Patients hospitalized with community acquired pneumonia (CAP) have a substantial risk of death, but there is evidence that adherence to certain processes of care, including antibiotic administration within 8 hours, can decrease this risk. Although national mortality data shows blacks have a substantially increased odds of death due to pneumonia as compared to whites previous studies of short-term mortality have found decreased mortality for blacks. Therefore we examined pneumonia-related processes of care and short-term mortality in a population of patients hospitalized with CAP.We reviewed the records of all identified Medicare beneficiaries hospitalized for pneumonia between 10/1/1998 and 9/30/1999 at one of 101 Pennsylvania hospitals, and randomly selected 60 patients at each hospital for inclusion. We reviewed the medical records to gather process measures of quality, pneumonia severity and demographics. We used Medicare administrative data to identify 30-day mortality. Because only a small proportion of the study population was black, we included all 240 black patients and randomly selected 720 white patients matched on age and gender. We performed a resampling of the white patients 10 times.Males were 43% of the cohort, and the median age was 76 years. After controlling for potential confounders, blacks were less likely to receive antibiotics within 8 hours , but were as likely as whites to have blood cultures obtained prior to receiving antibiotics (0.7, 0.3–1.5), to have oxygenation assessed within 24 hours of presentation (1.6, 0.9–3.0), and to receive guideline concordant antibiotics (OR 0.9, 0.6–1.7). Black patients had a trend towards decreased 30-day mortality (0.4, 0.2 to 1.0).Although blacks were less likely to receive optimal care, our findings are consistent with other studies that suggest better risk-adjusted survival among blacks than among whites. Further study is needed to determine why this is the case. Pneumonia (with influenza) is the leading infectious disease cause of death in the United States and the sixth leading cause of death overall  AccordinAs part of the Pneumonia Medical Quality Improvement Study (MQIS) a national expert panel established a set of process of care measures for patients hospitalized with CAP  Several The aims of this paper are to 1) examine whether there are significant racial differences in the processes of care that have been associated with mortality for patients hospitalized with CAP, and 2) to examine the relative risks of death within 30-days for blacks versus whites.KePRO, the Medicare Peer Review Organization for Pennsylvania, obtained these data as part of the Pneumonia MQIS project, whose goals is to assess and improve the quality of care for Medicare patients hospitalized with CAP.The study population was Medicare fee-for-service inpatients hospitalized at participating hospitals in Pennsylvania between 10/1/1998 and 9/30/1999. Inclusion criteria included having a primary ICD-9 diagnosis of pneumonia , or a primary diagnosis of respiratory failure (518.81) or sepsis (038. XX) with a secondary diagnosis of pneumonia. Only the first qualifying discharge was considered for each patient.Among the 204 hospitals functioning in PA during the study period, 101 agreed to participate in this study. For each hospital, a random sample of up to 60 discharges with qualifying ICD-9 codes was selected. For hospitals with fewer than 60 qualifying discharges, all charts were selected. In most cases, chart review data was collected by trained record abstractors either on site from the original record, or from photocopies sent to the offices of the Quality Improvement Organization. In two cases, data were collected by the hospital's own staff using an approved QIO data collection instrument (n = 2).Patients were excluded if they had no working diagnosis of pneumonia on admission or received care limited to comfort measures, left the hospital "against medical advice", or were transferred from another acute care hospital. Patients whose race was not white or black were also excluded.Chart review data included demographics, comorbid conditions, physical exam findings, laboratory data, and chest radiograph information. In addition, data on important processes of care for patients hospitalized with CAP were obtained by chart abstraction. These processes of care included: first antibiotics within 8 hours of admission, collection of blood cultures prior to antibiotic administration, oxygen saturation measurement within 24 hours of presentation, and concordance of antibiotic therapy with national guidelines. After initial training the abstractors performed data collection on charts were assessed using gold-standard cases that had been previously evaluated by multiple expert abstractors. If the abstractors did not achieve 95% accuracy, they underwent further training until they had an error rate of less than 5%. In addition, 10% of charts were reabstracted during the review process to monitor the accuracy of chart review. The error rate for these reabstracted charts remained less than 5%.The pneumonia severity index (PSI) was used to assess severity of illness at presentation  The PSI Due to the relatively small number of black patients in the cohort (n = 240) we performed a modified resampling