathione peroxidase, ll lines ,12 and ill lines  and has Bgn, is regulated by TGF-β signaling . We have previously shown down-regulation of several genes  regulated by the TGF-β signaling pathway, indicating a disruptive TGF-β signaling. This result further strengthens our hypothesis of a dysfunctional TGF-β pathway in rat endometrial tumors. Furthermore, it has been shown that exogenously administered BGN induced pancreatic cancer cells to arrest in the G1 phase of the cell cycle, indicating a direct inhibiting effect on proliferation of BGN in cancerous cells [Gene expression of Biglycan, us cells .Tgfb3 has been suggested to be stimulated by collagen 1 through the PI3K/ERK pathway [Tgfb3 in our model. Interestingly, it has been shown that TGFB3 expression is up-regulated in human EAC and that TGFβ-3 exerts promoting effects on invasiveness [Tgfb3 in rat EAC.In lung adenocarcinomas, the transcription of  pathway . Our pre pathway  which misiveness , which isiveness -19 and tIn conclusion, this work provides a unique data set of genes with distinct expression profiles that may serve as good candidates for further evaluations of genetic markers in EAC. Furthermore, by combining three different analysis approaches of the microarray data, we have identified a three-gene signature that seems to have underlying implications in EAC carcinogenesis. Table Cu3/Han, to produce an F1 progeny. Subsequently, an F2 offspring was produced by brother/sister mating of the F1 progeny and N1 progeny was produced by backcrossing the males to BDII females. The female progeny was palpated twice each week for identification of uterine tumors. Animals suspected to have tumors were euthanized and the tumor tissue surgically removed, subjected to pathological characterization and used to establish cell cultures . Here, we have investigated two groups of samples, i.e. cell lines established from tissues pathologically classified as endometrial tumors and cell lines established from tissues from normal/pre-malignant endometrium.The animal material was derived from crosses between BDII/Han females and males from two non-susceptible rat strains, BN/Han and SPRDThe raw expression data of the microarray experiments were obtained from experiments described in Karlsson et al. 2007. Briefly, total RNA was extracted from harvested EAC and non/pre-malignant cell lines and labeled with Cy3 during cDNA synthesis. Universal rat reference RNA (Stratagene) was used as a common RNA reference (transcribed and labeled with Cy5) and thus hybridized to all arrays. We have employed the two-channel cDNA microarray format using the 18K (6000 clones in triplicates) rat 70 mer oligonucleotide arrays. The arrays were printed at the Swegene DNA microarray resource center in Lund, Sweden, by a BioRobotics MicroGrid 2 arrayer  together with a split pin system. Each probe in the probe set  was printed in triplicates at random positions on the arrays. The arrays were scanned in an Agilent Microarray scanner . In total, 45 samples were arrayed. In the present work, we have focused the analysis on two groups of samples, constituting 29 arrays. These two groups represent early and more advanced stages of EAC and were therefore selected for the analyses in the present work, and is described more in detail in the discussion [t test with a significance threshold of 0.05 and correction for multiple testing. Comparisons were made between EAC cell lines vs non/pre-malignant endometrial cell lines. The p values were adjusted using the False Discovery Rate (FDR) procedure [The quality control of the GenePix Extraction results was performed using methods available from Bioconductor packages  in the statistical software package R. Intensities of negative and positive controls were inspected, as well as spatial effects, M/A plots and signal-to-noise distributions. No unexpected results were found, thus indicating an overall high quality of hybridizations and scanning. Subsequent to image acquisition and analysis, the microarray data were up-loaded into the BioArray Software Environment (BASE)  for analrocedure .In the classification analysis, fold change values extracted from the microarray data were imported into the Waikato environment for knowledge analysis  . For eacH-A)2/(H+A), where H is the number of heterozygous animals and A is the number of animals homozygous for the BDII allele. The test has a χ2 distribution with one degree of freedom. TDT statistics were calculated for markers adjacent to each gene in the EAC tumors versus non/pre-malignant lesions, and for differences between the two backgrounds, BDII/BN and BDII/SPRD, respectively.TDT statistics for the backcross progeny was performed on genotype data  from microsatellite markers located adjacent to chromosomal regions harboring the identified classifiers and the top 50 genes with the most significant differential expression between the two groups. The TDT statistic for backcross progeny is defined as  were verified in a previous study [Tgfb3, Inhba, Wfdc1, Gpx3, Acta1, Serpine, Cpe, Bgn, Ssg1, Lox1, Bnip3I and Cnn. Primer sets specific for these genes were constructed using the Primer3 program in the Center for Genome Research at the Whitehead Institute . The primers were designed to amplify across two or more different exons, thus ruling out DNA amplification. RT-PCR was performed on 500 ng of total RNA using Omniscript RT Kit according to the manufacturer's protocol . Total 500 ng of total RNA was retro-transcribed to cDNA using 1× RT buffer, 2 mM dNTP mix, 1 μM Oligo-dT primer, 10 units RNA inhibitor, 4 units Omniscripts Reverse Transcriptase and RNAse free water for volume adjustment to 20 μl. PCR Amplification was performed in a 25 μl reaction mix using 10 μl of the RT reaction. The running conditions were as follow: 94°C for 2 min, 24–28 cycles of 30 sec at 94°C, 30 sec at 59°C, and 1 min at 72°C and followed by final extension of 72°C for 5 min.Gene expressions for several genes associated with the TGF-beta pathway  and Bgn (seven classifiers).Click here for file


Tobacco use counseling interventions delivered in the primary care setting are efficacious, but limited evidence exists regarding their feasibility or efficacy in the Emergency Department (ED). ED randomized controlled trials evaluating referral for outpatient tobacco use counseling have not had a single subject in the intervention groups attend scheduled clinic appointments. Telephone counseling potentially affords the opportunity to provide this population with individual counseling more conveniently than traditional clinic counseling. The purpose of this preliminary study was to evaluate the intervention completion rate among cigarette smokers enrolled through the ED in a tobacco quitline (QL) and to assess the feasibility of a randomized controlled trial assessing the efficacy of this intervention.We conducted a prospective, randomized, controlled, un-blinded pilot study enrolling cigarette smokers presenting to a tertiary-care ED. Patients indicating a desire to quit smoking were randomized to receive either proactive telephone counseling through a QL (intervention) or a self-help manual (control).Of 212 smokers who indicated an interest in quitting, 20 subjects were randomized to the QL and 19 to control. Twenty-one did not meet inclusion criteria and 152 refused to participate. A total of 10 patients (50%) enrolled in the QL completed the full intervention. However, only a total of 20 patients (51%) were reached for follow-up at 3 or 6 months (10 in each arm). At 6-month follow-up a total of six subjects had either disconnected their phone, no longer lived at the provided phone number or had provided an incorrect number. Two declined to provide follow-up and the remainder could not be reached. Assuming all patients unavailable for follow-up were still smoking, the 7-day point prevalence smoking abstinence rate at 6 months was 20% (95% CI: 6 to 44%) for the QL group and 0% (95% CI: 0 to 15%) for the control group (p = 0.11).Compliance with the QL intervention was encouraging and may hold promise for providing needed tobacco use counseling to ED patients. Future studies are required, and should focus on more effective mechanisms to obtain outcome measures and a larger sample size.NCT00394420 Cigarette smoking is the most preventable cause of death and disability in the United States and accounts for approximately 435,000 deaths annually. Available evidence suggests that tobacco use counseling interventions delivered in the primary care setting are practical and efficacious, but limited evidence exists regarding their feasibility or efficacy in the Emergency Department (ED) .The ED is increasingly becoming the principal source of healthcare for patients unable to access primary care and preventive services. While the ED is recognized as a potential site to initiate preventive interventions such as smoking cessation counseling, resource constraints and low patient adherence pose significant barriers. Patients seen in the ED have high rates of cigarette smoking and many intend to quit . A multiOverall, the available evidence suggests a positive effect of proactive telephone counseling for increasing tobacco abstinence rates . The U.SIn a recent survey of patients presenting to the ED, 30% of current cigarette smokers were considering smoking cessation in the next month and 27% reported they would be interested in receiving telephone-based counseling for quitting tobacco use after the ED visit . TelephoFax-to-Quit model which has been demonstrated to both feasible [We undertook this pilot study to evaluate the intervention completion rate of ED patients who smoke cigarettes and are enrolled in proactive counseling through a QL. We used a feasible  and costfeasible  in the pThis pilot study was a prospective, randomized, controlled, un-blinded study using a convenience sample of cigarettes smokers recruited through the ED. Concealment of allocation was assured by remote randomization via a phone call to pharmacy after consent was obtained. The study protocol was approved by the Mayo Institutional Review Board prior to subject enrollment.The study was conducted in the ED of a tertiary referral center in the Midwest. The ED is located in a community of 85,000 people with approximately 70,000 patient visits per year. The admission rate is 30%.Subjects were enrolled between September 2003 and August 2004. We enrolled English speaking patients aged 18 or older who reported current daily cigarette smoking for at least one year and who indicated an interest in attempting to quit smoking.Attending ED physicians and residents identified