The main genomic region controlling the predisposition to type 1 diabetes is the Human Leukocyte Antigens (HLA) class II of the major histocompatibility complex. Association with different HLA types depends also on the studied populations. In our investigation, we tried to measure the phenotypic HLA class II association frequencies of DR3 and/or DR4 antigens, using a serologic method called microlymphocytotoxicity analysis, in diabetic and nondiabetic (ND) subjects originating from the west-Algerian region of Tlemcen. The aim of the present study was to determine which HLA DR antigens represent a high susceptibility to develop the disease in this area. Using a case-control retrospective study design, we randomly recruited ninety-one related subjects, 39 type 1 diabetics and 52 ND as controls, at the Internal Medicine Board of Medical Centre University of Tlemcen.p = 1.000, OR = 0.95), whereas DR4 and DR3DR4 antigens were associated with susceptibility to develop type 1 diabetes . Also, no incidence for DR3 (p = 0.2646) or DR3DR4 (p = 0.0699) antigen frequencies was related to the sex ratio. However, significant differences in HLA DR4 frequencies between type 1 diabetics and ND were found to be related to sex (p = 0.0085).DR3 antigen frequencies were comparable between the type 1 diabetics and the ND subjects and showed no association with the disease (Taken together, our investigation showed that the strongest association with type 1 diabetes was noticed in the presence of HLA DR4 antigens followed by DR3DR4 antigens. This study highlighted a characteristic of Tlemcen population; a history of consanguineous marriages. Association studies between the disease and genetic polymorphisms should be undertaken in a population where consanguinity is more limited to reduce confounding in result interpretations. INS) promoter situated on the chromosome 11p15 [The type 1 diabetes, previously called insulin-dependent diabetes mellitus (IDDM), is the consequence of progressive and selective destruction of pancreatic β cells by an immune-mediated process . It is wme 11p15 ,11. Otheme 11p15  etc. Theme 11p15 ,12. Due However, the different HLA types associated with diabetes depend also on the population. The purpose of our study is to measure HLA DR3 and/or DR4 antigen frequencies and their association in diabetic and nondiabetic subjects originating from the west-Algerian region of Tlemcen. Using a case-control retrospective study design, we attempt to determine which is the greatest HLA DR susceptibility contributing to developing type 1 diabetes. Ninety-one (91) eligible subjects (thirty-nine (39) type 1 diabetics and fifty-two (52) nondiabetics with relatives with type 1 diabetes as controls), were recruited at the Internal Medicine Board of the Medical Centre University of Tlemcen.Table DR4 and DR3DR4 antigens showed an association with susceptibility to type 1 diabetes , in contrast, DR3 antigens showed no association with the disease . It is important to notice that the strongest association is found in DR4 phenotype as indicated in Figure p = 0.0361 and p = 0.5887 respectively). On the contrary, the DR3 molecules frequency is slightly decreased in type 1 diabetic patients compared to the controls and presents no statistically significant difference (p = 1.000). Furthermore, no incidence was related to the sex criteria for the frequencies of DR3 and DR3DR4 molecules (p > 0.05). However, significant differences in HLA DR4 frequencies are linked to the female sex and present a value definitely higher in type 1 diabetic patients compared to those of nondiabetics for the same sex (p < 0.05)  and genFortunately, molecular epidemiology offers the hope of the possibility of preventing the disease in the future, by evaluating the potential factors of risk of developing this pathology . AlthougIt is well established that associations between type 1 diabetes and certain HLA antigens largely facilitate the identification of the subjects having a potential risk to develop the disease. Among the putative HLA molecules known to confer a high susceptibility are DQA1 (*0301)-DQB1 (*0302) (= DQ8), DQA1 (*0501)-DQB1 (*0201) (= DQ2), DRA-DRB1 (*0401) (= DR4) and DRA-DRB1 (*0301) (= DR3) . HoweverIn Algeria, and especially in its western region (Tlemcen), which is known for its history of consanguineous marriage, there is a high rate of consanguinity. This fact could increase the risk of developing type 1 diabetes by favouring the transmission of HLA haplotypes and recessive genes of susceptibility except for HLA antigens that are common in both parents. For these reasons, we were interested in checking whether phenotypically DR3DR4 antigens would involve less risk to develop the disease, in comparison with DR3 or DR4 antigens, knowing the fact that the presence of a probable consanguinity could reduce the frequency of DR3DR4 polymorph phenotype compared to that of DR3 or DR4 phenotype.On a purely comparative basis, similarities seem to be found between our results and those of other investigators ,13,15 wiIn Algeria, very few investigations have been undertaken to study the impact of the genetic background on the risk to develop type 1 diabetes in its population, where an annual average incidence of 4.7 per 100000 has already been listed . In a siIt is certain that screening of HLA class II sub-types and determination of DNA coding sequences allows more precise characterization of ethnic groups, especially, because the same coded molecules can differ by the position of few amino acids. For example, it is well established that DQA1 and DQB1 alleles (sub-types of HLA DQ) code respectively for the alpha and beta chain of the DQ molecule . Thus, tMoreover, it is true that the search of an association with a candidate gene allows a better characterization for most of the frequent multifactorial diseases, because the candidate genes are directly implied in the pathological processes. Today, it is reported that the CTLA-4 (cytotoxic T-lymphocyte antigen-4) allele, localised on the chromosome 2p33, former IDDM12, is largely associated with the susceptibility of numerous common complex diseases, such as the common autoimmune disorders Graves' disease, the autoimmune hypothyroidism and type 1 diabetes . CombineType 1 diabetes, or youth diabetes, is a multifactorial disease occurring on a genetic ground of predisposition and starts under certain environmental conditions. The most effective preventive strategy must be designed at the pre-diabetes stage, since immune and/or genetic markers can easily indicate subjects with high risk before the clinical symptoms. Thus, the genetic analysis of HLA class II associations has allowed a screening of the contributing molecules to the type 1 diabetes development and the selection of subjects, which are likely to contract the disease.In this study, we were confronted with difficulties in interpretation of our results which are mainly due to the presence of some indices of consanguinity in our studied population, showing a non excess of DR3DR4 phenotype compared to the DR3 or DR4 phenotype, and a no link of DR3 phenotype to the disease. This result could be due to an ethnic characteristic of Tlemcen population, a history of consanguineous marriages. Nevertheless, these preliminary results made it possible to answer the asked questions. Thus, the strongest type 1 diabetes association is statistically revealed with phenotypic expression of DR4 followed by DR3DR4 phenotype. Association studies between the disease and genetic polymorphisms should be carried out on the population having more limited consanguinity to reduce confusions in result interpretations.via a questionnaire. The patients' mean (± Standard Deviation) age at clinical onset was 12.28 ± 5.97 years with range of 5 to 22 years and median of 11 years. Subjects who were not first degree related and who were not originating from Tlemcen region were excluded. The use of first-degree relatives eliminates exposures of environmental factors such as food items and viruses since first-degree relatives usually share the same milieu. For execution of the protocol, the informed consent was obtained from all the participating subjects to the designated study.The study was conducted on ninety-one first-degree related subjects , randomly recruited at the Internal Medicine Board of the Medical Centre University of Tlemcen (west-Algeria). The sample included thirty-nine patients with type 1 diabetes , and fifty-two healthy subjects  selected from diabetes relatives as controls. Prior medical histories and personal characteristics were obtained from participants The applied serologic technique lies on the aptitude of the antibodies to recognize allotropic determinants of HLA molecules on cellular surface . This me2 vs. expected χ2 was employed to show significance of frequency differences with the sex ratio. A p value of less than 0.05 was considered statistically significant (two-by-two table: degrees of freedom (df) = 1, chi-square ≥ 3.84) [The comparison of phenotypic frequencies was obtained by using chi-square analysis with Yates' correction or by Fisher's Exact test, whenever appropriate. The application of the observed χ ≥ 3.84) . The ass ≥ 3.84) ,34 (confAM drafted the manuscript, performed statistical analyses and carried out the bibliography research. MS participated by coordinating and orienting the designated study. BA carried out HLA phenotyping and participated in the study design. KM recruited the eligible subjects. All authors read and approved the final version of the manuscript.