procedure of the white cohort with matching to the black patients on age and gender  We incluUnivariate statistics were used to compare sociodemographic and clinical characteristics between white and blacks patients. Categorical variables were analyzed using the Chi-square test and continuous variables were analyzed using Student's t-test.Separate discrete conditional logistic regression models were estimated for each of the individual process of care measures, and for 30-day mortality  The PSI Of 4889 charts requested, the complete medical record was available for 4823. Of these, 4034 patients, 240 of whom were black, were eligible for inclusion in the study. Patients were excluded because they were neither white or black (N = 231) or because they had no working diagnosis of pneumonia on admission (n = 413), their care was restricted to comfort measures (n = 173), they were transferred from another acute care facility (n = 37), or they left "against medical advice" (n = 14).For each of the ten resamplings 720 white patients were sampled and matched to the 240 black patients based upon the age and gender as previously discussed. The clinical and demographic characteristics of the study population are presented in Table In univariate analysis mortality at 30-days was 7.8% for whites and 5.8% for blacks (p = 0.3), and 82.1% of whites received antibiotics within 8 hours as compared to 75.7% of blacks (p = 0.04). Regarding blood culture performance, 96.4% of white and 97.1% of blacks had blood cultures obtained within 24 hours, and 84.8% of whites and 77.8% of blacks had blood cultures obtained prior to antibiotics (p = 0.03). Oxygenation saturation was assessed within 4 hours of 88.9% of whites and 93.9% of blacks (p = 0.03).Figures In the regression models, after adjusting for severity of illness with the PSI, black patients were significantly less likely to receive antibiotics within 8 hours with an odds ratio (OR) of 0.63 and 95% confidence interval (CI) of 0.41 to 0.97. Black patients also had a trend towards decreased all-cause mortality at 30-day with an OR of 0.4 and 95% CI of 0.16 to 1.0. There were no significant differences between whites and blacks in regards to obtaining blood cultures prior to antibiotics (OR 0.69, 95% CI 0.32–1.47), oxygenation assessment within 24 hours (OR 1.61, 95% CI 0.85–3.04), or use of guideline concordant antibiotics (OR 0.86, 95% CI 0.62-1.71).This study found significant racial differences in an important process of care for patients with CAP, specifically time to antibiotic administration. Our results support the previous studies of racial variation in pneumonia care which demonstrated racial variations in care for patients hospitalized with community-acquired pneumonia. -13Our study also suggests that these variations may have clinically important outcomes since the process measures used to assess quality of care in this study have been previously associated with increased 30-day mortality. -9Our study is also consistent with previous studies which found that blacks hospitalized with CAP have lower short-term mortality rates as compared to do whites ,6 It is Racial variations in CAP are important to assess since unlike coronary artery bypass surgery, hemodialysis, and many other conditions that have been studied, the inpatient treatment of CAP is largely outside of the control of the patient. Although the patient has input into being admitted to the hospital, after that point the patient has little input into the processes of care such as choice and timing of antibiotics, diagnostic testing or location of care. This has several advantages in studies where researchers seek to determine if racial differences in care reflect patient preferences, provider decisions or some negotiation between them.There are several possible explanations for our findings of racial variations in these processes of care. Besides the obvious conclusion that there may be biases that affect care there are several other possible factors that may be responsible that we are not able to examine. One possible factor is that there are geographic or other factors that results in blacks presenting for admission at hospitals with overall lower quality of care for patients with CAP. Although we were not able to control for this factor other studies have suggested that the reverse is usually true. ,13 That To attempt to adjust for imbalances between black and white patients we used a modified resampling technique to generate 10 samples of white patients, which were matched to the black population. We then pooled these results over the 10 samples. This approach allowed us to obtain a more robust estimate of the effect racial variation may have on mortality and processes of care then would be obtained from a single random matched sample  InterestThere are several limitations that should be acknowledged. First we did not have information on the physicians, hospitals, or the geographic locations of the providers so we were not able to adjust for clustering. Second our study was limited to Medicare patients hospitalized in Pennsylvania. It will also be important to examine whether patients with other types of insurance, such as Medicaid and managed care, and from other states have similar outcomes. In addition we were also unable to assess