potential study participants by asking smokers if they were interested in participating in a research study that might help them quit. Recruitment was limited to times when study personnel were available to consent and enroll subjects. Study personnel were available approximately 50% of dayshifts, 20% of evenings and 5 % of night shifts.Identified patients were then screened by study personnel to assess eligibility for the study. A templated interview form was utilized to ensure all inclusion/exclusion criteria were assessed. Patients were excluded if they were critically ill, in severe pain, presenting for psychiatric complaints, not available for telephone follow-up, admitted to the hospital, refused informed consent, or had no plans to quit. The final question presented to the prospective participants meeting all other criteria was "are you interested in stopping your smoking?" If they responded yes, the details of the study and the consent process were reviewed with them.Utilizing a computerized block randomization schedule, subjects were randomized in blocks of four to receive a proactive QL intervention through an established QL or a United States Public Health Service (USPHS) self-help manual. Proactive telephone counseling describes counseling that is initiated by a counselor rather than the subject. Both groups received strong advice to quit from study personnel.Names and telephone numbers of patients randomized to the QL group were faxed to the QL, which then initiated contact with the patient. Multiple contact attempts were made over the following week by telephone. Patients in the QL were instructed to call the QL if they had not been reached in one week. QL counseling involved an initial 45-minute telephone session followed by up to four 10 to 15 minute follow-up sessions around their identified quit date. QL patients not successfully contacted within one week of enrollment were sent a letter inviting them to call, as well as information on strategies to help them quit.Baseline demographic data, smoking history, discharge diagnosis, and scores on the Fagerström Test for Nicotine Dependence (FTND) and the Contemplation Ladder were obtained. Baseline data were collected by a study assistant.The 6-item FTND is a validated scale with scores that range from 0 to 10, with higher scores indicating greater levels of nicotine dependence . A scoreThe Contemplation Ladder is a measure of readiness to quit smoking . It consBoth groups were scheduled for follow-up telephone calls to assess self-reported smoking status at 3 and 6 months from enrollment. Multiple attempts to reach subjects were made between the hours of 7 am to 7 pm on weekdays and 10 am to 4 pm on Saturdays. In order to avoid reporting bias, a separate staff of evaluators blinded to participant group allocation interviewed subjects.The primary outcome was completion of the QL intervention by the intervention group in order to assess the willingness of an ED population to participate in this type of counseling. Completion was defined as completing the baseline QL counseling and at least one telephone call around their quit date. Feasibility of a larger randomized controlled trial for efficacy of the intervention was also evaluated by recording completion rates for telephone outcome calls at 3 and 6 months after enrollment. Secondary outcome was the 7-day point prevalence smoking abstinence  at 3 and 6 months. Analysis was intention-to-treat and all patients unavailable for follow-up were assumed to be smoking. The sample size of 40 participants was determined by funding available for the pilot.Data were summarized using medians with interquartile (IQR) or full ranges for continuous variables and percentages for categorical variables. For each percentage abstinence rate, the numerator and denominator are presented with 95% exact binomial confidence intervals. A 2-sided Fischer's exact test was used to compare abstinence rates between groups at 3- and 6-month follow-up. Discharge diagnoses were summarized as a crude measure of patient acuity.Two hundred twelve smokers were screened by study personnel Figure . Twenty Based on the discharge diagnoses, the overall acuity of illness in enrolled patients was low. The majority of patients had minor traumatic injuries or musculoskeletal complaints . Other diagnoses included minor upper respiratory illness, chest pain, headache, dysuria/urinary tract infection, dental pain, chemical exposure, deep venous thrombosis, renal colic and atrial fibrillation. Diagnoses did not differ by treatment group.A total of 10 patients (50%) in the QL group completed the intervention. The median number of phone calls required to complete the first counseling session was 3 (range 2–6). Seven patients could not be reached by telephone or mail and 3 declined the intervention when contacted. The median number of calls made in attempts to reach this group was 6 (range 1–16). Baseline demographic and clinical characteristics of these two groups were similar, except for a trend towards a lower median FTND score in the group that completed the intervention  versus the group that did not complete the QL .One QL subject declined both the intervention and any further contact for follow-up outcome telephone calls at the initial contact. Of the remaining 38 patients, only two patients (5%) could be reached for follow-up at both 3 and 6 months (both in QL). One reported smoking at both points and one had quit at both time points. Eighteen patients could be reached once at either the 3- or 6-month follow-up, resulting in a total of 20 (51%) contacted with 10 in each arm. One control group subject declined to provide any information when contacted for follow-up. The median number of calls made for patients who were eventually reached for follow-up was 3 (range 1–7) at 3 months and 1 (range 1–3) at 6-month follow-up. In contrast the median number of attempts for the patients we were unable to contact was 12 (range 1–29) at 3 months and 4.5 (range 1–21) at 6 months. At 6-month follow-up a total of six subjects had either disconnected their phone, no longer lived at the provided phone number, or had provided an incorrect number.The 7-day point prevalence smoking abstinence rate at 3-month follow-up was 10%  for the QL group versus 5%  for the control group (p = 1.00). At 6 months, the QL group had a 20% abstinence rate  compared to 0%  for the control group (p = 0.11).This feasibility pilot trial of a smoking cessation intervention initiated through the ED provides important information regarding the success and failures of ED-based interventions. First, we observed that the QL intervention completion rate 50%) was considerably higher than previous ED studies where no subjects followed-up with outpatient appointments for tobacco use counseling ,5. Given0% was coConvenience may be an important factor affecting compliance in the ED population and the completion rate for phone counseling intervention in this small population is encouraging given the evidence already available supporting the efficacy of proactive QL counseling. Proactive QL counseling  has been shown to increase abstinence rates compared to a reactive QL  in other populations , and proFax-to-Quit program developed by the Wisconsin Center for Tobacco Research and Intervention [Fax-to-Quit model in the primary care setting is feasible and cost-effective [Despite their proven efficacy and widespread availability, significant barriers to QL use clearly exist as most state QLs currently reach only 1% to 5% of their tobacco-using population . One of rvention . In thisffective . In the Fax-to-Quit model, however, has not been formally evaluated through the ED. Despite the promising completion rate for the QL counseling intervention in this pilot, we were only able to reach 51% of study participants for any follow-up smoking assessment. Further investigation of the efficacy of the Fax-to-Quit model in the ED population is warranted particularly given it has been encouraged by the American College of Emergency Physicians [The ysicians . HoweverOur pilot study was limited by small sample size, low rates of follow-up, and self-reported outcomes. We enrolled a convenience sample as we were not able to approach all eligible patients and study personnel were not always available to screen patients identified by attending staff. Therefore, we may have an unrecognized selection bias. The completion rate for the proactive quitline intervention is a descriptive outcome and is compared only to previous ED studies that have demonstrated low rates of follow-up with outpatient counseling referrals.Linking a QL to tobacco use interventions in the ED may be one alternative to providing needed tobacco use counseling to ED patients without significantly increasing demands on clinician time or resources. Compliance with the QL intervention in this pilot study was encouraging, but larger studies are required to determine if this is an accurate estimate of true participation rates for the ED population. Future studies should also focus on the development of effective protocols for identifying motivated smokers and more effective mechanisms to obtain outcome measures.ED, Emergency Department; QL, Quitline; CI, Confidence Interval; USPHS, United States Public Health Service; FTND, Fagerström Test for Nicotine Dependence; DVT, Deep Venous Thrombosis; UTI, Urinary Tract Infection; CDC, US Center for Disease Control and Prevention.The author(s) declare that they have no competing interests.NEES and JOE conceived the study, designed the trial, and obtained research funding. NEES supervised the conduct of the trial and data collection. NEES drafted the manuscript, and JOE contributed substantially to its revision. NEES takes responsibility for the paper as a whole. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Insulin resistance (hyperinsulinemia) is said to be the signal event and causal in the development of type 2 diabetes mellitus. Pulsatile arterial compression of the right anterolateral medulla oblongata is associated with autonomic dysfunction, including “driving” the pancreas, which increases insulin resistance causing type 2 diabetes mellitus. In this prospective study, we hypothesize that decompressing the right cranial nerve X and medulla will result in better glycemic control in patients with type 2 diabetes mellitus.Ten patients underwent retromastoid craniectomy with microvascular decompression for type 2 diabetes mellitus. Patients were followed for 12 months postoperatively by blood glucose monitoring and studies of glycemic control, pancreatic function and insulin metabolism. No changes in diet, weight or activity level were permitted during the course of the project.Seven of the 10 patients who received microvascular decompression for type 2 diabetes mellitus showed significant improvement in their glucose control. This was noted by measurement of diabetes markers and decrease of diabetes medication dosages. One patient was completely off diabetes medication, while attaining euglucemia. The other 3 patients did not improve in their glucose control. The body mass index of these 3 patients was higher  than those with better outcomes .Arterial compression of the right anterolateral medulla appears to be a factor in the etiology of type 2 diabetes mellitus. Microvascular decompression may be an effective treatment for non-obese type 2 diabetes mellitus patients. Over the past 43 years, we have studied pulsatile vascular compression of the cranial nerves. We demonstrated that a number of hyperactive dysfunction syndromes were not only caused by vascular compression but that they could be relieved without loss of function by mobilizing the offending blood vessel or vessels away from the nerve .2 It is nIn this series, we found that 86% of the patients could be improved or relieved of their hypertension by microvascular decompression of the left lateral medulla; half of the patients were able to discontinue all antihypertensives; and in the other half, the medication dosage was decreased significantly. This work has been verified by a number of investigators.–29 On thThe present study extends the above data regarding the cranial nerve and hypertension to include autonomic dysfunction (type 2 diabetes mellitus [DM]) as a result of arterial elongation and pulsatile compression of the right anterolateral medulla oblongata.Metabolic syndrome, insulin resistance and type 2 diabetes are all related to neural loop feedback dysfunction. Multiple metabolic changes are seen in these syndromes. Recent work revealed that the neuropeptides, especially the kinins and their G-protein–coupled receptors , are up-regulated in the medullary regions of the human brainstem. These changes are seen in both hypertensive and diabetic specimens. These daIn diabetic rats, glucose transporters (GLUT) and accompanying mRNA are increased in the medulla oblongata, especially in the area of the ninth cranial nerve level of that structure. In diabe33Our preliminary report was a retrospective study in which we performed MVD of the medulla in 15 consecutive patients with type 2 DM. We found that we not only had to relieve the pulsation but also the distortion of the brainstem due to the compression. Therefore, we developed a device which when placed against the medulla decreases both arterial pulsation and distortion.Ten patients with type 2 DM which was steadily progressive volunteered for this study, which was approved by the Institutional Review Board (IRB) on the basis of our prior preliminary study and our minimal morbidity and rare mortality (0.01%) in over 6,000 of these operations. The major decision we made was to operate on persons with no other symptoms such as facial pain, which if relieved might confuse the result of the medullary decompression on the DM.Ten patients with type 2 DM who were still making insulin, verified by C-peptide measurements, and had visible right lateral medullary compression by arterial loops on MRI scan underwent right retromastoid craniectomy and microvascular decompression of the medulla using previously described techniques of exposure  and an innovative, patented implant Figures . A smallThe presence or absence and degree of arterial compression at operation were evaluated and recorded independently by the principal investigator and independently by neurosurgical colleagues.The patients, 9 men and 1 woman, ranged in age from 43 to 63 years  at the time of operation. Known duration of DM ranged from 1 to 20 years .MRI grading was performed preoperatively and postoperatively. Postoperative scans were performed 1 year postoperatively. The preoperative result was blinded at the time of the postoperative grading.The scale used for grading was based upon which artery was compressing, designated by Roman numerals I-IV; and the amount of compression by the artery, designated by A-D, and O equaling ‘no artery adjacent to medulla’ — type I: vertebral artery (VA); II: posterior inferior cerebellar artery (PICA); III: both VA and PICA; IV: other; and compression grading O: no artery adjacent to medulla; A: artery proximate to medulla; B: artery mildly compressing medulla; C: artery moderately compressing medulla; D: artery severely compressing medulla.Two-hour postprandial (2-hr pp) blood glucose levels  were measured preoperatively while patients were taking their diabetes medications, and also after they had gone through a ‘washout’ period of 1 week while not taking their diabetes medications. The first 3 subjects did not have the preoperative testing done with medications as this test was added to the protocol after they had been operated upon. Follow-ups of the meal study with medications were done at 3 and 6 months postoperatively. The meal study without medications was repeated at 12 months postoperatively.The meal study that was done after a 1-week washout period of all diabetes medications was completed by all 10 participants preoperatively and 12 months postoperatively. Patients were admitted to the clinical research unit the night before their testing, where they received a balanced meal for dinner, based on their BMI, and then nothing by mouth after midnight. A radioactive glucose isotope 3-3H-glucose was infused intravenously for 3.5 hours (210 minutes) through “0” time. Standardized liquid breakfast of a commercial product, 7 kcal/kg, was consumed at 0 time. Blood samples were taken from an intravenous line (in the arm opposite the one that was used for the isotope infusion) every 15 minutes, starting at 30 minutes before ingestion of liquid meal through 120 minutes after ingestion. Samples were then taken at 30-minute intervals for the following 2 hours.1c), weight and medication audits were performed. Patients were instructed to make no changes in diet or exercise during the study period.At the preoperative, 3-month, 6-month and 12-month meal studies, serum insulin, Hemoglobin (A1c levels were measured using Bio-rad variant 21 and Phospholipase C (PLC).Plasma glucose levels were measured with the use of the YSI 2300 STAT glucose analyzer (Yellow Springs Instruments). Serum insulin levels were measured with the use of Immulite 2000  Hemoglobin A1c: 4.0%-6.4%; serum insulin: <25 μU/mL (microunits per milliliter); two-hour postprandial plasma glucose: <140 mg/dL.Normal nondiabetic levels that were used for the metabolic assessments: FPG: 70-110 mg/dL; An= 7) and ‘Failed Responders’ (n= 3). Good responders‘ inclusion criteria required that overall response with regard to glycemic control either improved or did not worsen at the 12-month postoperative follow-up. Failed responders had no slowing in the natural progression of diabetes [Tables Results for individual lab, weight and medication measurements have been segregated into two different groups of ‘Good Responders’ (MRI findings in 10 patients are tabulated in 1c (A1c) following MVD are collated in 1c was improved at 12 months in 7 patients and worse in 3 patients. Change in weight was minimal and had no effect on change in A1c. A body mass index (BMI) over 32 correlated with failure to improve in 3 patients. Graphs 1c changes.The changes in hemoglobin AFasting blood glucose levels are collated in Two-hour postprandial glucose (PPG) levels are collated in Fasting serum insulin levels are collated in Baseline BMI of responders was 27.9 ± 2.1 SD, while that of nonresponders was 34.4 ± 2.5 SD. There was little-to-no change of BMI in the participants at 12 months postoperatively.Of most significance is the finding that the normal course of disease progression was slowed in the majority of the participants. The reduction of diabetes medicines is also significant because of the decrease of the side effects associated with these medications that the patients may have to deal with . It is important to note that these improvements were seen while subjects made no behavioral changes, i.e., no weight change, no changes in diet or exercise/ activity levels. In clinical settings, life behavior changes of diet and exercise would be recommended and encouraged as is the standard of care. Body mass index appears to be an important factor in the outcome of this study. Those who had the best outcome from the intervention had BMIs identified as ‘overweight’, while those who did not respond had BMIs in the obese category, viz., 30 or greater. The significance of this autonomic/ metabolic abnormality spreads beyond type 2 DM. The implications are made with some surety that a number of problems of aging are due to arterial compression of the brainstem, specifically the medulla oblongata. The first of these problems is so-called essential hypertension, well known to neurosurgeons but with only early penetration into the general medical literature.Many studies have been performed in attempts to show a genetic contribution in the inheritance of hypertension and diabetes. None of these have ever been able to complete the syllogism. It appears to us that one does not inherit hypertension. One does not inherit diabetes. Rather, one inherits blood vessels: veins and, especially, arteries. We inherit the size, the location, the proclivity for deterioration and elongation of arteries. We may possibly inherit the sensitivity of our myelin to pulsatile vascular compression.As the senior author worked his way through the entities described above, caused by vascular compression of the cranial nerves and the brainstem, he found in the literature that many investigators had confused mechanism with etiology. The many excellent studies done on mechanism in diabetes are not mutually exclusive from the etiologic factors found here; rather they are complementary to this work.Type 2 diabetes mellitus is associated with arterial pulsatile compression of the right anterolateral medulla oblongata, which appears to be an important etiologic factor. This etiologic factor must not be confused with mechanism. Instead, the many studies of the mechanism of type 2 DM correlate with, and complement this, etiologic process. Microvascular decompression may be an effective treatment for non-obese type 2 diabetes patients.Source of Support: Highmark Blue Cross Blue Shield of Western Pennsylvania provided funding for laboratory and operative costs for this study.