Studies of the transcriptional transactivator (Tat), a key regulatory protein of HIV, have yielded insight into the control of eukaryotic transcription Viruses are intracellular pathogens that are subject to intense selective pressures during their ongoing battles within the host. To propagate successfully, they must exploit numerous machineries of the infected cell. Thus, studies of their replicative cycles have yielded fundamental insights into eukaryotic biology. A prime example is the human immunodeficiency virus (HIV), which is a lentivirus that causes the acquired immunodeficiency syndrome (AIDS). Unlike simpler oncoviruses that rely exclusively on host cell machinery, lentiviruses code for additional accessory and regulatory proteins that act as molecular switches at different stages of viral entry and exit from the infected cell. Studying the actions of these viral proteins has yielded understanding of diverse cellular functions such as the innate immunity against retroviruses, control of transcriptional elongation, export of macromolecules from the nucleus to the cytoplasm, and intracellular trafficking of proteins (reviewed in ).The transcriptional transactivator (Tat) is a key regulatory protein of HIV. It is expressed early after the virus integrates into the cell, and stimulates the elongation of RNA polymerase II (RNAPII). This type of transcriptional control had not been previously appreciated; thus, work on Tat established a new paradigm in the field of eukaryotic biology. Moreover, these findings impacted greatly studies of cotranscriptional processing of nascent mRNA. To understand these processes better, we need to start with the basics of transcriptional control.RNAPII is the enzyme that transcribes protein-coding genes in eukaryotic cells. Elegant studies in vitro first suggested that the simple recruitment of RNAPII to transcription units was not sufficient for the copying of genes and cotranscriptional processing of their transcripts. Rather, distinct steps could be defined, which began with the assembly of the preinitiation complex (PIC), promoter clearance, pausing, and arrest, and ended with efficient elongation of transcription (reviewed in ). The ceDrosophila melanogaster  of the RPB1 subunit of RNAPII (reviewed in ). This piewed in ) and humiewed in .The transition to robust elongation depends on the positive transcription elongation factor b (P-TEFb) (reviewed in ). P-TEFbPLoS Biology suggests that Tat and P-TEFb can also recruit TAF-independent transcription complexes to the HIV LTR [Tat is unique among transcriptional activators in eukaryotic cells in that it functions via RNA rather than DNA promoter elements . It bind HIV LTR  , myoblast determination protein (MyoD), and nuclear factor κ-B (NF-κB). The last one is of great interest as it explains how the HIV genome can be transcribed before the synthesis of Tat . CellulaRecently, important aspects of the regulation of P-TEFb have been revealed . Of inteSaccharomyces cerevisiae, there are two candidates for PTEFb, CTDK-1 and Bur1/2. CTDK1-negative but not Bur1/Bur2-negative yeasts still grow, albeit poorly and only on rich media (reviewed in [Caenorhabditis elegans, genetic inactivation of CDK9 or CycT1 and CycT2 resulted in the inhibition of all RNAPII transcription [D. melanogaster, following heat shock, PTEFb is recruited upstream of activated promoters [C. elegans, binds the histidine-rich stretch in CycT1, thus decoying P-TEFb away from RNAPII and blocking the elongation of transcription [How central is P-TEFb to eukaryotic transcription? In cription . Moreoveromoters . Althougromoters . Indeed,romoters , it mighcription .These are exciting findings and suggest a plethora of future experiments, including the genetic inactivation of subunits of P-TEFb and isoforms of HEXIM1 in the mouse. Of special interest are questions as to where to place this mechanism of transcriptional regulation in the hierarchy of competing or complementary processes. What roles do different P-TEFb complexes play in the transcription of specific genes? How central will the regulation of P-TEFb be to cellular growth, proliferation, and differentiation, and what roles will it play in normal development and disease states? As to HIV, how can we use our knowledge of P-TEFb to slow down viral replication and/or to eliminate the state of proviral latency in the host? Obviously, we are only at the beginning of this journey, which promises to change radically our view of eukaryotic transcription.


Serial Analysis of Gene Expression (SAGE) is a method of large-scale gene expression analysis that has the potential to generate the full list of mRNAs present within a cell population at a given time and their frequency. An essential step in SAGE library analysis is the unambiguous assignment of each 14 bp tag to the transcript from which it was derived. This process, called tag-to-gene mapping, represents a step that has to be improved in the analysis of SAGE libraries. Indeed, the existing web sites providing correspondence between tags and transcripts do not concern all species for which numerous EST and cDNA have already been sequenced. web site.This is the reason why we designed and implemented a freely available tool called Identitag for tag identification that can be used in any species for which transcript sequences are available. Identitag is based on a relational database structure in order to allow rapid and easy storage and updating of data and, most importantly, in order to be able to precisely define identification parameters. This structure can be seen like three interconnected modules : the first one stores virtual tags extracted from a given list of transcript sequences, the second stores experimental tags observed in SAGE experiments, and the third allows the annotation of the transcript sequences used for virtual tag extraction. It therefore connects an observed tag to a virtual tag and to the sequence it comes from, and then to its functional annotation when available. Databases made from different species can be connected according to orthology relationship thus allowing the comparison of SAGE libraries between species. We successfully used Identitag to identify tags from our chicken SAGE libraries and for chicken to human SAGE tags interspecies comparison. Identitag sources are freely available on Identitag is a flexible and powerful tool for tag identification in any single species and for interspecies comparison of SAGE libraries. It opens the way to comparative transcriptomic analysis, an emerging branch of biology. In order to characterize the molecular basis underlying self-renewal versus differentiation decision-making process we investigated the transcriptomic changes of various states related to this process, in two model systems : one derived from chicken and the other from human cells. We decided to use Serial Analysis of Gene Expression (SAGE)  to attaia priori regarding the genes to be studied. It can be used with mRNAs derived from cells of any eukaryotic species. SAGE is based on the isolation of a unique sequence tag from each individual transcript and on serial concatenation of several tags into long DNA molecules. Sequencing of concatemer clones reveals individual tags and allows quantification and identification of transcripts. Tag counts are digitally archived and statistically significant comparisons of expression levels can be made between tag counts derived from different populations of cells.Serial Analysis of Gene Expression is a comprehensive method for analyzing transcriptomes  without any An essential step in SAGE library analysis is the unambiguous assignment of each 14 bp tag to the transcript from which it was derived. This process, called tag-to-gene mapping, represents a step that has yet to be completed in the analysis of SAGE libraries. The automated version of this process mostly involves extracting "virtual tags" from sequence databanks : these virtual tags are predictions of the 14 bp sequences that might be produced by a SAGE experiment. The quality of the databanks from which the virtual tags are extracted represents a limiting step in this process. Ideally, the databanks should represent the complete collection of each and every transcript, fully sequenced and annotated. This clearly has yet to be achieved for most species, and therefore one must use the available information that comes mainly from large EST (Expressed Sequence Tags) projects.Arabidopsis thaliana, Bos taurus, Homo sapiens, Medicago truncatula, Meleagris gallopavo, Mus musculus, Pinus taeda, Rattus norvegicus, Sus scrofa, Triticum aestivum and Vitis vinifera). However, this site doesn't include tag to UniGene mapping for several other species for which numerous EST and cDNA have already been sequenced. This is the reason why we designed and implemented a freely available tool for tag identification that can be used in any species for which transcript sequences are available. It can include both complete cDNAs and EST cluster sequences and allow to interrogate the database according to the source of data, to assess the quality of virtual tags derived from different transcript sequences. In this paper we describe the use of this tool for the chicken  where a large EST sequencing effort was completed )Identitag is a flexible and powerful tool for tag identification in any single species and for interspecies comparison of SAGE libraries. It opens the way to comparative transcriptomic analysis, an emerging branch of biology.Project name: Identitag• Project home page: • Operating system(s): SUN, Linux, Mac OS X• Programming languages: Perl, Bourne Shell, MySQL• License: GNU GPL• CK participated to the design of Identitag, implemented Identitag, and participated to the biological validation of Identitag with SAGE libraries. FD constructed the first two SAGE libraries with which Identitag was tested and participated to the biological validation of Identitag with these data. LD and DM brought their expertise in the orthology area, in order to design the orthology relationship. OG supervised this work. All authors participated in the writing of the manuscript, read and approved the final manuscript.


The objective of this randomized prospective study was to compare the efficacy of 50 mcg vaginal misoprostol and 3 mg dinoprostone, administered every nine hours for a maximum of three doses, for elective induction of labor in a specific cohort of nulliparous women with an unfavorable cervix and more than 40 weeks of gestation.One hundred and sixty-three pregnant women with more than 285 days of gestation were recruited and analyzed. The main outcome measures were time from induction to delivery and incidence of vaginal delivery within 12 and 24 hours. Admission rate to the neonatal intensive care unit within 24 hours post delivery was a secondary outcome.The induction-delivery interval was significantly lower in the misoprostol group than in the dinoprostone group . With misoprostol, more women delivered within 12 hours  and 24 hours , spontaneous rupture of the membranes occurred more frequently , there was less need for oxytocin augmentation  and fewer additional doses were required . Although not statistically significant, a lower Caesarean section (CS) rate was observed with misoprostol  but with the disadvantage of higher abnormal fetal heart rate (FHR) tracings . From the misoprostol group more neonates were admitted to the intensive neonatal unit, than from the dinoprostone group . One woman had an unexplained stillbirth following the administration of one dose of dinoprostone.Vaginal misoprostol, compared with dinoprostone in the regimens used, is more effective in elective inductions of labor beyond 40 weeks of gestation. Nevertheless, this is at the expense of more abnormal FHR tracings and more admissions to the neonatal unit, indicating that the faster approach is not necessarily the better approach to childbirth. Induction of labor is carried out for maternal and fetal indications. One of the most common indications is prolonged pregnancy . Recent 2) and misoprostol (PGE1), are widely used in "induction of labor" practice for ripening the cervix and stimulating uterine contractions in order to achieve vaginal delivery. Although dinoprostone has been approved by the FDA for cervical ripening in women at or near term, misoprostol is not currently approved for such use by the FDA, although it has the advantages of lower cost, no need for refrigeration and probably higher efficacy.Prostaglandin analogues, dinoprostone  with that of vaginal dinoprostone (3 mg) when both are administered at an interval of nine