the robustness of our analysis using traditional techniques such as model cross validation on a new independent sample, or by randomly subdividing our current sample into a training and test samples, due to our small sample size. However we do have quasi-replication, at least in the white sample, by the multiple sampling that we performed. Finally we were unable to adjust for potential bias in pulse oximetry since this is a retrospective study. However recent work ,19 questDespite these limitations, we believe our results, and the results of other studies, allow us to conclude that blacks are less likely than whites to have processes of care that are considered to represent superior quality of CAP care. Despite this, blacks with CAP have similar, and perhaps lower mortality than whites. Further research should both investigate how to make sure all patients receive optimal CAP care, and identify the factors responsible for the paradoxical advantage in survival that is seen among blacks.None declared.EM and JW conceived the study. EM and JC were responsible for the analysis. EM was responsible for the initial draft of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Amborella, as comprising the basal-most grade of all other angiosperms. A major exception is the recent study by Goremykin et al. , whose analyses of 61 genes from 13 sequenced chloroplast genomes of land plants nearly always found 100% support for monocots as the deepest angiosperms relative to Amborella, Calycanthus, and eudicots. We hypothesized that this conflict reflects a misrooting of angiosperms resulting from inadequate taxon sampling, inappropriate phylogenetic methodology, and rapid evolution in the grass lineage used to represent monocots.Numerous studies, using in aggregate some 28 genes, have achieved a consensus in recognizing three groups of plants, including Acorus and added these plus previously sequenced Acorus genes to the Goremykin et al. (2003) dataset in order to explore the effects of altered monocot sampling under the same analytical conditions used in their study. With Acorus alone representing monocots, strongly supported Amborella-sister trees were obtained in all maximum likelihood and parsimony analyses, and in some distance-based analyses. Trees with both Acorus and grasses gave either a well-supported Amborella-sister topology or else a highly unlikely topology with 100% support for grasses-sister and paraphyly of monocots (i.e., Acorus sister to "dicots" rather than to grasses). Second, we reanalyzed the Goremykin et al. (2003) dataset focusing on methods designed to account for rate heterogeneity. These analyses supported an Amborella-sister hypothesis, with bootstrap support values often conflicting strongly with cognate analyses performed without allowing for rate heterogeneity. In addition, we carried out a limited set of analyses that included the chloroplast genome of Nymphaea, whose position as a basal angiosperm was also, and very recently, challenged.We used two main approaches to test this hypothesis. First, we sequenced a large number of chloroplast genes from the monocot Amborella (or Amborella plus Nymphaea), but not monocots, is the sister group of all other angiosperms among this limited set of taxa and that the grasses-sister topology is a long-branch-attraction artifact leading to incorrect rooting of angiosperms. These results highlight the danger of having lots of characters but too few and, especially, molecularly divergent taxa, a situation long recognized as potentially producing strongly misleading molecular trees. They also emphasize the importance in phylogenetic analysis of using appropriate evolutionary models.These analyses show that  Amborella) are themselves the "earliest" angiosperms, but rather that they belong to the lineage of angiosperms that resulted from the first evolutionary split in angiosperm evolution. When the term "sister" is used to refer to a phylogenetic placement it refers to the sister group to the rest of the angiosperms unless otherwise specified.] A breakthrough in the seemingly intractable problem of identifying the earliest lineages of angiosperms occurred in 1999 and 2000, when each of many multigene studies identified the same three groups as the earliest branching angiosperms [Amborella, a vessel-less shrub with unisexual flowers endemic to New Caledonia, as the sister-group to all living angiosperms  were analyzed. In 31 of 33 phylogenetic analyses this study found that "Amborella is not the basal angiosperm and not even the deepest branching among dicots" .In sharp contrast stands the study of Goremykin et al. , in whicdicots"  as the other two angiosperms sampled , a phenomenon known -39 to giAmborella-sister topology will be recovered instead. In addition, we carried out a similar, but much more limited set of analyses in response to a follow-up paper by Goremykin et al. [Nymphaea as a basal angiosperm.The current study was undertaken to test whether the grasses-sister topology is indeed an LBA artifact. We hypothesize that, by analyzing the Goremykin et al.  dataset n et al.  that appAcorus, and added it to the 13 taxa, 61 gene first- and second-position alignment matrix of Goremykin et al. [Acorus was chosen for two reasons. First, it is well supported as the sister to all