Osteopontin (OPN) is a multifunctional cytokine that has been primarily investigated in Th1 diseases. Recently, it has also been implicated in Th2-mediated allergic diseases, such as asthma. The expression of OPN in allergic rhinitis (AR) is currently unknown, as is the effect of intranasal glucocorticosteroids (GCs) on that expression.Subjects with AR were randomised to receive treatment with fluticasone propionate (FP) (n = 12) or a placebo (n = 16) over the grass pollen season and nasal biopsies were taken prior to, and during the season. OPN expression in the nasal mucosa was examined with immunohistochemistry. Healthy non-AR controls (n = 5) were used as a comparator.OPN expression was detected in epithelial cells, subepithelial infiltrating/inflammatory cells and cells lining the vessels and glands of all subjects. Comparison of the pre- and peak-pollen season biopsy sections in placebo treated patients revealed no increase in OPN expression during the grass pollen season (5.7% vs 6.4%). Treatment with a local glucocorticosteroid did not alter the expression of OPN during pollen exposure (6.2% vs 6.7%).OPN has been increasingly associated with the pathogenesis of various Th2-mediated diseases. However, our finding that the OPN expression in the nasal mucosa of AR patients is not significantly affected by allergen exposure and is comparable to that of the healthy controls, suggests that intracellular OPN is not directly involved in the pathogenesis of allergic rhinitis. The inflammatory process in allergic rhinitis (AR) involves many different inflammatory cells, cytokines, chemokines and other regulatory molecules . It is wOPN is a pleiotropic cytokine normally expressed by many cell types , which hThe aim of the current study was to determine the level and tissue distribution of OPN expression in the nasal mucosa of patients with AR and to determine whether OPN expression is affected by allergen exposure during a grass pollen season in allergic individuals. We also aimed to investigate whether a nasal glucocorticoid affected local OPN expression. The nasal biopsies used in the current study have previously been evaluated in other studies showing clear clinical effects of pollen exposure, as well as effects of a nasal glucocorticoid treatment on both symptoms of rhinitis, eosinophilia and expression of several cytokines ,5.Nasal biopsy samples were obtained as a part of a previously published clinical study . Briefly2 anti-mouse IgG ). The positive staining was detected using the Liquid DAB Substrate-Chromogen System (DAKO), followed by counterstaining with Mayers Hematoxylin  A matched isotype control, mouse IgG2B  (Sigma), was used at the same concentration as the primary antibody. Representative pictures were recorded prior to destaining the slides of hematoxylin with 1% HCl in 70% ethanol.Nasal biopsy sections were immersed in OCT compound in cryomoulds , snap frozen in liquid nitrogen and stored at -80°C prior to processing. Tissue sections of 5 μm thickness were prepared, wrapped in aluminium foil and stored at -80˚C. Thawed sections were fixed in 2% formaldehyde and treated with pre-heated PBS containing 0.0064% sodium azide, 0.18% glucose, 0.1% saponin, 1:3750 glucose oxidase, for 40 mins to inhibit endogenous peroxidise. Unspecific binding was blocked for 30 mins using 10% rabbit serum . Sections were incubated with mouse anti-osteopontin monoclonal antibody (clone 190312. MAB1433)  for 2 hrs, followed by incubation with a secondary antibody (peroxidase conjugated rabbit F(ab')The hematoxylin destained samples were assessed in a blinded fashion using a Leica DC 300F microscope  at a magnification of ×200. The positively stained area of the entire tissue section was determined using quantitative imaging software (Leica QWin) with the same threshold used for all sections. The data was expressed as a percentage of total tissue area.GraphPad 5  was used for the non-parametric statistical analysis of the percentage of positive staining in tissues. A Mann Whitney test was used to compare the controls with the patient groups and to compare the changes (Δ change) between the 2 patient groups over the season. The Wilcoxon signed rank test was used to compare the changes over the season. A P value of < 0.05 was considered statistically significant. The data are presented in a scatter plot with mean and SEM of the treatment group.Immunohistochemistry revealed clear expression of intracellular OPN in epithelial cells Figure  and vasc+ cells over the pollen season and no increase with FP treatment. To determine if the expression of OPN is related to the change in eosinophil numbers previously seen in the AR nasal biopsies, the staining was compared to the EG2+ cell counts previously obtained [+ staining was distinctly different to that of the OPN staining (data not shown). Additionally, no correlation between the EG2+ cell counts (epithelium or subepithelium) and OPN staining was observed (Spearman R 0.049-0.274).Previous studies on these nasal biopsies have shown a clear increase in the number of EG2The mean OPN expression (% area) was similar in nasal biopsies from healthy controls (4.1 ± 0.8%) compared to pre-season patient samples . During the grass pollen season, no significant change in OPN expression was seen in AR patients treated with placebo or FP Figure . CompariThere is an increased scientific interest in the putative role of OPN in several diseases, including asthma, allergy and rhinosinusitis ,10,14,15While OPN is known to be produced by epithelial and endothelial cells in other healthy tissues , our datOPN has been seen to be expressed as a secreted form (s-OPN) and an intracellular form. The s-OPN is commonly measured in different culture supernatants or body fluids. It is extensively modified post-translationally and acts like a Th1 cytokine. Intracellular OPN is less well characterised and has been reported as a translational alternative to s-OPN. It has been shown to be involved in the modulation of cytoskeletal processes  as well + cell numbers induced by a pollen season, which was inhibited with the use of FP. Eosinophils have recently been discussed in relation to OPN [+ cell numbers was analysed. However, the increase in EG2+ cell numbers induced by season was not mirrored with an increase in OPN expression, confirming that eosinophils are not the only cells producing OPN.A previous study of the nasal biopsies reported here, looking at the expression of eosinophils , showed n to OPN ,13 and aThis study shows for the first time that the expression of OPN in the cells of the nasal mucosa of AR patients does not increase over a natural pollen season, although an increase in several cytokines and cells in the nasal mucosa over the pollen season has been reported ,5,6.et al [et al [The scope of this study is limited to the intracellular expression of OPN in the nasal mucosa of AR patients. It should be emphasised that although no differences in intracellular OPN expression were observed, this may not be the case for s-OPN. The expression of OPN in the nasal lavage fluid of AR patients should be measured to determine if soluble OPN expression is changed by natural pollen exposure or glucocorticoids. In the lower airways, Takahashi et al  found thl [et al  reported+ cells [et al [It has been previously seen that glucocorticoids are quite effective in inhibiting both the expression of inflammatory cells and clinical symptoms  induced + cells  in the n+ cells  the expr+ cells . Erin ets [et al  has prevs [et al . The lacIn conclusion, despite the large role that OPN plays in many diseases, including Th2 diseases, like asthma, OPN in allergic rhinitis has been shown here to not be directly involved in the nasal mucosa changes over the pollen season, or in the response to glucocorticoid treatment. This suggests that intracellular OPN in AR patients cannot be used as a marker of disease.The authors declare that they have no competing interests.SO performed the immunohistochemistry and analyses, and drafted the manuscript. KS and CM helped in design of experiment. AB and JO conceived of the study. JO and KS helped draft the manuscript. TP provided the data from a previous eosinophils study. All authors read and approved the final manuscript.


In reactive nodes and in follicular lymphomas, MVD was higher in the paracortex than in germinal centres or in neoplastic follicles. However, in neoplastic follicles an increase in αVβ3-positive endothelium suggested the activation of an angiogenic pathway different from that present in the reactive follicles. In large B-cell lymphomas, MVD was higher than in reactive and neoplastic follicles but lower than in the reactive paracortex. The number of immature vessels (LH39 negative) and of αVβ3-positive vessels was higher than in reactive lymph nodes and follicular lymphoma suggesting that a switch to a different angiogenic pathway has occurred. Finally, we have demonstrated that within reactive and neoplastic follicles vascular regression is occurring, perhaps constraining the growth of reactive follicles alongside other phenomena such as apoptosis. Vascular regression was previously believed to occur in adults only in ovarian and endometrial tissue. We conclude that different types of angiogenesis are present in follicular lymphomas and large B-cell lymphomas. This has implications for possible future therapies.The few studies published on angiogenesis in lymphoma have raised the question of whether or not microvessel density (MVD) is associated with more aggressive disease and have reported the observation that in follicular lymphomas, vessels are mature rather than immature. We investigated MVD and the vascular phenotype within follicular or diffuse large B-cell lymphomas, reactive nodes and tonsils. Vascular phenotype was defined by the expression or loss of reactivity to the antibody LH39 (detecting the LH39 laminin epitope of the basement membrane in mature vessels) and by detection of  Evidence supporting a role for neoangiogenesis in haematological malignancies has been found in lymphoid  is associated with more aggressive lymphomas. A group of authors  or compaet al  , 21 patients with large B-cell non-Hodgkin's lymphoma and 17 with reactive hyperplasia 12 lymph nodes and five tonsils. Fresh tissue was collected in the theatre and frozen in liquid nitrogen. The diagnosis was performed on routine formalin-fixed paraffin-embedded material with the support of appropriate immunostainings according to the WHO classification . Staining for CD3 and CD20 was performed as well. We chose CD34 as endothelial marker as it does not cross-react with lymphoid cells on frozen tissue, as CD31, and stains a wider range of intratumour vessels than anti-Factor VIII antibodies  staining endothelial cells , the antilamina lucida antigen LH39 antibody and the anti-All immunostainings were performed on frozen tissue sections. In the single immunostainings, the primary antibody was incubated for 1 h at room temperature. Labelling was performed with an avidin–biotin peroxidase system (DAKO Duet). The appropriate secondary antibody was applied for 35 min, after which the Dako streptavidin–biotin complex was applied followed by DAB solution to develop the staining reaction.The double immunostaining was performed as follows using the anti-CD34 and anti-LH39 antibodies. Firstly, the anti-LH39 antibody was incubated for 1 h at room temperature. Labelling was performed with an avidin–biotin peroxidase labelling system (DAKO Duet): the appropriate secondary antibody was applied for 35 min, after which the Dako streptavidin–biotin complex was applied and the staining was developed by applying DAB solution. After rinsing, the sections were incubated overnight with the anti-CD34. After further rinsing sections were incubated with rabbit-anti-mouse immunoglobulins and, finally, with APAAP complexes.All the cells showing staining were scored as positive, independent of the staining intensity. Staining without the primary antibody was routinely carried out as negative control.In the present study, microvessel is defined, according to the classic definition of In reactive tissue and in follicular lymphomas, the evaluation of vascularity was performed separately for the follicles (either reactive or neoplastic) and for the paracortical area. In follicular lymphoma cases in which focal large cell lymphoma transformation was present (five cases), the area with a diffuse pattern was scored separately. In diffuse large B-cell lymphomas only the scoring of the neoplastic component was performed.We counted on each section the five vascular hot spots (i.e. the five areas with the higher number of vessels). Having put the vascular hot-spot area under a high-power field (×400 objective), all the vessels present in the field were counted and the value of the sum of the vessels counted in the five spots was calculated.To evaluate the proportion of mature vessels, at least 200 vessels were counted from each slide on which double immunostaining for CD34 and LH39 had been performed. If less than 200 vessels were present in the section, they were all counted. The vascular maturation index (VMI) defined by αVβ3 to all the CD34-positive vessels. In this case, the staining was performed on serial sections and the count for CD34 on a section was related to the count for αVβ3 on the same areas on the following section. Also in this case, we counted at least 200 CD34-positive vessels from each biopsy. If less than 200 vessels were present, they were all counted. In this case, the index indicates the percent of the vessels expressing αVβ3.Similarly, an index was derived comparing vessels positive for Vessels were scored as regressing according to the criteria described by The number of regressing vessels was counted compared with the total number of vessels. All the vessels were scored within the follicles present on each section (either reactive or neoplastic) and the vessels present in five randomly chosen high-power fields in the interfollicular areas (reactive tissue and follicular lymphoma cases) or in the areas of diffuse lymphoma.αVβ3+) and comparison of regressing vessels, between follicles and paracortex in reactive hyperplasia and follicular lymphomas, were carried out using paired sample t-tests since all vessel values were referring to the same sample. There were 17 reactive samples from nodes and tonsils, and 23 follicular lymphomas. All comparisons gave a significant P-value at the 95% significance level  and putative immature vessels  no statistical analysis was attempted.In reactive lymphoid tissues the staining with LH39 not only demonstrates the presence of positivity in the basal membrane of vessels and lymphatics, but also highlights an LH39 meshwork present throughout the tissue. Both vessels and lymphatics are within this meshwork. Regression of this meshwork is present in the germinal centres. In lymph nodes the meshwork terminates at the capsule while in tonsil, where the lymphatic tissue is extranodal, no discrete boundaries are present.In follicular lymphomas the LH39 meshwork is similar to that seen in reactive tissue. As in reactive germinal centres, regression of this meshwork is seen within the neoplastic follicles  and was higher than the number of αVβ3-positive vessels seen in reactive tissue (follicles and paracortex) (The proportion of LH39-positive vessels within the large cell lymphomas was lower than that observed in reactive tissue and follicular lymphoma (both in follicles and paracortex). The number of acortex) .Results of the scoring for vascular regression are shown in Table 3The discrepancies in previous studies  concerniTherefore, we did not confirm the data suggesting that MVD in lymphomas is higher than in reactive lymphoid tissue  newly formed LH39 vessels are observed at the edge, probably recruited from the surrounding stroma.The observation of vascular regression in the germinal centre, associated with neoangiogenesis is the second novel finding. Active vascular remodelling and angiogenesis in adult normal tissues were until now thought to be limited to endometrium, ovary and breast, under the control of cyclic hormonal changes . The occαVβ3, the presence in the paracortex of a few immature (LH39-negative) vessels and the expression of VEGF on the neoplastic cells. The observations by αVβ3-positive vessels suggest that in the paracortex of follicular lymphomas a second angiogenic pathway is appearing.This situation described in reactive nodes is maintained in follicular lymphomas as summarised from the literature  and Lymphatic vessels have not been investigated in this study. Efforts have been made to exclude them on morphological examination but the recent identification of the molecule Lyve 1 and the raising of a specific antibody against this protein (αVβ3 expression, the humanised anti-αVβ3 antibody Vitaxin (The heterogeneous patterns of vascularity observed in this study suggest that different types of approach may be needed to exploit anti-angiogenic treatment in lymphomas. In follicular lymphomas, where vessels are mostly mature but with In conclusion, our data support the hypothesis that follicular lymphomas grow in a fashion very similar to that of reactive lymph nodes while large B-cell lymphomas have angiogenic patterns similar to that seen in many epithelial tumors.


Bacillus subtilis genes dnaD and dnaB are essential for the initiation of DNA replication and are required for loading of the replicative helicase at the chromosomal origin of replication oriC. Wild type DnaD and DnaB interact weakly in vitro and this interaction has not been detected in vivo or in yeast two-hybrid assays.The dnaD mutation and two different dnaB mutations. Five different intragenic suppressors of the dnaD23ts mutation were identified. One intragenic suppressor was a deletion of two amino acids in DnaD. This deletion caused increased and detectable interaction between the mutant DnaD and wild type DnaB in a yeast two-hybrid assay, similar to the increased interaction caused by a missense mutation in dnaB that is an extragenic suppressor of dnaD23ts. We isolated both intragenic and extragenic suppressors of the two dnaBts alleles. Some of the extragenic suppressors were informational suppressors (missense suppressors) in tRNA genes. These suppressor mutations caused a change in the anticodon of an alanine tRNA so that it would recognize the mutant codon (threonine) in dnaB and likely insert the wild type amino acid .We isolated second site suppressors of the temperature sensitive phenotypes caused by one The intragenic suppressors should provide insights into structure-function relationships in DnaD and DnaB, and interactions between DnaD and DnaB. The extragenic suppressors in the tRNA genes have important implications regarding the amount of wild type DnaB needed in the cell. Since missense suppressors are typically inefficient, these findings indicate that production of a small amount of wild type DnaB, in combination with the mutant protein, is sufficient to restore some DnaB function. In addition to DnaA, the proteins needed to load the replicative helicase at the origin are also needed for replication initiation. In Bacillus subtilis, these include DnaD, DnaB (not to be confused with the E. coli replicative helicase DnaB), and DnaI. These proteins are conserved in low GC content Gram-positive bacteria and in some cases are known to be required for replication initiation {e.g., B. subtilis, all are needed to load the helicase (DnaC in B. subtilis) at oriCpriA in replication restart have been described in both E. coliB. subtilisE. coli, these mutations are typically in the gene for the helicase loader (E. coli dnaC) B. subtilis, these mutations are in dnaB, part of the helicase loading machinery Initiation of DNA replication is an important event in the cell cycle. In bacteria, several proteins are required for initiation, but not elongation, of replication. DnaA is the most highly conserved replication initiation protein and is found in virtually all bacteria dnaD and dnaB gene products are both essential proteins that interact with each other in vitro B. subtilis and dnaB is needed for enrichment of the oriC region in membrane fractions dnaD and dnaB cause a block in replication initiation at the non-permissive temperatures. Four temperature sensitive mutations in B. subtilis dnaB have been described: dnaB134ts , dnaB1ts, dnaB27ts, and dnaB19tsB. subtilis dnaD, dnaD23ts, has been described The dnaD23ts and dnaB134tsdnaB, dnaBS371P, that causes a serine to proline change at amino acid 371. That is, dnaBS371P is an extragenic suppressor of the temperature sensitive phenotype of dnaD23ts cells, and an intragenic suppressor of the temperature sensitive phenotype of dnaB134ts cells dnaBS371P, also called dnaB75, had been isolated previously based in its ability to bypass the need for PriA in replication restart dnaD23tsB. subtilis cells, indicating an increased interaction between these proteins in vivo Previously, we described the isolation and characterization of suppressors (temperature resistant revertants) of the temperature sensitive phenotypes caused by dnaD23ts mutation, three different intragneic suppressors have been described dnaD23ts mutation. We found that one of the intragenic suppressors caused increased interaction between the mutant DnaD and wild type DnaB in a yeast two-hybrid assay, similar to the increased interaction between wild type DnaD and the mutant DnaBS371P In addition to the extragenic suppressor of the dnaBts mutations, dnaB19ts and dnaB134ts. In both cases, intragenic and extragenic suppressors were isolated. None of the extragenic suppressors were in replication genes and most appeared to be informational suppressors.We also isolated suppressors of two different E. coli and B. subtilis. Transformations were done using standard procedures mls marker.Media and growth conditions were as previously described E. coli strains used for cloning and procedures used for strain constructions were as previously described B. subtilis strains are listed in trpC and pheA mutations. Suppressor strains are listed in dnaB and dnaD alleles discussed here are summarized in dnaD23ts causes an alanine to threonine change at amino acid 166 (A166T) dnaB19ts causes an alanine to threonine change at amino acid 379 (A379T) and dnaB134ts  causes a lysine to glutamic acid change at amino acid 85 (K85E) DnaD is 232 amino acids and 7 or 108dnaD23ts (KPL73), dnaB19ts (MER271), or dnaB134ts (KPL69) cells (grown in LB medium at 30°C) onto LB plates and incubating overnight at either 45°C or 48°C, essentially as described Spontaneous suppressors were isolated by plating approximately 10dnaB19ts following mutagenesis with ethyl methanesulfonate (EMS). For the mutagenesis, dnaB19ts (MER271) cells were grown in LB medium at 30°C to an OD600 = 0.4. Cells were washed twice with LB and resuspended to an OD600 = 1.0. Seven independent pools of cells were mutagenized in LB with 1.2% EMS (Sigma) for 30 minutes at 30°C and then washed twice with LB. Cells were grown for four generations at 30°C in LB without mutagen for recovery, and plated on LB at 45°C or 48°C for selection of temperature resistant suppressors.We