hours between repeated doses in a well-homogenized cohort of full term pregnancies (nulliparous women with an unfavorable cervix and without pregnancy complications).th day of gestation. A sequence from a computerized random number generator was used for the allocation of patients to each group. The vaginal administration of prostaglandins was performed by one of the resident doctors on duty, who was not involved in managing these women in labor or delivery. The study was double blind, since the patients were not aware of which type of medication was used, and the deliveries were then performed by two gynecologists blinded to the induction regimen utilized.Between March 1, 2001 and July 16, 2003, 163 women were recruited for the study: 80 women in the misoprostol group and 83 women in the dinoprostone group. All of the women were recruited at Ioannina University Hospital, a tertiary referral center for high-risk pregnancies, with about 1600 deliveries a year. The Ethics Committee of the University of Ioannina approved the study and all participants gave their written informed consent after they had been made aware of the purpose of the study. Although the main indication was prolonged pregnancy, some of the inductions were performed at the patient's request after consultation at 40 weeks of gestation,  and only if they had not delivered by the 285Inclusion criteria were: 1) age>18 years old 2) nulliparity, 3) accurate dating of gestation, including crown rump length (CRL) measurements in the first trimester of pregnancy, 4) singleton viable pregnancy, 5) gestational age ≥ 285 days, 6) cephalic presentation, 7) unfavorable cervical status defined as a Bishop score (BS) of ≤5, 8) intact membranes, 9) reactive non-stress test (NST). Exclusion criteria were: 1) known contraindications to receiving prostaglandins, 2) placenta previa, 3) prior uterine surgery and 4) any antenatal complications.GA was estimated by ultrasound biometry (via CRL measurements in the first trimester of pregnancy) in cases where there were more than 3 days difference from that obtained from the last menstrual period (LMP) . UterineA NST to ensure the well-being of the fetus was performed for each patient at the time of recruitment and admission to the hospital (at least 285 days of gestation) and one hour before the application of the prostaglandin. After the reassessment of the cervical BS, either 50 mcg misoprostol, or 3 mg dinoprostone was administered in the posterior vaginal fornix at 23:00 hours. The NST was repeated (duration of two hours) after 1 h and 5 h. If the woman was in active labor, the membranes spontaneously ruptured or the FHR not reassuring, the patient was transferred to the labor room. Otherwise, a second BS evaluation was carried out the next morning at 08:00 am (after 9 hours). If the cervix was favorable, (BS ≥ 5), the patient was admitted to the labor ward where oxytocin augmentation was carried out if the uterine contractions were unsatisfactory and amniotomy was performed when appropriate. If the cervix was still unfavorable, a second dose of misoprostol or dinoprostone was given and the same evaluation steps as described above were followed. After a total of 18 h had elapsed, non-responders were given a third dose of prostaglandin. When the third dose was insufficient for initiating spontaneous labor, a trial of labor was offered with oxytocin infusion and if no progress was achieved within 6 hours , the patient underwent a CS.The outcome measures were divided into "obstetrical" and "neonatal". The primary outcome measures were time from induction to delivery and incidence of vaginal delivery within 12 and 24 hours; the secondary outcomes were the CS rate, the need for oxytocin augmentation, the incidence of meconium stained amniotic fluid, the incidence of uterine tachysystole, abnormal FHR tracings, maternal morbidity, the admission to neonatal intensive care within 24 hours and neonatal arterial cord ph, base deficit.Statistical analysis was performed using SPSS version 11 software. The Chi square test and Fisher's exact test were used to analyze nominal variables in the form of frequency tables. Normally distributed (Kolmogorov-Smirnov Test with Lilliefors correction) metric variables were tested by the T-test for independent samples, while non-normally distributed metric variables were analyzed by the Mann-Whitney U test. All tests were two-tailed with a confidence level of 95% (p < 0.05). Values are expressed as mean ± standard error (SEM).The two groups were comparable in terms of patients' age  and indication for induction  in the misoprostol and dinoprostone groups, respectively. Gestational age  and the preinduction BS (2.7 ± 0.1) in the misoprostol group were also comparable to the dinoprostone group  and (2.9 ± 0.1), respectively.The induction-delivery interval was significantly shorter  in the misoprostol group, with even less need for a second or third dose  compared to dinoprostone. With misoprostol, more women delivered within 12 h  and almost all of the women delivered within 24 h . In addition, spontaneous rupture of the membranes occurred more often after the administration of misoprostol (p < 0.05) and there was a reduced need for oxytocin augmentation in labor: 65.8% vs. 80.7% with dinoprostone (p < 0.05). However, uterine tachysystole (p < 0.05)) and meconium stained amniotic fluid (p > 0.05) occurred more often in the misoprostol group as did abnormal heart rate tracing  , or needed neonatal resuscitation  but none of the babies had birth asphyxia . The meaA 28-year-old woman at 41 weeks of gestation had an unexplained stillbirth after receiving a single dose of dinoprostone. Seven hours later she had a cardiotocogram without abnormal FHR patterns and regular contractions of the uterus were evident. We decided to move her to the labor ward and within half an hour of entry, no cardiac activity of the fetus was found. During these 30 minutes FHR monitoring had been discontinued, as it was not included in the study design. We decided to let her attempt vaginal delivery. An amniotomy was performed and the amniotic fluid was found to be clear and a vaginal delivery was achieved within 6 hours. Direct examination of the fetus, the placenta and the umbilical cord (UC) showed only a thin UC with excess twisting around its axis. The anatomopathology examination of the fetus revealed no abnormality except a microscopically decreased Wharton's jelly.Nowadays, induction of labor is more widely used than ever before ,25. ReceTo the best of our knowledge, the present study is the only one that compares misoprostol and dinoprostone in such well-homogenized groups. All of the women were nulliparous with intact membranes and at more than forty weeks' gestation with no antenatal complications and all had an unfavorable cervix. In these carefully selected patients, misoprostol at the dose used not only shortened the time between induction and delivery (11.9 vs. 15.6 h), but it also was significantly more effective than dinoprostone. The positive point was that this result was achieved with a very low CS rate even in the dinoprostone group, , respectively. A difference of 5% in favor of misoprostol, although not statistically significant, might have clinical importance in terms of patient health and cost effectiveness. Although in the recent large meta-analysis . In addiEven though misoprostol improves the kinetics of labor during induction in a more efficient way than dinoprostone, concerns persist with respect to intrapartum fetal "wellbeing". In order to avoid uterine hyperstimulation and abnormal FHR tracings, we used for first time in the literature, a 9 h interval between the prostaglandin doses. Although we indeed achieved a low rate of uterine hyperstimulation syndrome , we still noticed a trend towards a high rate of abnormal FHR tracings during induction with misoprostol. Our findings, in accordance with the previous Cochrane metanalysis [Attempting an explanation to the aforementioned side effects of misoprostol use and taking into account other reports ,31,32, iIt still has to be mentioned that in many of our participants, the vertex was not engaged in the pelvic inlet on the day of admittance and this should have been included as an independent risk factor in the initial study design. The exact cause of the stillbirth in the dinoprostone group remains unclear, emphasizing thus, the need for continuous FHR monitoring during labor induction if regular uterine contractions persist ,36.To conclude, 50 mcg misoprostol at a 9 h interval is more highly effective in promoting cervical ripening and in inducing labor, compared to dinoprostone. However, certain aspects concerning fetal well being during labor induction remain questionable. Larger prospective studies comparing elective induction to expectant management after a completed 40-week gestation (on the basis of early ultrasound biometry) might reveal a subgroup of women, such as nulliparous with an unfavorable cervix, who might benefit from an elective induction, preferably with a 25 mcg misoprostol initial dose.E.P: conceived of the study, participated in the sequence alignment, performed the statistical analysis and drafted the manuscript.N.P: conceived of the study, and performed the labor inductionsA.D: performed the neonatal examination and follow upS.A: performed the neonatal examination and participated in the neonatal data analysisC.V: was the midwife involved in women allocation, medications preparation and labour data registrationT.S: conceived of the study and data analysisE.P: conceived of the study and coordinated the studyK.Z: conceived of the study, performed the labor inductions and coordinated the study


The mechanism(s) of nociceptive dysfunction and potential roles of opioid neurotransmitters are unresolved in the chronic pain syndromes of fibromyalgia and chronic low back pain.6-Phe7 (MEAP) and nociceptin immunoreactive materials were measured in the cerebrospinal fluid by radioimmunoassays.History and physical examinations, tender point examinations, and questionnaires were used to identify 14 fibromyalgia, 10 chronic low back pain and 6 normal control subjects. Lumbar punctures were performed. Met-enkephalin-ArgFibromyalgia  and low back pain  groups had significantly higher MEAP than the normal control group . MEAP was inversely correlated to systemic pain thresholds. Nociceptin was not different between groups. Systemic Complaints questionnaire responses were significantly ranked as fibromyalgia > back pain > normal. SF-36 domains demonstrated severe disability for the low back pain group, intermediate results in fibromyalgia, and high function in the normal group.Fibromyalgia was distinguished by higher cerebrospinal fluid MEAP, systemic complaints, and manual tender points; intermediate SF-36 scores; and lower pain thresholds compared to the low back pain and normal groups. MEAP and systemic pain thresholds were inversely correlated in low back pain subjects. Central nervous system opioid dysfunction may contribute to pain in fibromyalgia. Fibromyalgia (FM) is an enigmatic condition characterized by increased complaints of widespread pain with tenderness to palpation . The tenThe pain present in FM may induce antinociceptive neural mechanisms with the release of opioid peptides. This hypothesis was tested by measuring opioid peptides in cerebrospinal fluid, and comparing these levels to systemic pain thresholds, subjective complaints, and quality of life measures in 3 sets of volunteers. FM and Chronic Fatigue Syndrome often overlap . Chronic6-Gly7-Leu8, and Met-enk-Arg6-Phe7 (MEAP) [Two peptides were selected for measurements because they were involved in antinociceptive responses. Alternatively, dysfunction of their release could predispose to chronic pain. Preproenkephalin A is the precursor for leucine-enkephalin, methionine-enkephalin (Met-enk), Met-enk-Arg7 (MEAP) ,10. Noci7 (MEAP) .FM, LBP and Normal control subjects between the ages of 18 and 70 years were recruited to this IRB-approved protocol from rheumatology and orthopedics clinics, advertisements, and word of mouth. Normal subjects were pain and chronic fatigue free, had no diabetic, neurologic, inflammatory, autoimmune, or other chronic disorder that could predispose to pain, alterations in sensation, or known variations in cerebrospinal fluid composition. LBP inclusion criteria were (i) dominant pain complaint of low back pain, and (ii) imaging studies within the past 6 months. Exclusion criteria were: (a) evidence of a lumbar fracture or tumor to explain the pain, (b) any chronic illness that may affect functional status such