other monocots [Acorus plus grasses represent monocot diversity about as well as any two groups of monocots. Second, unlike grasses, its chloroplast genome does not appear to have evolved at unusually high rates [Acorus dataset consisted of 40 protein gene sequences, 22 newly determined in this study and 18 from preexisting databases. This corresponds to 65.6% (40/61) of the genes and 71.4% (32,072/44,937) of the nucleotide characters analyzed by Goremykin et al. [We gathered new sequence data for an additional monocot representative, n et al.  to give monocots -43. Thusgh rates ,44. The n et al. .Acorus matrix, containing data for all 61 genes and including gaps where data for Acorus were not available, and a "truncated" matrix, containing only those 40 genes where Acorus sequences were available. Inspection of the resulting trees revealed no topological incongruences and no significant change in bootstrap support (BS) between the full and truncated analyses [see Acorus are based on the full matrix dataset. This allows us to include all available relevant data, allowing the fullest and most direct comparisons to the Goremykin et al. [A number of initial analyses were conducted in parallel on the "full" yses see  and 2. Tn et al.  analysesAcorus to the Goremykin et al. [Acorus instead of grasses to represent monocots has a major effect on the results. This is especially dramatic for equal-weighted maximum parsimony (MP) analyses of both nucleotides and amino acids, where there is a shift from 100% BS for monocots-sister when only grasses are used to represent monocots . Transversion parsimony (RY-coding) of the original dataset . Substituting Acorus for grasses improves the support for Amborella-sister to 100% . On the other hand, equal-rates ML  to strong (98%) support, in which monocots are monophyletic with equivalent support.Inclusion of both grasses and des Fig.  and amindes Fig. , gives a ML Fig.  and tran ML Fig.  give an Amborella-sister topology when Acorus was substituted for grasses were the NJ methods without a ML model. When the PAUP* [Amborella, Calycanthus, and Acorus form a monophyletic group with 100% BS. Importantly, however, this same grouping is obtained, with all 12 distance settings, even when grasses are included, such that, as in equal-weighted parsimony analyses , Ambes Figs.  and 2F, Amborella-sister when only grasses represent monocots, continue to do so, but with higher BS, when Acorus is added, either with or without grasses [see Finally, it should be noted that ML and NJ methods using models (see next section) that give sses see  and 2.Amborella is sister to the rest of angiosperms, as the analyses with Acorus strongly indicate, why do so many of the Goremykin et al. [If the lineage leading to n et al.  analysesAmborella-sister topologies using ML with all 56 combinations of the 14 substitution models and four rate-heterogeneity conditions specified by the MODELBLOCK script provided by MODELTEST [Amborella-sister topology was preferred when either invariant sites or gamma rate categories were used but not when they were used in combination. For the other 12 models, the Amborella-sister topology was preferred for all three conditions that allowed for rate heterogeneity across sites (Table We first compared the relative likelihood of the grasses-sister and ODELTEST . The fouODELTEST  model (aAmborella- or grasses- sister) were used to calculate the likelihood of the other topology . For both topologies, the model chosen by MODELTEST using either the hierarchical likelihood ratio tests or the Akaike information criterion was the 5-substitution-type-transversion (TVM) + I + G model, where the probability of going between A and G is equal to that of C and T. With this model, using parameter estimates from either topology, a heuristic search found the Amborella-sister topology with 98% BS, and the SH-test [These results held when the parameters estimated on one topology  was also recovered over a wide range of α values . Using the estimated PInvar = 0.58 without gamma-distributed rate categories, we obtained the ogy Fig.  with 97%995 Fig. ]. The BSses Fig. . The SH-Amborella and Calycanthus is identical  with that for grasses-sister across the entire range of both α and PInvar values, while both of these BS values always equal 100 minus the BS value for Amborella-sister  when constant sites are removed [see There are 20,071  constant sites in theGoremykin et al.  matrix. ster see , Fig. A.oved see , Fig. B.Amborella-sister with low BS of 58% [see Amborella-sister with stronger support . The most noteworthy shift towards stronger support involves ML analysis with equal rates, where inclusion of third positions changes the topology, from grasses-sister , only two genes put monocots sister (and both with low, 13 and 34%, BS), whereas 11 of 40 genes put Amborella sister [see Acorus added and grasses removed, 21 genes place Amborella sister and 1 places Acorus sister [see The single gene trees also illustrate the effect of taxon sampling. When ster see . In contNymphaea alba became available (released to EMBL database on July 13, 2004). This sequence was generated as part of a very recent study, also by Goremykin et al. [Nymphaea and Amborella recovered as sister taxa . In their abstract, Goremykin et al. [While