also isolated suppressors of dnaA, dnaB, dnaC (helicase), and dnaD using antibiotic-resistant markers linked to the wild-type allele of each gene. For dnaA, dnaB, and dnaD, a Tn917 insertion containing the chloramphenicol resistance gene (cat) from the plasmid pTV21Δ2 linked to each gene was used as a selectable marker 917ΩHU163 is linked to dnaA, zhb-83::Tn917 is linked to dnaB, and Tn917ΩHU151 is linked to dnaDdnaC (helicase) was done with the spectinomycin resistance gene associated with a dnaC-myc fusion from strain KPL314 dnaD and dnaB were amplified by PCR and the products either sequenced directly or cloned and then sequenced. For suppressors thought to be in alanine tRNA genes, the relevant tRNA genes were amplified by PCR and either sequenced directly or cloned and sequenced. The rrnO locus was cloned into the integrative vector pGEMcatSuppressor mutations were classified and mapped essentially as described dnaD alleles dnaD23Δ154-155, dnaD23W188L, dnaD23T148M, and dnaD23A138G were amplified by PCR from the appropriate chromosomal DNA and inserted into the vector pGBDU-C3 to create in frame fusions of DnaD23Δ154-155, DnaD23W188L, DnaD23T148M, and DnaD23A138G to the DNA binding domain (DBD) of the Gal4 transcription factor dnaB that cause altered interaction between DnaB and DnaD indicate that these proteins are likely targets for regulatory factors. One approach to study DnaB and DnaD, and to possibly identify regulators of their functions, is to identify second site mutations that suppress phenotypes caused by dnaB and dnaD mutations. In the case of intragenic second site suppressors, this approach also has the potential to provide structure-function insights. To these ends, we isolated and characterized second site suppressors of a temperature sensitive mutation in dnaD and two different temperature sensitive mutations in dnaB. Temperature resistant revertants were isolated and characterized phenotypically to determine which were most likely to be second site suppressors and not simple back mutations. Isolates that were more temperature resistant than the original mutant, but also more temperature sensitive than the true wild type were chosen for further analyses. Most of the second site suppressors were intragenic and the dnaD and dnaB alleles discussed in this work are listed in DnaD and DnaB are involved in multiple protein and DNA interactions. The existence of mutations in dnaD23ts mutation  causes an alanine to threonine change at amino acid 166 of DnaD dnaBS371P mutation (We isolated 25 independent spontaneous suppressors (temperature resistant revertants) of the temperature sensitive phenotype caused by the mutation , line 1 dnaD+ cells. They grew as well as wild-type through the full range of temperatures at which wild-type grows. We chose not to characterize these because it was not clear that they represented second site suppressors.The remaining 20 apparently intragenic suppressor mutants fell into six classes based on differences in growth and colony phenotypes on LB plates at a series of temperatures. The phenotypes of one group of 9 independent revertants were indistinguishable from those of wild type dnaD  leads to deletion of two amino acids in DnaD, amino acids 154 (asp) and 155 (gln). We refer to the allele of dnaD that contains both this deletion and the original mutation that causes the temperature sensitivity as dnaD23Δ154-155 , dnaD325 causing a leucine to isoleucine change also at amino acid 193 (dnaDL193I), and dnaD326 causing a deletion of glutamine and aspartate at amino acids 155 and 156 (dnaDΔ155-156). All three of these suppressors are different from the alleles described here, although the 2 amino acid deletions are remarkably similar. That we isolated different alleles in our experiments indicates that none of the selections have been saturated.Three intragenic suppressors of dnaDts selection, dnaBS371P, allowed for detection of an interaction between DnaB and DnaD in a yeast two-hybrid assay dnaBS371P is located at the region of DnaB that is similar to a family of phage replication proteins dnaDts were also located in the phage homology region of DnaD. We used yeast two-hybrid analysis dnaDts intragenic suppressors allowed for a detectable interaction between mutant DnaD and wild-type DnaB.We previously found that the only extragenic suppressor isolated in the B. subtilis, the dnaD23Δ154-155 mutation allows for increased or unregulated interactions to occur between DnaB and DnaD, just as dnaBS371P does We found that DnaD23Δ154-155 and DnaB interact in the yeast two-hybrid assay. We fused DnaB to the activation domain (AD) and DnaD23Δ154-155 to the DNA binding domain (DBD) of the yeast transcription factor Gal4. In this system, a physical interaction between the Gal4 AD and DBD domains will drive expression of ADE2, thereby allowing for growth of the yeast on medium lacking adenine dnaD23A138G did not yield reproducible results, and the intragenic suppressor dnaDA166S was not tested.In contrast to the yeast two-hybrid results indicating increased interaction between DnaD23Δ154-155 and DnaB, we did not detect physical interactions between the other mutant forms of DnaD and DnaB. Wild-type DnaB did not detectably interact in the two-hybrid assay with either DnaD23T148M or DnaD23W188L . The intThere are multiple mechanisms by which intragenic suppressors could partly restore function to the DnaDA166T (DnaD23ts) mutant protein. Wild type DnaD is an oligomer The DnaDA166T (DnaD23ts) mutant protein has decreased binding to ssDNA in vitro, and decreased cooperativity of binding dnaD23ts that is in dnaB. This extragenic suppressor, dnaBS371P, causes increased interaction with DnaD dnaD23ts restore the normally cooperative DNA binding and/or cause increased interaction with DnaB, although any potential increase in interaction with DnaB was not detected in a yeast two-hybrid assay.One of the intragenic suppressors, DnaD23Δ154-155, caused increased interaction with DnaB, possibly contributing to the mechanism of suppression. The loss of these two residues likely uncovers an otherwise hidden DnaB binding site. This phenotype is similar to that caused by the extragenic suppressor of dnaDA166T (dnaD23ts) by altering some other aspect of DnaD function. For example, the suppressors could alter the oligomeric state of DnaD, affect interaction with DnaA, or possibly make DnaD less sensitive to uncharacterized negative regulatory factors affecting replication initiation. The suppressors are not in the N-terminal region of DnaD that contains the main oligomerization activity (aa 1–128) It is also possible, although probably less likely, that the suppressors compensate for the defect caused by dnaB134ts (dnaBK85E). In addition, we isolated a total of 31 independent suppressors of dnaB19ts (dnaBA379T), some spontaneous and some following EMS mutagenesis  As reported previously, dnaB134ts fell into four groups based on growth and colony phenotypes at a series of temperatures. All groups contained members that were linked to dnaB and the sequence of at least one representative from each group was determined   or DnaBA379T (DnaB19ts). Little is known about the mechanistic defects caused by these dnaB mutations, so it is difficult to know if the suppressors restore normal function or compensate for decreased function by increasing some other aspect of DnaB function. Wild type DnaB is found in membrane fractions of cells and is involved in the enrichment of oriC in membrane fractions dnaB134ts, it is plausible that some of the other intragenic suppressors could work similarly.As with dnaB19ts were extragenic, as they were not linked to dnaB. All of these mutations caused allele-specific suppression and the mutants formed very small colonies on LB plates. Cells appeared normal in phase contrast microscopy (data not shown). Suppressors from one of these groups were linked to Tn917ΩHU163 (near dnaA), but did not appear to be in dnaA. A ribosomal RNA and tRNA gene cluster (rrnO) is also linked to this transposon, and based on the growth phenotype and linkage, we suspected that the suppressors might be informational. We PCR-amplified and sequenced trnO-ala from the rrnO operon of one of the suppressor mutants and found a mutation in the tRNA-ala gene that changes the alanine anti-codon to one that will recognize a threonine codon . We found that, in the three additional mutants that were characterized, each had a mutation that changes the anti-codon from recognizing an alanine codon to recognizing a threonine codon. In total, two sequenced isolates were in trnA-ala, and one each was in trnB-ala and trnO-ala (Based on the finding that a mutation in dnaB134ts (dnaBK85E) suppressors were not linked to dnaB and thus were extragenic. These caused phenotypes similar to those of the suppressors of dnaB19ts that were in tRNA genes. That is, all of the extragenic suppressors of dnaB134ts were allele-specific and caused slow growth. One group of suppressors (917ΩHU163 (near the rrnO-trnO operon) and suppressors in the other group were not linked to the rrnO operon and were not characterized further.Two groups of the pressors , line 6 rrnO operon from one suppressor mutant into the integration vector pGEMcat. When integrated into the chromosome of a dnaB134ts (dnaBK85E) mutant, this clone was able to suppress the temperature sensitive phenotype, indicating that it contained the suppressor mutation. We sequenced the two tRNA genes in the rrnO operon, trnO-ala and trnO-ile, from all four suppressors in this class, i.e., the cloned rrnO operon and the other three that were not cloned. In all four suppressors, trnO-ala and trnO-ile were wild type. This was not completely surprising since the mutation in dnaB134ts is in a lysine codon (changing it to glutamate) and not in an alanine or isoleucine codon.We cloned the dnaB134ts were not likely to be in any regulatory factor affecting replication initiation. Rather, we suspect that, while not in trnO-ala or trnO-ile, they might still be informational suppressors, and at least the one we cloned is likely to be in one of the rRNA genes of the rrnO operon (dnaB134ts that appears to be in the rrnO cluster, but that is not in either of the tRNA genes in the rrnO operon, is in the 16S rRNA gene and might be causing translational misreading, thereby allowing for synthesis of some wild type DnaB protein.These extragenic suppressors of O operon , line 6.dnaDts and dnaBts mutants did not result in the identification of new regulators of DNA replication. Most of the revertants were intragenic and some were allele-specific informational suppressors. The nature of these mutations have functional implications for DnaB and DnaD.The isolation of temperature resistant revertants of dnaB19ts mutation has some interesting implications regarding DnaB function. The efficiency of suppressor tRNAs that suppress missense mutations is generally very low dnaB19ts cells implies that the amount of wild-type DnaB necessary for the cell is much lower than the amount of DnaB found in wild-type cells. It may be that there is excess DnaB in wild-type cells, and thus decreasing the concentration of functional DnaB is not harmful to cells. In addition, since DnaB is a tetramer, it is possible that mixed tetramers are functional and perhaps only a single wild type protein in a tetramer is sufficient for function and restoration of temperature resistant growth to dnaB19ts cells. We have not tested these possibilities.The isolation of mutations in tRNA-ala genes that are informational suppressors of the temperature sensitive phenotype caused by the dnaD23ts that increase interaction with DnaB, and mutations in dnaB that increase interaction with DnaD and suppress dnaD23ts, together indicate that increased interaction between these proteins might be an important part of suppression. Perhaps DnaB and DnaD naturally interact in vivo, as indicated by weak interactions in vitro. The mutations that increase this interaction could either strengthen the interaction or increase the frequency of interaction, perhaps by bypassing a normal regulatory step. The increased interaction could restore a function defective in the mutant protein or enhance a different function, thereby compensating for the defect in the mutant protein. The mechanisms by which these mutations affect DnaD and DnaB should become clearer with more structural analyses of these essential replication initiation proteins.Intragenic suppressors of