as diabetes, cancer, chronic obstructive pulmonary disease, chronic inflammatory diseases, renal insufficiency or similar debilitating disorders, (c) previous back or neck surgery, and (d) FM or Chronic Fatigue Syndrome. FM subjects had a prior clinical diagnosis of FM including widespread pain affecting all 4 quadrants and the axial skeleton lasting at least 3 months that was not explained by any other chronic illness, and the presence of at least 11 of 18 tender points when manual, digital pressure of ~4 kg was applied [This self – report questionnaire containing 44 queries grouped into the following modules: (i) Fatigue; (ii) Musculoskeletal: morning stiffness, muscle pain, muscle spasms, dry eyes, dry mouth, fingers sensitive to the cold, fingers turn blue and/or white in the cold, swollen lymph nodes, swollen joints, fever; (iii) Chest: shortness of breath (SOB), SOB when hurrying on level ground or walking up a slight hill, SOB when walking with other people of own age on level ground, stop for breath when walking at own pace on level ground, SOB when washing or dressing, rapid heart rate, chest pain, irregular heart rate, palpitations; (iv) Headaches: migraine or tension type; (v) Neurological: numbness or tingling of hands or legs, inability to concentrate, problems with memory, dizziness; (v) Ear, Nose and Throat (ENT): problems with balance, hearing loss, ear pain, sensation of ear blockage or fullness, ringing in the ears, sinus pain; (vi) Bladder: urinary urgency, pelvic discomfort / pain / pressure, persistent bladder fullness after urination, dysuria; and (vii) Irritable Bowel Syndrome (Rome I criteria): abdominal pain relieved with bowel movement, abdominal pain with a change in frequency or consistency of stool, changes in stool consistency, changes in stool form (hard or loose/watery), changes in passing of stool, bloating or feeling of abdominal distention, passage of mucus, nausea or vomiting . SubjectMOS SF-36[Subjects completed the MOS SF-36. The dom2 rubber stopper making contact with the skin  [2 recorded. The average pressure causing pain was the Average Pain Threshold. FM and Normal subjects had manual digital pressure examinations of these points [All subjects had pressure testing at 9 bilateral sites 18 total) using a hand held dolorimeter  with a 1 cm8 total uLumbar punctures were performed using local anesthetic and 23G spinal catheters. Volumes of 4 to 8 ml were obtained, centrifuged, aliquoted, and immediately frozen at -80°C. Samples were shipped on dry ice to Dr. Lars Terenius for measurement of neuropeptides. Neuropeptides were extracted from 1 ml aliquots using C-18 SepPak cartridges, eluted, dried, and resuspended for validated radioimmunoassays for MEAP ,14 and nGeometric mean and 95% confidence intervals were determined for each neuropeptide, with arithmetic means and 95% CI's for all other variables. Differences between groups were assessed by ANOVA. Differences between means for each pair of groups were assessed by 2-tailed unpaired Student's t-tests with Bonferroni corrections for multiple comparisons. Significance was ascribed for p < 0.05.Lumbar punctures were performed on 14 FM , 10 LBP  and 6 Normal  subjects. The averages and ranges of ages for these 3 groups were similar . There were 4 African-Americans in the FM group, and 1 each in the LBP and Normal groups, and 1 Asian in the FM group. The remainder was Caucasian.Significant differences were found between FM and Normal results by 2-tailed unpaired Student's t-tests with p < 0.05 after Bonferroni corrections for multiple comparisons of this data. All of the FM subjects complained of fatigue figure . Other hDomain Scores for the Normal group were all near the predicted values of 100 figure . FM scor2) compared to the LBP  and Normal  groups . The normal volunteers had significantly lower geometric mean concentrations  than the FM , and LBP  , LBP  and Normal  groups.2, which was coincidentally the lower 95% CI for the Normal group. MEAP – irm concentrations had a wide range in the FM subjects, but the geometric mean was significantly higher than for Normal subjects. Pain threshold and MEAP – irm concentrations did not have linear correlations in either the FM or Normal group. These data suggested that FM subjects were fundamentally different from Normal. When the pain threshold was below 2 to 2.3 kg/cm2, MEAP – irm levels increased approximately 4-fold compared to Normal. The combination of the widespread pain, systemic complaints, low pain threshold and high MEAP – irm concentrations in CSF was distinct from the low level of symptomatic complaints, normal (high) pain thresholds, and lower MEAP – irm levels found in the Normal group.These 2 variables were correlated  when all subjects were examined as a single group figure . This coThe LBP group had a chronic regional pain syndrome, no fibromyalgia, normal pain thresholds and systemic complaints, but severe disability SF-36 scores). Only the LBP group showed a linear correlation between pain threshold and MEAP – irm concentrations  of CFS were probably independent of those responsible for the elevated MEAP – irm levels in FM. Unfortunately, CFS subjects without FM could not be simultaneously tested to determine if their MEAP – irm levels were normal (as would be predicted).Nociceptin – irm levels were the same in our three groups. The levels were about 10% of that found in women during labor . It was The Normal group had Systemic Complaints and SF-36 scores in the normal ranges, high pain thresholds, low numbers of manual and dolorimetry-derived tender points and low cerebrospinal fluid MEAP and nociceptin concentrations. The LBP chronic regional pain group had similar Systemic Complaints scores, systemic pain thresholds and dolorimetry-defined tender point counts. However, most of their SF-36 results were near zero indicating the worst level of impairment of the 3 groups. MEAP – irm was just significantly elevated compared to the Normal group, and was correlated to the systemic pain threshold. The FM group was distinct since they had widespread pain complaints, the highest Systemic Complaints scores, the lowest pain thresholds, and highest numbers of tender points of the 3 groups. Their SF-36 scores were intermediate between the LBP and Normal groups. MEAP – irm concentrations were significantly higher in the FM than Normal group. The co-existence of Chronic Fatigue Syndrome with FM did not alter the MEAP – irm concentrations. This suggested that Chronic Fatigue Syndrome mechanism(s) did not involve preproenkephalin dysfunction. Nociceptin – irm levels were not different between these groups, and were lower than previously reported results from pregnant women in labor. Significant differences in MEAP – irm concentrations from previous studies may be due to the highly controlled definition of patients in this study, selection of control groups, and differences in peptide extraction methods.6-Phe7; SF-36, short-form of 36 questions with the following domains: PF, physical functioning; SF social functioning; PP, role limitation due to physical problems; EP, role limitation due to emotional problems; MH, mental health; E/F, energy / fatigue; P, pain; GP, general perception of health; CH, change in health; Systemic Complaints domains: MS, musculoskeletal; HA, headache; Neuro, neurological; ENT, ear, nose & throat; IBS, irritable bowel syndrome (Rome I criteria); SOB, shortness of breath.CSF, cerebrospinal fluid; FM, fibromyalgia; fMRI, functional magnetic resonance imaging; irm, immunoreactive material; LBP, low back pain; MEAP, Met-enk-ArgThe author(s) declare that they have no competing interests.JNB compiled the results and wrote the manuscript. DJC oversaw the clinical investigations that were performed by JC. GW was responsible for the sample repository, shipping, and collection of data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Inhaled side-stream tobacco smoke brings in all of its harmful components impairing mechanisms that protect the airways and lungs. Chronic respiratory health consequences are a complex multi-step silent process. By the time clinical manifestations require medical attention, several structural and functional changes have already occurred. The respiratory system has to undergo an iterative process of injury, healing and remodeling with every exposure.To have a better understanding of the initial changes that take place when first exposed to environmental tobacco smoke, we have developed an exposure model, using the frog palate that closely represents the features of obstructive airways where ciliary dysfunction and mucus hypersecretion occur.Mucus transport was significantly reduced, even after exposure to the smoke of one cigarette (p < 0.05) and even further with 4-cigarettes exposure (p < 0.001). Morphometric and ultrastructural studies by SEM show extensive areas of tissue disruption. Gelatinase zymography shows activation of MMP9 in mucus from palates exposed to tobacco smoke.The clearance of mucus on the frog palate is significantly reduced after exposure to environmental tobacco smoke. Cilia and the extracellular matrix are anatomically disrupted. Tobacco smoke triggers an increased activity of matrix metalloproteinases associated with a substantial defoliation of ciliated epithelium. These studies enhance the knowledge of the changes in the mucociliary apparatus that occur initially after exposure to environmental tobacco smoke, with the goal of understanding how these changes relate to the genesis of chronic airway pathologies in humans. Respiratory diseases, infectious and non-infectious, are a prime cause of morbidity, mortality and health system utilization in many countries. Exposure to cigarette smoke is an important factor in causing as well as increasing complications in several pulmonary disorders. The mucociliary clearance constitutes the first line of defense to maintain the airways as free as possible of foreign bodies . ImpairmIn laboratory studies we have shown that the physical properties of mucus in nonsmokers are not altered by age or by restrictive pulmonary pathology. However, mucus properties show alterations when exposed to tobacco smoke and these alterations are noticeable in the very early stages of smoke exposure, even at an exposure level in the range of 1 to 5 cigarettes per day .The majority of studies on the outcome of tobacco use have been performed in the late phases of the process -7; the ain vivo and ex vivo epithelial injury models that replicate the features of airway diseases [Therefore our laboratory has been developing and using diseases -12. The ex vivo model, and in later studies use in vivo models.We also aim to have a better understanding of the mechanism by which ciliated epithelial cells are exfoliated after being exposed to tobacco smoke, as this may relate directly to impaired mucus clearance in several human airway diseases including chronic bronchitis and chronic obstructive pulmonary disease (COPD). Hence we decided first to test our novel Rana catesbiana, the upper portion of the head is removed following the procedures described in previous work [From a bullfrog, ous work ,11, by c2, and 15 NaHCO3 (220 mosm/l). The palate was placed inside the frog chamber, a wooden box with a glass top and fitted glass front, and manipulated trough glove openings. The humidity inside the box is maintained at 100% using an ultrasonic Pari nebulizer and the temperature is kept between 22° to 24°C by a rheostat-controlled, externally mounted light source. Before carrying out any measurement, the palate was allowed to stabilize inside the box for 15 minutes before testing.The FRS was prepared by mixing standard Ringer injection with sterile water (2:1). The composition of standard frog Ringer (in mmol/l) is 90 NaCl, 3 KCl, 2 CaClThe exposure chamber Figure  with a vThe exposure chamber was designed to allow a steady flow of side stream tobacco smoke to reach the epithelial tissue. The concentration of cigarette smoke inside the exposure chamber was maintained constant throughout the experiment by the presence of a mixing baffle on the outflow of the chamber. The concentration of tobacco smoke inside the chamber was not directly measured, but is estimated that the half palate was exposed to the smoke of each cigarette delivered, diluted in 180 liters of fresh air. As much as possible, the conditions  in the frog chamber and in the exposure chamber were maintained similar, with cigarette smoke being the independent variable.The palate was divided longitudinally in two halves along the midline as shown in Figure The palate was placed under a dissecting stereomicroscope