this manuscript was in the final stages of preparation, the chloroplast genome sequence of n et al. , in whicn et al.  and subjn et al.  present n et al.  that monNymphaea that our main results and conclusions were entirely unchanged by its inclusion/exclusion. Using the Goremykin et al. [Amborella alone did not emerge as sister to all other angiosperms in any of these analyses. Most commonly , whereas an equal-rates ML analysis found Nymphaea deepest  and Amborella next deepest . Hendy and Penny [That long branch attraction can be a serious problem in phylogenetic inference has long been known to the systematics community, ever since this phenomenon was first explored by Felsenstein . Felsensnd Penny  showed tnd Penny  showed tAmborella-sister topology and 2) the idea that the grasses-sister topology is a consequence of LBA causing a misrooting of angiosperms. When the monocot Acorus was directly substituted for grasses, strong support for Amborella-sister was obtained . Con. ConAmboce  and better analytical methodologies as they become available. At the same time, we must face up to two serious limitations arising from extinction. First, nd years ,62  the posn et al.  and somen et al. ) that thn et al. ,72-74) ms for most genes between Pinus and angiosperms was between 0.50 and 1.50 substitutions/site), which they felt could lead to "misleading" phylogenetic results. However, because our analyses with all three positions or only third positions gave such similar results to those using only first and second positions, we believe that for this particular dataset third positions are not contributing "excessive" homoplasy and leading to spurious affiliations. This conclusion is consistent with a considerable body of literature dealing with the phylogenetic utility of third positions in organellar genes [Many of our analyses, including all but one of the 61-gene concatenate analyses shown, included only first and second codon positions. This is because Goremykin et al.  chose toar genes -80, whilar genes ,24. Cautar genes ), and evar genes ,73,82.Our findings, and those of others -70,83, hAcorus gramineus Soland. (a voucher specimen is deposited at the IND herbarium) for 22 of the 61 chloroplast genes analyzed by Goremykin et al. [psbC-D and psbE-J operons; from rpl2 to 3'-rps12 gene), as well as the primers described in [rbcL, atpB, trnL-F, and trnE-D region, respectively, were used for amplifications and/or sequencing. For the most part, however, based on the initial sequences, a number of sequencing primers were designed and used for chromosome walking with long PCR products. Primer sequences are available upon request from SS. PCR products were separated by electrophoresis using 0.8% agarose gels, visualized with ethidium-bromide, and cleaned using Qiagen columns . Cleaned products were then directly sequenced using the BigDye™ Terminator cycle sequencing kit (PE Applied Biosystem, Foster City, CA, USA) on an ABI 3100 DNA automated sequencer (PE Applied Biosystem, Foster City, CA, USA). Sequence data were edited and assembled using Sequencher™ 4.1 (Gene Codes Corporation, Ann Arbor, MI, USA). The Acorus sequences for these 22 chloroplast genes (atpA, atpE, clpP, cemA, lhbA, 3'-petB, petD, petG, petL, psaB, psaI, rpl20, rpoA, rpoB, rpoC1, rpoC2, rps2, rps14, rps18, rps19, ycf3, ycf4) are deposited in GenBank (accession numbers AY757810-AY757831). These were combined for phylogenetic analyses with full-length or partial Acorus sequences already available in GenBank for 18 other chloroplast genes [AF123843 (psbB, psbT, psbN, psbH), AF123771 (rps7, 3'-rps12), AF123828 (psbE, psbF, psbL), AF123813 (psbD, psbC), AF123785 (rpl2), D28866 (rbcL), X84107 (rps4), U96631 (psbA), AB040155 (matK), AF197616 (atpB), and AJ344261 (psaA)]. The 40 Acorus genes used here come from two closely related species – A. calamus (14 genes) and A. gramineus (26 genes) – and correspond to 65.6% (40/61) of the genes and 71.4% (32,072/44,937) of the nucleotide characters analyzed by Goremykin et al. [We used long PCR to generate full-length or partial sequences from n et al. . Long PCn et al. , which cribed in -87 for tAcorus, the Acorus genes were individually aligned with the individually extracted gene alignments from the Goremykin et al. [Acorus. Using the same procedure, Acorus was also added to the amino acid matrix provided by V. Goremykin. The relevant 61 chloroplast genes of Nymphaea [For all first and second codon position analyses, the data matrix provided by V. Goremykin was used without modification. For analyses that included n et al.  dataset n et al. , and theNymphaea  were likWe also constructed a new matrix consisting of all three codon positions by extracting genes from 13 sequenced chloroplast genomes of land plants (GenBank numbers: AP002983, AP000423, AJ271079, Z00044, AJ400848, AJ506156, AJ428413, X86563, AB042240, X15901, D17510, AP004638, X04465), aligning them, and hand editing apparent mistakes. The first and second position version of this matrix was nearly identical to the Goremykin et al.  matrix, Phylogenetic analyses were performed in PAUP* 4.0b10 . Unless Bootstrap support  was estiThe Shimodaira-Hasegawa (SH) test  was perfBS – bootstrap support; LBA – long branch attraction; ML – maximum likelihood; MP – maximum parsimony; NJ – neighbor joining; Ti/Tv – transition:transversion; NT – nucleotides; Plnvar – proportion of invariant sitesAcorus) used in this study and conceived and drafted the first and last figures. DWR carried out the phylogenetic analyses and made all other figures. All three authors contributed to the overall design of the study, drafted parts of the manuscript, and read and approved the final manuscript.SS generated the new sequences , 2. (NA but same topology and similar BS, 100 vs. 100), 3. (86 vs. 88, 84 vs. 90).Click here for fileAcorus includedTrees from truncated RY-coded matrix with . This are the same analyses as in Additional file 1 except the DNA is RY-coded. For comparison, the corresponding BS values for the Amborella sister relationship in the full matrix, along each row, are: 1. (100 vs. 100, 100 vs. 100), 2 (98 vs. 100, 100 vs. 100), 3. (100 vs. 100, 100 vs. 100). Click here for fileComparison of gamma-distributed rates with two versus four rate categories. This figure shows that using two rate categories gives essentially the same results as using four rate categories with this dataset. The dataset is the first- and second-position, 61-gene matrix with grasses, Acorus, or both used to represent monocots. The ML HKY85 model was used and parameters were estimated as described in Methods.Click here for fileTrees when constant sites are removed from the first- and second-position matrix of Goremykin et al. [19]. A. ML HKY85 and equal rates. B. NJ with distances calculated using an ML HKY85 model and equal rates.Click here for fileNJ analysis using ML proportion of invariant distances. Distances were calculated using the ML HKY85 model, the estimated proportion of invariant sites, and the first- and second-position matrix of Goremykin et al. [19].Click here for fileML trees using third positions only. A. HKY85 model with equal rates. B. HKY85 model with four gamma-distributed rates.Click here for fileAcorus Sister group to the rest of angiosperms found in individual gene analyses using first- and second-position data without Top, ML HKY85 with four gamma-distributed rates. Bottom, Parsimony analysis.Click here for fileAcorus addedSister group to the rest of angiosperms found in individual gene analyses using the ML HKY85 model with four gamma-distributed rates and . Top, all three positions. Bottom, first and second positions.Click here for fileAcorus added and grasses removedSister group to the rest of angiosperms found in individual gene analyses using the ML HKY85 model with four gamma-distributed rates with . Top, all three positions. Bottom, first and second positions.Click here for fileSister group to the rest of angiosperms found in individual gene analyses using parsimony on all three positions. Top, Acorus added. Bottom, Acorus added and grasses excluded.Click here for file


Schistosoma mansoni infected subjects can reduce bleeding, thereby alleviating the pathology caused by schistosomiasis.Management of patients with bleeding oesophageal varices comprises of mainly diagnostic endoscopy, sclerotherapy and band ligation. One of the major problems to do any of the above is the active bleeding which makes any intervention difficult. The neuropeptide hormone somatostatin administered exogenously has caused a reduction in portal hypertension and variceal bleeding in patients suffering from liver cirrhosis. We believe that the symptomatic use of somatostatin for variceal bleeding in Schistosoma mansoni with bleeding from oesophageal varices in the last 24 hours. One group of schistosomiasis patients will be treated with somatostatin and praziquantel, the other with propanolol and praziquantel. Survival graphs will be set up to correlate somatostatin administration with survival time. A two part questionnaire will be set up to control treatment outcomes. The pre-treatment part of the clinical questionnaire will identify inclusion criteria questions, the post-treatment part of the questionnaire will identify treatment outcomes.We herein present a study protocol for establishing this neuropeptide as a potential therapeutic agent in schistosomiasis. Adolescent subjects, age range varying from 12–17 years will be selected, based on several inclusion criteria, most important being infection with We expect that the administration of somatostatin as a bolus followed by a 24 hour long infusion, will stop bleeding immediately, delay rebleeding as compared to the control study group and delay mortality in the somatostatin treated subjects. S. mansoni infected patients. In such patients portal hypertension leads to the formation of portal-systemic collaterals, specifically gastro-oesophageal collaterals (varices). Bleeding of these oesophageal varices can occur depending on the severity of fibrosis, and can be fatal. Praziquantel, the most commonly used anti-schistosomal drug, is effective against the worm stages of the parasite, but has no activity against the pathology (fibrosis) caused by the egg stages or the variceal bleeding that can be fatal in its outcome. These observations have stressed the need to combine praziquantel treatment with symptomatic treatment like with somatostatin.Complications due to severe schistosomiasis include fibrosis, hepatomegaly, splenomegaly, haematemesis, varices, portal hypertension, ascites