Sham-operated animals showed no significant differential expression. A subset of the differentially expressed genes was validated using quantitative RT-PCR. Distinct sets of TFBS could be identified that were significantly enriched in each one of the different temporal gene expression clusters. These included binding sites for transcription factors that had previously been recognized as contributing to the onset of regeneration, including NF-κB, C/EBP, HNF-1, CREB, as well as factors, such as ATF, AP-2, LEF-1, GATA and PAX-6, that had not yet been recognized to be involved in this process. A subset of these candidate TFBS was validated by measuring activation of corresponding transcription factors  in nuclear extracts from the remnant livers.Analysis of the gene expression time series data using ANOVA resulted in a total of 309 genes significantly up- or down-regulated at This analysis revealed multiple candidate transcription factors activated in the remnant livers, some known to be involved in the early phase of liver regeneration, and several not previously identified. The study describes the predominant temporal and functional elements to which these factors contribute and demonstrates the potential of this novel approach to define the functional correlates of the transcriptional regulatory network driving the early response to partial hepatectomy. Thesecle (G1) -12. Thesis group ,14. Previs group ,16.Characterizing changes in gene expression using microarray technology has provided new insight into the regulation of liver regeneration -20. Nota1 phase [[In this study we used cDNA microarrays to monitor changes in gene expression at 1, 2, 4, 6 h after PHx in remnant livers in the rat. These time-points provide information on the course of events during the initiation of the regenerative response accompanying the emergence of hepatocytes from the quiescent state and the onset of the G1 phase [,22. The 0 and the early G1 phase in hepatocytes [Differences in gene expression in rat liver were analyzed at 1, 2, 4 and 6 h after PHx corresponding to the transition from Gatocytes . Thus, tany of the four time points at a 30% local false discovery rate threshold which corresponded to ~20% false discovery rate (FDR) threshold  approach detailed in the Methods section. Six clusters provided maximum information on distinct temporal patterns and were well distinguishable from randomized data partitioning. Partitioning beyond six clusters reduces the confidence in the clustering result, as the performance is closer to that of randomized data Figure . It shouIn order to validate the differential expression of key genes across all the observed gene expression clusters QRT-PCR was performed on a total of 17 genes that represent the response profiles in the different clusters. The findings, shown in Figure , following a manually curated assignment process as detailed in the supplemental data  is a software program designed to identify transcription factor binding sites in the promoter region of coordinately regulated genes . PAINT aIn order to corroborate the PAINT analysis, we obtained time series data on the DNA binding activity detected in nuclear extracts from remnant livers for several of the transcription factors implicated by our PAINT analysis Figure . We sele0 and the transition to the G1 phase of the cell cycle [It is notable that for many of the transcription factors analyzed that show an early transient increase, we observed a significant resurgence in the DNA-binding activity between 4 h and 6 h. Such a profile has been observed previously in time course studies of TF activation. For instance, Rudnick et al.  reportedIn addition to the transcription factors identified by our PAINT analysis, we also monitored activation of STAT-3. The activation of this transcription factor after PHx was reported in the literature ,28,46,47Further insight into the functional consequences of the transcriptional regulatory network is obtained from Figure In this study, microarray gene expression data obtained during the initial 6-hour period after partial hepatectomy were used to characterize the transcriptional regulatory network that drives the onset and early progression phase of liver regeneration. Following clustering of the gene expression data, PAINT analysis was used to characterize significantly enriched TF binding sites in the different clusters to identify TFs that might have contributed to the temporal profile of gene expression obtained. TF activation could be directly confirmed by analysis of nuclear extracts. Insight into the functional role of the genes regulated by these TFs was obtained from the gene ontology analysis of TF-gene relationships. Not unexpectedly, the analysis suggests that multiple TFs coordinate to control a wide range of functions during the early phase of liver regeneration Fig. . ImportaSeveral previous studies reported microarray studies of gene expression changes in rodents after partial hepatectomy using a variety of platforms. The majority of these studies presented data on mice, including some that included early time points -20,48. HThese results demonstrate that relevant functional information on the transcriptional control of the early response to partial hepatectomy can be obtained from the PAINT analysis of clustered microarray data. Each of the six temporally distinct gene expression clusters is characterized by a unique pattern of significantly over-represented binding sites for TFs. Activation of a selection of the candidate TFs was confirmed by oligonucleotide binding assays of nuclear extracts. Notably, there was relatively little overlap in the TFs driving the response in different temporal clusters. This is not to say that transcription factors involved in one cluster didn't play any role in the response in other clusters , but those associations didn't reach statistical significance in our analysis. It is possible that these factors contribute to the fine-regulation of the gene expression responses within clusters, but the number of differentially expressed genes in these studies was too low to identify such combinatorial control by multiple TFs with sufficient statistical weight.As with any computational approach, it is important to note that our unbiased discovery approach using PAINT, while informative in predicting a role for novel TFs, is subject to false negatives, i.e. not all the currently known TFs in liver regeneration were present in the computational predictions. For example, STAT-3 is known to play role in liver regeneration ,46,47 anOur study highlights significant candidate mechanisms for transcriptional control of specific genes and gene clusters and classifies these by functional category, but does not identify individual genes as actively being controlled by these TFs. Further studies using chromatin immunoprecipitation (ChIP) or related methodologies will be required to validate the role of each TF in individual gene responses, which will further clarify the role of individual TFs in the functional changes occurring after PHx. Nevertheless, the study points not only to the complexity of the transcriptional control of the early response to PHx, but also suggests that there is a clear underlying organization to the temporal response of genes in different functional categories that is driven by transcriptional regulation. The data reported here should provide a basis for a more detailed analysis of the role of each of these transcription factors to the regulation of individual genes and gene categories. However, these findings also emphasize the fact that the study of any individual factor will not capture the systemic nature of the regulatory machinery that drives the regenerative response of the liver to partial hepatectomy.This conclusion is also relevant for the recognition that multiple cell types contribute to regenerative responses in the remnant liver. The analysis of gene expression profiles in total tissue extracts from the remnant liver incorporates contributions from parenchymal cells, Kupffer cells, endothelial cells stellate cells and other non-parenchymal cells. Although parenchymal cells contribute approximately 70% of the total cell number and 90% of tissue mass in the liver, robust gene expression responses in non-parenchymal cells may occur that reach the threshold for detection in our microarray studies. Similarly, differences in zonal distribution across the liver acinus exist that are difficult to capture in such in vivo studies. However, the response to partial hepatectomy (and by extension the response to other forms of liver injury) is by nature a systemic response of the whole tissue, in which the contributions of different cell types are integrated to generate the coordinated temporal pattern of regeneration. Our analysis is an effort to capture this integrated response profile by focusing on the system-wide gene expression and regulation by transcription factors. A better understanding of this systemic response profile will ultimately be a critical step in mobilizing the regenerative potential of the liver for therapeutic purposes.Adult male Sprague-Dawley rats 275–350 g) were anesthetized and subjected to two-thirds PHx by ligation and resection of the median and left-lateral lobes, following standard procedures [ g were aEscherichia coli containing individual sequence verified rat cDNA clones were purchased from Research Genetics . cDNA clone inserts were amplified by PCR directly from the clones in culture with primers specific to the vector sequences flanking the insert cDNA. All cDNA clones spotted on the microarray were generated by PCR using GF200 primer pairs, therefore all clones contains 110 bps vector sequence on their GF200 forward primer side. Vector probes generated with a GF200 forward primer on an empty pT7T3Pac vector in RT reaction were used as a universal reference for all clones as a control for cDNA on the microarray [MicroGrid II microarrayer . The array contains 9084 target clones that had annotation in the Unigene database linking them to known genes  and GSE9137 (sham).MIAME compliant microarray data are deposited at (1) partial hepatectomy (PHx or control), (2) time following PHx . These variables were considered as fixed effects, whereas the biological variability  was considered as a random effect in the ANOVA response model. We estimated the parameters in the ANOVA response model to these fixed- and random-effect variables and sought genes with statistically significant parameters. For each gene, we evaluated the statistical significance of the observed expression against the null hypothesis that PHx has no effect on the gene expression at any of the four time points. Multiple testing was accounted for with the overall false