provided with a reticulated eyepiece. Mucociliary clearance was determined by observing the movement of particles of charcoal powder gently deposited on a sample of mucus on the palate surface; its clearance was visually monitored and MTV determined. The displacement of 3 – 5 μL of endogenous frog mucus sample was calculated by dividing the distance traveled by the transit time across the 0.3 inch (7.62 mm) segment marked between 0.1 and 0.4 inches in the graduated eyepiece. At least five measurements of the time required for the mucus sample to travel the defined distance were made every time to obtain control and smoke exposure mucus transport velocity.We used cigarettes regularly available at commercial outlets of a representative brand in terms of customer preference and toxic emissions: nicotine 0.5 – 2.1 mg, tar 4 – 24 mg, carbon monoxide 5 – 25 mg, hydrogen cyanide 0.04 – 0.21 mg, benzene 0.025 – 0.069 mg, formaldehyde 0.018 – 0.1 mg. One cigarette took 17 minutes on average to burn completely in this preparation.After being exposed to one cigarette, the half palate was brought back to the frog chamber and MTV was measured in both control and exposed halves. A sample of mucus was collected from each half, and placed in separate containers and frozen at -80°C in liquid nitrogen followed by storage in a -80° freezer until analysis. The exposed half, brought back to the exposure chamber was further exposed to 3 more cigarettes . Following tobacco exposure, the half palate was again brought back to the frog chamber to measure mucus transport and collect tissue and mucus sample in both halves, control and exposed. One set of half palates (control and exposed to four cigarettes) was stored at 4°C overnight, and transport of mucus was reassessed the next day.Sample of mucus from both halves were immersed in glutaraldehyde 2.5%, and placed in properly identified separate containers for storage at 4°C for later Scanning Electron Microscope (SEM) studies. The epithelial tissue was carefully dissected and separated from the palate musculature. Samples of the palate tissue from each half were sectioned, frozen in liquid nitrogen and stored at -80°C for gelatinase zymography studies.2 0.5 mM, EDTA 10 mM, Tris-HCl 1 M, pH 6.8) was added to the ground samples  that were sonicated for 30 seconds on ice and then centrifuged at 8000 rpm for 5 minutes at 4°C. The supernatant was collected for protein assay . Normalizing the protein content as 5 μg, different amount of samples were loaded into the 7.5% separating zymography gel  and were run at 100 volts for 20 minutes, 150 volts for 40 minutes. After electrophoresis, the gel was washed 3 times (20 minutes per a time) in 2.5% Triton X-100 at room temperature followed by incubation for 96 hours in zymography development buffer . The gel was then stained for 2 hours with stain solution  followed by de-staining (ethanol: acetic acid: H2O= 1: 2: 22) overnight. The expression of the gelatinases was shown on the gel as clear bands against the dark background stained with Coomassie blue. The bands were compared with the gelatinase standards for MMP 2 and 9 running in the first lane of each gel. The density of the gels was measured in the Bio-Rad scanning densitometer.Samples of tissue, as well as mucus, were taken out of the freezer and ground to a powder in a mortar and pestal. Liquid nitrogen was added to keep the samples frozen. Homogenizing buffer , ten minutes at each step, followed by two additional periods of absolute ethanol (10 minutes each). The samples were further dehydrated by critical point drying at 31°C for 5 – 10 minutes, then mounted on a specimen holder for SEM and dried overnight in vacuum desiccators. In the final stage of preparation for viewing, the samples were sputter coated with gold . Samples were viewed using SEM (Hitachi S-2500). Images were scanned directly to a computer and stored as image files for subsequent viewing and analysis.Data are expressed as mean ± standard deviation unless otherwise stated. A paired Student-T test was used for simple comparison. The level of significance was set at 5 %.The modified fresh frog palate exposure model was relatively easy to prepare and practical to handle. On gross examination, the surface of the palate exposed to side stream tobacco smoke did not show any macroscopic change in appearance after cigarette smoke exposure (CSE) compared to the control halves maintained in the normal chamber.MTV) was measured in both half palates prior to exposure of one half palate to cigarette smoke, and were identical (19.5 ± .03 mm/sec). Two additional MTV determinations were carried out in the control half after one and four cigarettes and after 24 h period of recovery during which time the palate was kept at 4°C. A paired T-test showed no statistical difference in mucus transport velocity among the control measurements during the entire experiment  immediately after exposure to the side stream smoke of one cigarette compared with the non-exposed half palate. Further exposure to the side stream smoke of three more cigarettes  significantly reduced MTV (p < 0.001), with no signs of recovery after 24 hours.We obtained a sample of mucus from the half palate exposed to four cigarettes and used it on the control half palate to measure MTV. Clearance was within the normal range. Immediately after, the same sample of mucus was tested on the exposed half palate and mucus clearance was again seen to be very slow. Mucus transport had basically ceased in some areas of the tobacco-smoke exposed palate.Scanning electron microscopy (SEM) studies to assess the integrity of the epithelium after exposure to side stream smoke of one cigarette showed areas where the layer of cilia looked disordered. However, we did not see loss of cilia or exfoliation of ciliated epithelial cells after careful examination under the SEM in lower and high power of the entire surface of the sample after this level of exposure.In Figure Gelatinase zymography showed increased activity of MMP-9 in mucus collected from the palates exposed to tobacco smoke of four cigarettes compared to mucus from control palates as shown in Figure Major findings in this study include: a) our amphibian mucociliary clearance model appears to be appropriate to study the early effects of tobacco smoke exposure; b) the clearance of mucus is significantly reduced after exposure to side stream tobacco smoke associated to dose response; c) ciliated cells are anatomically and physiologically acutely affected therefore drastically impairing mucus clearance. Acute irritation and inflammation of cilia due to exposure to tobacco smoke could explain this alteration; d) mucus seems to not be physiologically affected at this stage; and e) tobacco smoke triggers an increased activity of matrix metalloproteinases (in particular MMP9) associated with disruption of the extracellular matrix, most likely affecting cellular attachments to the basal membrane generating a substantial exfoliation of the ciliated cells.These findings might assist us in enhancing the knowledge of the changes in the mucociliary apparatus that occur early after exposure to environmental tobacco products, with the goal to understand how these changes relate to the development of chronic airway pathologies in humans.The frog palate has been used for several decades as a model to assess mucociliary clearance -17. Diffet al [This exposure model is an isolated system, theoretically free of interferences from other agents or systemic physiological responses resulting from other internal or external influences. In this injury model we randomly exposed either half palate to side stream tobacco smoke and used the opposite half palate as the control . Since there was no statistical difference in mucus transport between them, previously established in different sets of pairs of palates, one side was arbitrarily selected for control and the opposite half for exposure to tobacco smoke. This study follows an approach utilized previously by Zayas et al  that comet al [In assessing the mucus transport rate in both half palates before any experimental procedure and to insure that they functioned identically, we established baseline data for our experiment. This helped to reduce error variability due to sex, weight, age, as well as control for any seasonal effect on the mucus transport rate in the frog palates as demonstrated by Rubin et al .Since the palate is excised from the frog we may assume that any observed effect or response is a local and direct effect of exposure to side stream tobacco smoke, which is an advantage of our model. From the data obtained, we would conclude that our frog palate exposure model is suitable to study the acute effects of second hand tobacco smoke.We may also conclude that clearability of mucus seems not to be altered at this stage, at this level of exposure and in this particular exposure preparation or design. Therefore, at this stage mucus transport seems not to be functionally affected for being cleared in a normal fashion by non-exposed cilia. However, after more prolonged tobacco exposure there will be modifications in mucus properties, as seen and measured in smoking dogs where after two months of tobacco exposure, the galactose content and the viscoelastic properties of mucus were observed to present alterations. After further exposure the viscosity and elasticity properties of mucus were reversed to quasi-normal levels, but galactose content did not . Hence tMucus clearance is a function of cilia beat frequency and mucus viscoelastic properties. A change in mucus clearance may be due to one or the other component or both. In future studies, incorporating high speed digital imaging of the palate surface to determine cilia beat frequency will allow us to further differentiate between cilia related effects versus mucus related effects.Exposure to tobacco smoke with all its noxious agents and components will possibly allow us to separate and study in detail, the timing and appearance of different phases of the organism response. Our exposure model may allow us to individualize and study the inflammation phase resulting from the tobacco exposure. We can then focus on the injury phase occurring in the mucociliary system. Later we will explore mechanisms involved in the healing process. The remodeling of the ciliated epithelium following acute injury will be an important component of our studies and particularly how the remodeling is mediated. The time frame of the injury and recovery phases needs to be determined.Cilia do not work alone, but in association with other cilia to produce metachronal waves. Several metachronal waves may contribute to the propulsion of the mucus layer to flow over irregularities or non-ciliated areas. Our data indicates that acute tobacco exposure may have an initial and early irritation effect, possibly mediated by an unknown mechanism on the exposed palate that leads to inhibition of ciliary beat frequency or discoordination of metachronal waves. Such factors adversely affect clearance by imposing stasis or local eddies resulting in erratic clearance that may be the reason why scanning electron micrographs of palates after one cigarette showed cilia somewhat disordered, but not visibly disrupted.We have shown that tobacco smoke exposure interferes with mucociliary clearance. Sustained exposure may lead to loss of ciliated epithelium associated with activation of matrix metalloproteinases. Significant loss of cilia or ciliated epithelial cells results in disruption or even cessation of mucociliary clearance. Matrix metalloproteinases may be implicated in this injury through disruption of epithelial cell-to-cell or cell-to-basement membrane connections. Further clarification of the mechanisms involved will be undertaken in subsequent studies.Our exposure model can assess physiological, ultrastructural and molecular parameters in response to the initial deleterious effects of acute exposure to side stream tobacco smoke in an epithelial model homologous to the human airways. In these preliminary studies we did not attempt to mimic "human smoking conditions". However, our results show that MTV is affected even after exposure to one cigarette. Although concentration of smoke used in the present study is higher than likely encountered in typical environmental tobacco exposures, they are within an order of magnitude of those computed for exposure in poorly ventilated cars or homes. Future studies will try to replicate real conditions faced by non-smokers exposed to environmental tobacco smoke and to characterize the mediation and effectors of this acute injury.