formation and death . ComplicSomatostatin is emerging as the ideal vasoactive drug for the control of variceal bleeding, and is as effective as sclerotherapy -4. In a Schistosoma caused morbidity. In endemic regions, at any given time, only a fraction of infected patients develop severe hepatic fibrosis. There may be a direct correlation between the development of severe fibrosis and the inability to generate required somatostatin levels. Our ongoing research at the Laboratory of Pathology give evidence to this fact, since somatostatin levels in Senegalese patients with severe morbidity  are significantly lower than that in patients with no severe morbidity [We have studied the potential role of somatostatin in modulating orbidity -10. ThisComplications linked to hepato-intestinal schistosomiasis are increasing in the area of Richard-Toll, Northern Senegal. These cases are being identified at the local health centre. However, considering the high prevalence of schistosomiasis in this region, it is likely that the number of severe cases is much higher. Clear guidelines for the management of such severe complications and criteria for referral and surgery are required in this region. The establishment of an algorithm on how to treat these patients and create the appropriate infrastructure is urgently needed. The priority is how to take care of these patients and more so what is the best way of doing this under local conditions. One of the first steps is to critically evaluate the Niamey-Belo-Horizonte ultrasound methodology in patients with severe periportal fibrosis. Efficient management of bleeding varices in the afflicted patients is imperative.Schistosoma mansoni infection. Moreover using this neuropeptide may increase time to failure of drug treatment, decrease incidences of early re-bleeding (day 4, 8) and incidences of death during the follow up period. Decreased frequencies of late rebleeding (days 30, 60, 90) may occur, all indicating the safety of using somatostatin. Praziquantel cover would be given to all study patients.Given the background that somatostatin is an ideal vasoactive drug in the field of liver pathology, it is our opinion that somatostatin will be more efficacious and safe as compared to currently used beta blocker drugs like propanolol, in the control of acute oesophageal variceal bleeding due to Age and Morbidity criteria – Adolescent subjects, age range varying from 12–17 years will be selected. The inclusion criterion will be schistosomiasis patients with bleeding from oesophageal varices in the last 24 hours. A random selection will be made to form two groups, a study group and a control group. Control of active infection will be done by means of CAA-strips on urine or blood. Subjects will be asked to fill in an informed consent form and the pre-treatment part of a questionnaire.The inclusion criteria will be established fibrosis due to schistosomiasis of clinical history, physical examination and laboratory findings . Clinically active upper gastrointestinal bleeding (haematemesis of fresh or semi fresh blood and/or melena and/or haematochezia) with or without haemodynamic instability (systolic blood pressure < 80 mm Hg and heart rate > 120 bpm) will be selected. Subjects must be male or non-pregnant, non-lactating female subjects. Females of childbearing potential will have to utilize contraception for the duration of the study. Written or verbal documented informed consent will be needed from all subjects.Exclusion criteria will include participation by subjects in another investigational study within the last 14 days. Subjects may not undergo treatment with endotherapy, i.e. band ligation, sclerotherapy or other . Treatment with somatostatin, vasopressin or their analogues will also be a exclusion criteria. Subjects with end stage liver disease with hepatorenal syndrome, diffuse hepatocellular carcinoma, patent porto-systemic shunts, known diagnosis of non-fibrotic portal hypertension, severe cardiovascular diseases, i.e. acute myocardial infarction and heart failure will be excluded. Concurrent use of metoclopramide is also not advised.Conduct of trial – Active bleeding episode (haematemesis, haematochezia, melena) from a potential variceal source should be confimed by a medical team (the ER physician, the ICU physician, the investigator). Patients may be outpatients or already hospitalised patients. Patients will be randomised to either arm in a sequential manner. Randomisation, and the start of study drug infusion if in adjunctive therapy arm, should be accomplished as soon as possible following identification of a patient qualifying for the study and following the conduct of pre-randomisation study procedures.Sample Size Criteria – Assuming a 80% chance of finding a significant difference <0.05 between the two study cohorts, the following statistics were established:(A) If 99% of the untreated subjects and –10% of somatostatin treated subjects bled -5 volunteers per group were sufficient;20% treated subjects bled – 7 volunteers per group were required;30% treated subjects bled-9 volunteers per group were needed.(B) If 90% of untreated subjects bled, and –10% of somatostatin treated group bled – 7 volunteers in each cohort were sufficient;20% of somatostatin treated group bled – 9 volunteers in each group were required;30% of somatostatin treated group bled – 10 volunteers in each group were needed.