discovery rate (FDR) controlling procedure of Benjamini and Hochberg [The normalized gene expression data was analyzed using a mixed-effects ANOVA response model for each gene using the statistical software package in R following Pavlidis and Scholtens ,54. We eHochberg  and a loHochberg . The locsilhouette coefficient (SC) is utilized to assess the quality of the clustering results: the closer the SC is to 1, the better the quality. Our CNC approach takes this a step further and assesses the performance of the clustering results by comparing SC from clustering original data with that from the randomly permutated data (destroying any inherent structure): the larger the difference between the quality metric between the original data clustering vs. randomized data clustering, the higher the confidence in the resulting clusters. This information was utilized to explore different parameters in the clustering algorithm, i.e., number of clusters specified in PAM. We sought the largest number of meaningful clusters that are distinct from random.We employed the Partitioning Around Medoids (PAM)  clusteri® Profession 10.1 database and associated MATCH® tool [® tool.We employed PAINT  to analyCH® tool . In eachCH® tool . The oveQRT-PCR analysis was performed to verify the microarray data. The cDNA templates were synthesized from total RNA using SuperScript II reverse transcriptase and oligo(dT) primer . Quantitative analysis of gene expression was performed on ABI Prism 7000  using 2× SYBR Green master mix . The primer pairs used in this analysis are shown in Table Activation of HNF-1, NFκB, STAT-3, CREB, GATA-1 CEBP/α and CEBP/β was identified as changes in DNA binding activity of the transcription factors in nuclear extracts using kits from Active Motif, Carlsbad, CA, as per the manufacturer's instructions. AP-2α and PAX-6 activity was assessed using the TransFactor Universal Colorimetric Kit  with biotinylated oligos containing binding sequence 5'-ACC GCC TGA GGC GTT A-3' (AP-2α) 5'-CTG ACC TGG AAC T-3' (PAX-6) and AP-2α and PAX-6 antibodies .EJ carried out gene expression experiments and transcription factors activation assays, participated in drafting the manuscript and statistical analysis. RV performed statistical and PAINT analysis. JBH conceived of the study, and participated in its design and coordination, and drafting the manuscript. All authors read and approved the final manuscript.Table S1. Liver regeneration associated changes in gene expression revealed by cDNA microarray analysis.Click here for fileTable S2. Sham surgery associated changes in gene expression revealed by cDNA microarray analysis.Click here for fileTable S3. TF binding sites enriched (FDR < 30%) in individual clusters.Click here for fileSupplemental text. Analysis of functional gene categories changing after partial hepatectomy.Click here for fileTable S4. Distribution of TF binding sites in the functional differentially expressed gene categories.Click here for fileTable S5. Clones spotted on the array.Click here for file


Previous pharmacokinetic studies have demonstrated bioequivalence of Imusporin  to the innovator product Neoral . This study was done to evaluate the clinical efficacy and safety of Imusporin in patients who have already undergone renal transplant and have stable graft function maintained on cyclosporine preparation other than Imusporin.t-test. Safety of the drug was assessed by the type and severity of adverse events developed during the therapy. Cost analysis was done assuming an average maintenance immunosuppression dose of 150 mg/day of cyclosporine.Twenty-two renal allograft recipients , with stable graft function, previously on Neoral or Bioral were switched over to Imusporin after recording their relevant baseline clinical and biochemical parameters. These were repeated on 1, 4, 7, 15, 30 and 90 days after the start of therapy. Change in dosage required to maintain C2 levels at each visit were analyzed by paired sample P>0.30). Change in dosage required to maintain C2 levels was not significantly different from the baseline dose of 2.34 mg/ kg body weight (P>0.1). No patient developed graft rejection after switchover therapy at a median follow-up of 16 months (14-18 months). Mean baseline SCr was similar to SCr at day 90 . No severe adverse events were reported. Mild side-effects included headache (4), somnolence (2), dry mouth (5) and generalized fatigue (6). Use of Imusporin  results in an annual savings of Rs. 19892 over Neoral  and Rs. 2263 over Bioral .Twenty-one patients completed the study. One patient was lost to follow-up. Mean C2 value before switchover was 894 ± 208 ng/ml, which was not significantly different from the mean values of C2 after switchover therapy (Imusporin is clinically as safe and efficacious as other cyclosporine preparations available while significantly reducing the cost of treatment. In developing countries like India, the cost of immunosuppression following renal transplantation is a major issue. There has been a constant attempt to evolve native cost-effective alternatives without compromising the safety of the graft.Cyclosporine has stood the test of time and has emerged as a major pillar for any immunosuppression regime. Neoral  was introduced as an equally efficacious and bioequivalent cyclosporine microemulsion formulation at reduced costs. However, to the best of our knowledge, there is no published clinical study on transplant patients.The obvious need for an alternative safe and efficacious cyclosporine preparation which is equivalent to the original product, yet cost-effective, cannot be denied. Imusporin  is a microemulsion formulation of cyclosporine at a lesser cost and could be economically beneficial to our patients in the long run.Although the bioequivalence and safety of Imusporin has already been established in healthy young volunteers at Dunedin, New Zealand in a study conducted by Cipla, there is enough evidence to show that CsA pharmacokinetics is different in transplant patients treated chronically with CsA. FurthermThis study was therefore designed on the native stable transplant population to evaluate the safety and efficacy of Imusporin and to assess its cost-effectiveness.Twenty-two renal allograft recipients , with stable graft function, previously on Neoral or Bioral were switched over to Imusporin after recording their baseline serum creatinine (SCr), cyclosporine levels after two hours of drug administration (C2 levels), dose of cyclosporine, body weight and relevant hematological and biochemical parameters including complete blood counts, renal function tests, liver function tests and blood sugar levels. Subject inclusion criteria were: (a) patients of either sex aged more than 18 years with renal transplantation and stable graft function for at least six months (b) subjects who were able to give informed consent and agreed to regular follow-up.Subject exclusion criteria were: (a) patients with significant hepatic disease, uncontrolled hypertension/diabetes mellitus (b) patients on concomitant drug therapy which is likely to increase or decrease cyclosporine levels or enhance its nephrotoxicity (c) patients with severe or unstable angina pectoris within the previous one month (d) history of hypersensitivity to the trial medication (e) doubts on patient compliance.Thirteen patients were on Bioral and nine were on Neoral prior to the start of the study. Mean duration of transplantation at the start of Imusporin was 28.59 months (range 6-53 months). Parameters were repeated on 1, 4, 7, 15, 30 and 90 days after the start of therapy.The efficacy of Imusporin was assessed by evaluating the following parameters:(a) Primary efficacy parameter was the change in dosage of cyclosporine (Imusporin) required to maintain C2 levels (b) Secondary efficacy parameters were (i) incidence of graft rejection episodes during the study and (ii) graft function maintenance based on any change in serum creatinine.Change in dosage required to maintain C2 levels at each visit was analyzed by paired sample t-test.Safety of the drug was assessed by the type and severity of adverse events developed during the therapy.Cost analysis was done assuming an average maintenance immunosuppression dose of 150 mg/day of cyclosporine.P>0.30). Change in dosage required to maintain C2 levels was not significantly different from the baseline dose of 2.34 mg/ kg body weight (P>0.1) [P=0.930) [Twenty-one patients completed the study. One patient was lost to follow-up. Mean C2 value before switchover was 894 ± 208 ng/ml, which was not significantly different from the mean values of C2 after switchover therapy  . No patiP=0.930) . No seveP=0.930) .et al[There could be potential implications of substitution of generic cyclosporine formulations for the original product Neoral due to the narrow therapeutic index of cyclosporine. Whereas Carnahan and Cooper et al have repet al6–8Based on the available literature, it is clear that acute rejections are more common in the initial six months following transplantation. We therefore included only those patients who had stable graft function following at least six months of renal transplantation, in order to minimize any possible adverse effects on graft function.Ideally, the study should have included only those patients who were on Neoral, this being the innovator product. However, some patients who were on Bioral were also included as a large proportion of the study population was on this drug due to its lower cost and it being an established product in the country. Further, the patients who were on Neoral were generally more reluctant to switch over to a generic preparation due to inherent fear.Throughout the study period, we monitored serum creatinine, C2 levels and the incidence of hospitalization for graft rejection or CsA toxicity as primary markers of safety and efficacy of our therapeutic conversion. We also monitored the need for dose adjustments after conversion to Imusporin as a marker for therapeutic equivalence of the two products.et al have also reported similar findings in a well-designed study from Tennessee, USA.[Our data documents no significant differences in any monitored parameters following conversion from Neoral / Bioral to Imusporin. Carnahan see, USA. We also As economics was the major driving force behind this study, the cost analysis was done very meticulously and projected as annual savings. It is clear from our data that there could be substantial savings on an annual basis if Imusporin is used and it could translate into mammoth amounts if calculated for a five or 10-year period.Some limitations of this study include the small sample size which increases the probability of making a type II error; and the short duration of follow-up which limits the ability to assess long-term safety and graft survival data. The study population includes patients only from the northern and central parts of the country and the study subjects were not randomized.Our results indicate that Neoral / Imusporin and Bioral / Imusporin are therapeutically equivalent CsA formulations and renal transplant patients maintained on Neoral can be safely and effectively switched over to Imusporin based on a 1:1 conversion ratio.In addition, Neoral to Imusporin conversion presents a substantial cost-saving opportunity.However, the present study has demonstrated the clinical safety and efficacy of Imusporin in only a limited number of stable renal transplant recipients on maintenance immunosuppression and the results cannot be generalized to the population of recipients for either prevention of rejection  or for initial therapy.