Systemic lupus erythematosus is an age- and gender-associated autoimmune disorder. Previous studies suggested that defects in the hypothalamic/pituitary axis contributed to systemic lupus erythematosus disease progression which could also involve growth hormone, insulin-like growth factor-1 and somatostatin function. This study was designed to compare basal serum growth hormone, insulin-like growth factor-1 and somatostatin levels in female systemic lupus erythematosus patients to a group of normal female subjects.Basal serum growth hormone, insulin-like growth factor-1 and somatostatin levels were measured by standard radioimmunoassay.2 = 3.03) in the entire group of normal subjects (i.e. 20 – 80 years). In contrast, serum insulin-like growth factor-1 levels were inversely correlated with age (adjusted r2 = 0.092). Of note, serum growth hormone was positively correlated with age (adjusted r2 = 0.269) in the 20 – 46 year range which overlapped with the age range of patients in the systemic lupus erythematosus group. In that regard, serum growth hormone levels were not significantly higher compared to either the entire group of normal subjects (20 – 80 yrs) or to normal subjects age-matched to the systemic lupus erythematosus patients. Serum insulin-like growth factor-1 levels were significantly elevated (p < 0.001) in systemic lupus erythematosus patients, but only when compared to the entire group of normal subjects. Serum somatostatin levels differed from normal subjects only in older (i.e. >55 yrs) systemic lupus erythematosus patients.Serum growth hormone levels failed to correlate with age (rThese results indicated that systemic lupus erythematosus was not characterized by a modulation of the growth hormone/insulin-like growth factor-1 paracrine axis when serum samples from systemic lupus erythematosus patients were compared to age- matched normal female subjects. These results in systemic lupus erythematosus differ from those previously reported in other musculoskeletal disorders such as rheumatoid arthritis, osteoarthritis, fibromyalgia, diffuse idiopathic skeletal hyperostosis and hypermobility syndrome where significantly higher serum growth hormone levels were found. Somatostatin levels in elderly systemic lupus erythematosus patients may provide a clinical marker of disease activity in these patients. Systemic lupus erythematosus (SLE) is the protean autoimmune disorder with strong familial penetrance. Immunologically, SLE is characterized by aberrations in T cell and B cell function ,2, over-We have shown that diverse rheumatic and musculoskeletal disorders, including osteoarthritis (OA) -12, diffInsulin-like growth factor-1 (IGF-1) synthesis is coupled to growth hormone via its capacity to stimulate hepatocyte IGF-1 production . In seveSomatostatin is a 14 amino acid polypeptide whose principal function is to regulate growth hormone release from the pituitary . ElevateThe present study was performed to determine the extent to which serum growth hormone, IGF-1 and somatostatin levels were modulated in patients with SLE. A linear regression analysis was performed to determine the relationship between age and serum growth hormone and IGF-1 levels in a group of normal female subjects so that these values could be employed for comparison to a group of predominantly young, female SLE patients.All studies were performed at University Hospitals of Cleveland (UHC) and the Wade Park Veterans Administration Medical Center (VAMC), Cleveland, Ohio. The UHC and VAMC Institutional Review Boards approved the study design with the research protocol, which included informed consent, being in keeping with the Declaration of Helsinki. Normal subjects and SLE patients were all volunteers. SLE Patients met the clinical and laboratory criteria for the diagnosis of SLE according to previously published classifications . PatientBlood drawn by venipuncture was clotted at room temperature, centrifuged, serum aliquots separated and stored at -70°C until assayed. Blood samples were generally collected during an identical 3–4 hr morning period to normalize the potential contribution of growth hormone pulses and serum glucose levels to serum growth hormone determinations -10. SeruBasal serum growth hormone and IGF-1 levels were determined by standard radioimmunoassay (RIA)  as previously described -10. The 2-value and regression line, respectively.The 2-tailed T-test was employed to analyze the differences in means of serum growth hormone, IGF-1 and somatostatin concentrations in groups of unequal size where p < 0.05 was significant. The population sample size was sufficient to detect a 20% difference in serum growth hormone and IGF-1 levels between control subjects and SLE patients and a 15% difference in serum somatostatin levels. The relationship between serum growth hormone and IGF-1 levels as a function of age was analyzed from scatter plots by linear regression analysis employing SPSS 11.1  and SigmaPlot 8.0  to calculate the adjusted rGlucose and insulin concentration was determined in serum from normal female subjects and from patients with SLE. No normal female subjects were excluded from the study as a result of detecting hyperglycemia or hyperinsulinemia However, 2 SLE patients were excluded from the statistical analysis on this basis (data not shown).Over the course of this study, SLE patients received medical therapy with NSAIDs, prednisone (10–60 mg/day), hydroxychloroquine sulfate or methotrexate as well as combinations of these drugs. No SLE patients were treated with azathioprine or cyclophosphamide during this study.2 = 0.269) in this group of normal female subjects was found  between age  and basal serum growth hormone levels in the young female normal subjects was found  in SLE remains to be elucidated. In this regard, van Hagen  showed tThe therapeutic implications and diagnostic utility of serum growth hormone, IGF-1 and somatostatin measurements in SLE as well as in other musculoskeletal disorders appears central to assigning a role for these factors in disease progression. Serum growth hormone remained elevated in some DISH and OA patients where clinical symptoms were significant ,11,12. TThe authors declare that they have no competing interests.CWD participated in the design of the study and performed the clinical analysis. CJM participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The available methods are mainly based on the annotation of genome sequences including two successive steps, the prediction of coding sequences (CDS) and their function assignment. The annotation process takes time. The available methods often encounter difficulties when dealing with unfinished error-containing genomic sequence.A necessary step for a genome level analysis of the cellular metabolism is the in silico reconstruction of metabolic networks. Instead of using predicted genes or CDSs to query public databases, entries from public DNA or protein databases are used as queries to search a local database of the unannotated genome sequence to predict CDSs. Functions are assigned to the predicted CDSs simultaneously. The well-annotated genome of Salmonella typhimurium LT2 is used as an example to demonstrate the applicability of the method. 97.7% of the CDSs in the original annotation are correctly identified. The use of SWISS-PROT-TrEMBL databases resulted in an identification of 98.9% of CDSs that have EC-numbers in the published annotation. Furthermore, two versions of sequences of the bacterium Klebsiella pneumoniae with different genome coverage  are examined. The results suggest that a 3.9-fold coverage of the bacterial genome could be sufficiently used for the in silico reconstruction of the metabolic network. Compared to other gene finding methods such as CRITICA our method is more suitable for exploiting sequences of low genome coverage. Based on the new method, a program called IdentiCS (Identification of Coding Sequences from Unfinished Genome Sequences) is delivered that combines the identification of CDSs with the reconstruction, comparison and visualization of metabolic networks (free to download at ).In this work a fast method is proposed to use unannotated genome sequence for predicting CDSs and for an The reversed querying process and the program IdentiCS allow a fast and adequate prediction protein coding sequences and reconstruction of the potential metabolic network from low coverage genome sequences of bacteria. The new method can accelerate the use of genomic data for studying cellular metabolism. In silico reconstruction of metabolic networks from genome sequences of organisms represents a starting point for a systematic analysis of metabolism [Knowledge about the metabolic network of an organism is essential for understanding its physiology and phenotypic behavior. A comprehensive understanding of the metabolic network at the system level is particularly important for both biotechnological and biomedical research and is now made possible by rapid advances in genome sequencing and functional genomics. tabolism -3. The ftabolism .Several methods and tools have been recently developed for the reconstruction, visualization and analysis of metabolic networks. These include general static metabolic network tools such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) , the BoeThe three-step method starting from gene finding has several drawbacks for the reconstruction of the metabolic network from incomplete or unfinished genome sequences. In unfinished sequences of a genome, especially in sequences with a low genome coverage (e.g. less than 4 fold), there may be many sequencing errors that do not warrant an accurate prediction of genes . For exain silico reconstruction of the metabolic network. The method is demonstrated with genome data from two organisms. A program combining automatic prediction and function assignment of CDSs with a visualization and comparison of metabolic networks of different organisms is also delivered.To avoid these problems, alternative methods are required for directly reconstructing metabolic networks from unfinished genome data. Sequencing and annotation are still time and resource consuming. An as early as possible exploitation of the genome data is of importance for functional genome research. In this work, we propose a method to identify coding sequences for proteins (particularly for metabolic enzymes) directly from unannotated low-coverage genomic data for The principle of the new method is schematically shown in Fig. S. typhimurium LT2 [S. typhimurium LT2 has 4449 CDSs, out of which 1218 have been annotated with enzyme EC numbers. These CDSs encompass 656 different unique EC numbers. The reliability of CDS prediction by the program IdentiCS is evaluated by using a nucleotide database (KEGG genome) and a combined protein database (SWISS-PROT + TrEMBL + TrEMBL update) separately. The annotated coding sequences or proteins of S. typhimurium were filtered out of these databases before sequence alignment. All CDSs having an E-value less than 10-10 were accepted and submitted for comparison with the KEGG annotation of S. typhimurium. The results are summarized in Table S. typhimurium are identified by using the KEGG genome database and the whole protein database SWISS-PROT and TrEMBL, respectively. The sensitivity on the nucleotide level (91.1% and 98.2% for the two databases respectively) is similar as on the CDS level. These results suggest that the SWISS-PROT-TrEMBL based approach is more preferable than the KEGG genome based approach for our method. It is understood that the combined protein database SWISS-PROT and TrEMBL contains almost all of the known protein sequences available in public databases (including proteins in silico translated from nucleotide sequences) while the KEGG database contains only a limited number of sequenced and annotated genomes. The difference becomes more significant if the organism studied is evolutionarily far from any organism whose genome is completely sequenced and annotated.To examine the applicability of the method proposed, the well-annotated genome sequences of rium LT2  are usedThe specificity of the method is about 81–82% on the CDS level and 87.2–94.9% on the nucleotide level for the KEGG genome database and the whole protein database SWISS-PROT and TrEMBL. The moderate specificity on CDS level is due to the relatively high amount of additionally predicted CDSs . It should be mentioned that all the additionally predicted CDSs have quite strong statistic significance . These additional CDSs may be missed in the original annotation and could in fact represent good candidates for an improved annotation of the genome.The inconsistence rate by IdentiCS is as low as 0.35% for the KEGG genome database and 0.64% for the SWISS-PROT and TrEMBL protein database, indicating the reliability of our method.In the above mentioned evaluation of the method, the cut-off E-value is less than 1E-10 for the CDS prediction. Further the effects of different scoring parameters  and their cut-off values on the CDS prediction by using the database SWISS-PROT and TrEMBL are examined. Fig. 2A,2B,2C) show the distribution of the true positive CDSs, false positive CDSs, sensitivity and specificity as function of bits score, E-value and identities respectively. The major part of the false positive CDSs is found in the regions of bits score less than ca. 80 and E-value larger than 1E-15. The specificity increases with the bits score and E-value in a form of a saturation curve while the sensitivity decreases almost linearly. This indicates that the prediction performance can be further optimized by choosing appropriate cut-off parameters. If the CDSs with bits score less than 75  are rejected, the false positive will decrease by 40% (from 987 to 602) while the false negative will increase from 110 to 144  of annotated EC-CDSs of E. coli based on the KEGG genome database. With the method proposed by Ma and Zeng [K. pneumoniae is found to be most similar to those of E. coli and S. typhimurium (data not shown). Thus, the predicted number of enzyme-encoding sequences for K. pneumoniae appears to be reasonable. With the same 3.9-fold coverage genome sequences of K. pneumoniae, the method of WIT predicted 2650 EC-CDSs which are twice the number of EC-CDSs in E. coli and S. typhimurium. The EC-CDSs predicted by WIT are significantly smaller and fragmented, possibly because of the presence of too many errors in the unfinished genome sequences. The fragmentation problem was overcome in our method that leads to a significant reduction in the number of identified EC-CDSs. The less false positive EC-CDSs will further simplify experimental design such as for microarray to examine the metabolic network.Both the KEGG genomes and SWISS-PROT-TrEMBL databases are used to identify enzyme-coding sequences from the 3.9-fold and 7.9-fold coverage genome sequences of and Zeng , the strin silico reconstruction of the metabolic network of K. pneumoniae. It would be of interest to examine if this also applies to other organisms or even lower genome coverage. The use of lower genome coverage sequences for studying cellular metabolism will greatly accelerate the exploitation of genome sequencing projects.A comparison of the unique EC numbers of EC-CDSs identified from the two different versions of genome sequences and by the different approaches reveals that the results of these different combinations share over 80% of common EC numbers Table . The WITK. pneumoniae as identified by the combination of 7.9-fold genome sequences and SWISS-PROT and TrEMBL databases are summarized in Table K. pneumoniae . From tFor a straightforward visualization of the biochemical reactions related to a specific organism and especially for comparing the metabolisms of different organisms, the KEGG metabolic maps act as a blueprint for visualization of metabolic networks in this work. Reconstruction, comparison and visualization of the metabolic network have been integrated in the program IdentiCS as a built-in component. It can work directly with the coding sequences and their functions predicted by IdentiCS or with existing annotation files such as the those in Microsoft Excel format or in GenBank flat file format. To use the KEGG maps for metabolic reconstruction, comparison and visualization information from the KEGG metabolic pathways is reformed into an Excel template. Metabolic network reconstruction is realized by mapping the identified enzymes (EC numbers) to the KEGG metabolic maps. Different similarity levels of the identified enzymes can be displayed in different colors. All identified enzymes in the map are marked and linked to their annotations. Web links to other Internet databases such as IUBMB , BRENDA S. typhimurium and K. pneumoniae demonstrated the applicability and reliability of the new method proposed for in silico identification of protein coding sequences from unannotated genome sequences. The use of protein sequence databases SWISS-PROT and TrEMBL is more favorable than the use of KEGG genome database for identifying coding sequences and thus for metabolic network reconstruction. Furthermore, the method allows an adequate reconstruction of the potential metabolic network from sequence data with low coverage (e.g. < 4 fold) of the bacterial genome as shown for K. pneumoniae. Together with the algorithms for the automatic annotation of sequences, the visualization and comparison of metabolic networks, the method and program developed in this work can accelerate the use of genomic data for studying cellular metabolism.The use of genome sequences from Salmonella typhimurium LT2 and Klebsiella pneumoniae. The genome of S. typhimurium LT2 has been completely sequenced and well annotated [S. typhimurium LT2 serve as a reference to evaluate the accuracy of the proposed method. The sequences and annotation for S. typhimurium LT2 were downloaded from KEGG [K. pneumoniae has been recently sequenced and the annotation is still in progress. Two different versions of the raw genome data of K. pneumoniae (3.9-fold whole genome shotgun coverage in 920 contigs and 7.9-fold coverage in 341 contigs) obtained from the Genome Sequencing Center of Washington University [The applicability of the method proposed above was examined with the genome sequences of two organisms, namely nnotated . Thus, trom KEGG  were obtained from the Swiss Institute of Bioinformatics . Not "faThe annotation process is based on similarity comparison as normally used in other annotation processes. The difference is that in our approach the gene or protein sequences from public databases are used as queries to search and locate similar ones in the raw genome sequences. When proteins from public database are used as queries, the tblastn algorithm in the BLAST program is applied that compares the query to all six translation frames of the unannotated DNA sequences. The dynamic translation of a small genomic database takes much less system resource than the translation of a large public database as in the conventional methods. Our method can thus be realized on a common PC system, especially when merely a subset of the public database is considered, for example for the purpose of identifying metabolic enzymes for metabolic network reconstruction.Because of sequence errors  and the local alignment nature of the BLAST algorithm, the BLAST research may report several small alignments between different parts of the query protein or gene and different parts of a genomic contig even if there should be only one alignment in the reality. In this situation, the tfasty34 program in the FASTA3 suite  should g1. they are coded on the same strand of the same genomic contig as the highest score fragment. In other words, all of these fragments must be translated either in positive or in negative frames.2. the alignments have an identity level not lower than 80% of the identity level of the highest score alignment.3. the generated larger sequence region has alignment gaps or extensions not more than 20% of its length.Since many queries can be similar to the same region on a genomic contig and sometimes they may have different function annotations, the program must judge and choose one annotation for this region. The decision is made by applying the following criteria:1. Each region normally has only one function. Here the region represents a piece of nucleotides either on the positive strand or on the negative strand of a DNA molecule. The same physical position on different strands of a DNA molecule can belong to different regions, and can therefore have a different function assignment. Although there are examples in some viruses that a region can code different proteins depending on the transcription frame, it happens very rarely in other organisms. The user can assign a tolerance value (e.g. 60 bp) to allow two successive regions to overlap each other to some extent.2. Highest similarity principle. If a query gene or protein has a similarity to a CDS higher than other queries, then the function of this query gene or protein is assigned as the annotation of the CDS. Bits score is used as a measure for similarity first. If two queries have the same bits score, then the identity level in percentage is taken as a second measure for similarity. If both bits score and identity are the same and these two entries have different function annotation (rarely occurred) then both of their functions are assigned to that region.3. Closest evolutionary relationship. If two or more query genes or proteins are comparably similar (e.g. the difference between their identity levels is lower than 5%) to a CDS but have different function, the evolutionary relationship between these organisms is further considered. The annotation of the organism that is mostly related to the studied organism from the viewpoint of metabolic evolution is transferred to the unknown CDS. The evolutionary relationship between different organisms and the one studied is established with the method of Ma and Zeng  after thIdentification of Coding Sequences from Raw Genome Sequences) can reconstruct the metabolic network of an organism with about 5 million base pairs of raw genome data. The computing time is less than 8 hours on a PC with 2.8 GHz Pentium 4 CPU and 512 MB memory. This program works together with Microsoft Excel under Windows environment.In this way, the coding sequences of a genome are identified and annotated at the same time. No second large-scale sequence alignment is needed. Once all the software and databases are prepared, our program which is called IdentiCS , false negative (FN) and false positive (FP) are used to calculate the sensitivity and specificity of CDS prediction in comparison with CDSs in the original annotation. The terms "sensitivity" and "specificity" are defined according to Burset and Guigo :We also evaluated the terms TP, FN and FP on nucleotide level according to Burset and Guigo  and calcJS is the developer of the method and the program. APZ supervised the study. Both authors read and approved the final manuscript.


The molecular basis of severe acute respiratory syndrome (SARS) coronavirus (CoV) induced pathology is still largely unclear. Many SARS patients suffer respiratory distress brought on by interstitial infiltration and frequently show peripheral blood lymphopenia and occasional leucopenia. One possible cause of this could be interstitial inflammation, following a localized host response. In this study, we therefore examine the immune response of SARS-CoV in human peripheral blood mononuclear cells (PBMCs) over the first 24 hours.in vitro with SARS-CoV and the viral replication kinetics was studied by real-time quantitative assays. SARS-CoV specific gene expression changes were examined by high-density oligonucleotide array analysis.PBMCs from normal healthy donors were inoculated We observed that SARS-CoV was capable of infecting and replicating in PBMCs and the kinetics of viral replication was variable among the donors. SARS-CoV antibody binding assays indicated that SARS specific antibodies inhibited SARS-CoV viral replication. Array data showed monocyte-macrophage cell activation, coagulation pathway upregulation and cytokine production together with lung trafficking chemokines such as IL8 and IL17, possibly activated through the TLR9 signaling pathway; that mimicked clinical features of the disease.The identification of human blood mononuclear cells as a direct target of SARS-CoV in the model system described here provides a new insight into disease pathology and a tool for investigating the host response and mechanisms of pathogenesis. Coronaviridae, and have the largest known single-stranded viral RNA genomes (27 to 32 kb). Coronaviruses, have both "early" and "late" phases of gene expression. Regulatory proteins are synthesized as "early" non-structural proteins, while the structural proteins are synthesized as "late" proteins. "Late" structural proteins are usually required in greater amounts thus, there is a necessity to regulate the expression of the viral genes quantitatively. After the viral entry via endocytosis or through specific receptors, the 5'-end of the viral genome is translated directly giving rise to twenty-three viral proteins, including the RNA dependent RNA polymerase (RdRp), and other functional products involved in transcription, replication, viral assembly and cell death. Coronaviruses can be classified into species and three major antigenic groups based on, serology, natural hosts, monoclonal antibody recognition and nucleotide sequencing [The causative agent for SARS has been identified as a novel coronavirus -3 with ggenomes 2 to 32 kbgenomes 2 to 32 kbquencing , althougHowever, SARS has rapidly caused a world-wide problem. The earliest known cases of SARS was reported in Guandong Province, China in November 2002, becoming more widespread by March 2003, when it was introduced to Canada, Singapore, Taiwan and Vietnam via Hong Kong. The largest number of infected patients has been in China with a worldwide incidence totalling more than 8,400 by July 2003. Infection by the virus induces high morbidity and mortality, the latter being estimated at 15% by the World Health Organisation. SARS is characterized by high fever, non-productive cough or dyspnea and in many cases may progress to generalized, interstitial infiltrates in the lung, thus needing intubation and mechanical ventilation , togethein vitro model system that will in the future allow detailed investigations of the host response to be made.We postulated that the pulmonary damage in SARS may not be a direct effect of the virus on the alveoli, but represents a secondary effect of cytokines or other factors proximal to but not from the lung tissue mediated by the host either as the primary or secondary response ,14. In t5 PBMCs were seeded into each well of a 24-well culture plate, 0.5 ml of complete RPMI-1640  added to each and cultured overnight at 37°C (5% CO2). A seed stock of SARS-CoV (strain SIN 2774) passaged in Vero E6 cells was used for infection. Vero E6 culture supernatants were added to each well in 50 μl volume at a concentration of 0.1 or 0.01 MOI (based on plaque forming units) and a control plate (media only). Each culture was set up in duplicate. After 4 hours of incubation, one set of the duplicate wells of the control plate and a 4 hours incubation plate were harvested while the rest received 0.5 ml media top-up and incubated for a further 2, 4, 6 and 8 days.PBMCs were obtained by Ficoll-Hypaque separation of whole blood. 2 × 10Harvesting was performed by gently flushing the wells with a Pasteur pipette to removed non-adherent cells, followed by a rinse of 1 ml RPMI. The rinsed fraction was pooled with the first harvest aliquot and spun at 1500 rpm. The cell pellet was washed twice with 2 ml RPMI to remove virus in the supernatant. 1.5 ml Trizol  was then added to the adherent cell fraction as well as the non-adherent cell fraction to lyse cells and stabilize the RNA. Extraction of total RNA was then performed following manufacturer's protocol and the resultant RNA dissolved in 40 μl water.® MGB probe: 5'-(6-FAM)AGAGCCATGCCTAACAT(NFQ)-3') in a one step PCR using master mix from Applied Biosystems (USA) according to manufacturers' recommendations. Reactions were performed using an ABI PRISM 7900 sequence detection system  and quantitation achieved using standard curves generated from in vitro transcribed RNA.The amount of SARS-CoV in each cell fraction was measured by real-time quantitative PCR assay. 2 μl of RNA was reverse transcribed and amplified in 20 μl using 0.9 μM each of forward (5'-GGTTGGGAT TATCCAAAATGTGA-3') and reverse (5'-AGAACAAGAGAGGCCATTATCCTAAG-3') primers, and 0.25 μM of TaqMan7 cells of mock-infected and infected cells were harvested and lysed using Trizol . Total RNA was isolated according to the manufacturer's recommendation. Quality of the total RNA was judged from the ratio between 28S and 18S RNA after agarose gel electrophoresis. 20 μg of total RNA was labeled with Cy-3 or Cy-5 using the Superscript II reverse-transcription kit  and hybridization was carried out overnight (16 hours) at 42°C on high-density oligonucleotide arrays  using universal human reference  as a reference. Hybridized arrays were scanned at 5 μm resolution on a GenePix 4000A scanner (Axon Instruments) with variable photo-multiplier tube voltage to obtain maximal signal intensities, and the resulting images were analyzed via GenePix Pro v4.0 (Axon Instruments) as described in the manual.At each time point , 5 × 10Raw data were analyzed on GenePix analysis software version 4.0 (Axon Instruments) and uploaded to a relational database. The logarithmic expression ratio for a spot on each array was normalized by subtracting the median logarithmic ratio for the same array. Data were filtered to exclude spots with a size of less than 25 μm and any poor quality or missing spots. Since the correlation of the overall data from reciprocal labeling was good, values obtained from reciprocal labeling experiments were averaged. In addition, the data were distilled to the set of gene features that were present at all 4 time points in both the viral infected samples and the negative controls. The results were represented as the logarithmic ratio of gene expression between the viral infected samples and their corresponding negative controls at the various time points. Application of these filters resulted in the inclusion of ~12,900 of the total ~19,200 gene features in subsequent analyses.f were translated so that their means were zero. Similar genes, whose translated gene-features exhibited same induction-repression pattern, were grouped together. Genes gi, gj were said to be similar if they satisfied the following condition:To discover patterns of gene expression, the values associated with each gene feature , where U(x) = 1 if x > 0, U (x) = 0 otherwise,git and gjt denote the translated values of gene features gi, gj at time t respectively; and N is the number of time points for which the expression of a gene was observed. Similar genes, based on the above criteria, are grouped together. Within each group, the genes were ordered in the descending order of their expression range (defined as the difference between the maximum and minimum ratios of gene expression). This algorithm is a special case of the Friendly Neighbor algorithm currently under development. The final plots were generated using the original expression ratios while preserving the clustering and ordering discovered by the above algorithm. To determine whether a gene observed to be responsive could appear merely by chance, 100,000 expression profiles were generated by randomly sampling the expression ratios from the entire dataset with replacement. The P value of a gene is the fraction of the random profiles whose logarithmic expression range is as good as, or better than that of the selected gene.where 3  of 0.1. The first sampling, taken from cells infected for 4 hours, showed an average copy number of 32 × 103 Fig.  and reprpatients  and cellpatients . An indipatients , in whic3 copies per well which is 4 times more than that attained by donor e, with 120 × 103 copies per well. Such variation strongly suggests that there is an underlying host-pathogen interaction influencing the kinetics of SARS-CoV replication efficiency. These in vitro observations may reflect the wide range of patient outcomes after SARS-CoV infection [The kinetics of viral replication was variable among the 6 donors Fig. . There wnfection .Antibody blocking experiments were also performed in which SARS-CoV was pre-incubated with convalescent patient sera for 30 minutes before introduction to the PBMCs and after a 4 day incubation period, the adherent cell fraction was harvested and assayed for SARS-CoV viral titer. Results clearly showed that even at high dilution, convalescent sera inhibited SARS viral replication (data not shown), presumably by blocking viral entry. This supports other reports indicating that SARS-CoV is not endocytosed through antibody mediated mechanisms and confirms a protective role for antibodies elicited either by the infection or through immunization ,20.in vitro with SARS-CoV (0.1 MOI) and harvested at 4 hours, 8 hours, 12 hours and 24 hours time intervals post-infection. As controls, uninfected aliquots of the same PBMCs were also harvested at the corresponding time points. Total RNA extracted from the PBMCs of the 3 individuals were pooled, labeled and hybridized to human oligonucleotide arrays consisting of ~19,200 gene features. Reciprocal dye swap replicate hybridizations were performed to minimize technical noise. Analysis of variance in expression levels for each gene across all the time points indicated the ~1200 genes which showed the largest variability  and their receptors (CCR4 and CCR7) were highly expressed in response to the infection Fig. , indicatin vitro infection  to be investigated. In future developments of the model, it will be interesting to compare the host response to different SARS-CoV isolates with inactivated preparations of the virus. In other diseases, in vitro models have revealed a number of key processes relevant to the clinical diseases [The emerging picture from this study implicates a central role for the immune response in the pathobiology of a SARS infection. While detailed diseases ,12,13 andiseases -42, furtNone declared.LFPN, MLH, ETL and REC conceived the study, its design and coordination, results analysis and drafted the manuscript. EEO and KFT carried out the virus infections. SYN, KRKM, VNV and JMC were involved in the array and statistical analysis. JT carried out the real-time PCR assays.The pre-publication history for this paper can be accessed here:List of all immune related genes after SARS-CoV infection Comprehensive list of 1087 immune related genes that were altered in PBMCs in response to SARS-CoV infection at 4 hours, 8 hours, 12 hours, and 24 hours. Genes were grouped and ordered using the algorithm described in Methods. Rows represent individual genes, columns represent individual time points. Each cell in the matrix represents the mean expression level from 3 subjects for a gene feature at a particular time point (non-infected PBMCs responses have been subtracted from infected responses). The red and green color bars reflect high and low expression levels respectively, while black indicates equivalent expression level. The magnitude of the log-transformed ratio is reflected by the degree of color saturation. The line graph indicates the average expression ratios for each group. The area above the axis indicates upregulation, while the area under the axis means downregulation.Click here for file