(C) If 70% of untreated subjects bled, and:10% of somatostatin treated group bled – 12 volunteers per group were sufficient;20% of somatostatin treated group bled – 18 volunteers per group were required;30% of somatostatin treated group bled – 28 volunteers per group were needed.In cirrhotic patients with bleeding oesophageal varices somatostatin administration controls bleeding in more than 80% of the treated patients. Based on this report, we propose to start this pilot study with 10 subjects/group.Two groups of 10 schistosomiasis patients each will be identified. Group (1) will be treated with Somatostatin  + Praziquantel (40 mg/kg). For somatostatin treatment the i.v. infusion will be started first; 3 mg somatostatin will be dissolved in the 1 ml of saline provided. This solution will be added to the saline transfusion unit and administered to the patient for the next 12 hours. Once finished the second packet of 3 mg somatostatin will be used similarly for a second saline transfusion unit for the remaining 12 hours. The bolus dose of 250 μg will be dissolved in the 1 ml of saline provided and administered over 90 seconds soon after the start of the i.v. infusion.Group (2) will be treated with Propanolol + Praziquantel.Survival graphs will be set up to correlate somatostatin administration with survival time.OR presence of a 20 mmHg drop in systolic blood pressure from the highest post resuscitation value AND achieving a heart rate of 120 bpm OR a 20 bpm increase from highest post resuscitation value OR Inability to achieve and maintain a Hct of – 27% of Hb of – 9 g/dl despite blood transfusion of 2 units or more.The primary efficacy variable is the number of patients meeting the failure of therapy definition during the infusion period. Failure criteria are defined as death during infusion, persistence of active bleeding , hematochezia, melena.A two part questionnaire will be set up to control treatment outcomes. The pre-treatment part of the clinical questionnaire will identify inclusion criteria questions:(1) When was the last haematemesis incidence?(2) When did the present incident start?The post-treatment part of the questionnaire will identify treatment outcomes. The following questions will be answered:(1) What was the reaction to somatostatin infusion?(2) When did bleeding stop after somatostatin infusion?(3) GI disturbances: Is there abdominal pain, nausea, diarrhea, after somatostatin infusion?(4) Control of early rebleeding: Is there early rebleeding in the 8 days following somatostatin administration?(5) Control of late rebleeding: When is the next rebleeding incident? Subjects will be followed up on a monthly basis, by home visits.(6) Control of mortality: Is there any difference in mortality time between the two groups.(7) Control of fibrosis: Does ultrasonography detect anti-fibrotic effect of somatostatin after treatment?It is expected that the administration of somatostatin as a bolus followed by a 24 hour long infusion, will stop bleeding immediately, delay rebleeding as compared to the control study group and delay mortality in the somatostatin treated subjects. Our protocol that is based on a pilot study will help to establish the importance of somatostatin in schistosomiasis.The author(s) declare that they have no competing interests.Both authors participated in the design of the study.The pre-publication history for this paper can be accessed here:


There are three major B-cell compartments in peripheral lymphoid organs: the germinal center (GC), the mantle zone (MNZ) and the marginal zone (MGZ). Unique sets of B-cells reside in these compartments, and they have specific functional roles in humoral immune response. MNZ B cells are naïve cells in a quiescent state and may participate in GC reactions upon proper stimulation. The adult splenic MGZ contains mostly memory B cells and is also known to provide a rapid response to particulate antigens. The GC B-cells proliferate rapidly and undergo selection and affinity maturation. The B-cell maturational process is accompanied by changes in the expression of cell-surface and intracellular proteins and requires signals from the specialized microenvironments.CCL20 and CCL18 respectively. The MGZ was characterized by high expression of many chemokines genes e.g. CXCL12, CCL3, CCL14 and IFN-associated genes, consistent with its role in rapid response to infections. A stromal signature was identified including genes associated with macrophages or with synthesis of extracellular matrix and genes that influenced lymphocyte migration and survival. Differentially expressed genes that did not belong to the above categories include the well characterized BCL6 and CD10 and many others whose function is not known.We performed laser microdissection of the three compartments for gene expression profiling by cDNA microarray. The transcriptional program of the GC was dominated by upregulation of genes associated with proliferation and DNA repair or recombination. The MNZ and MGZ showed increased expression of genes promoting cellular quiescence. The three compartments also revealed distinct repertoires of apoptosis-associated genes, chemokines and chemokine receptors. The MNZ and GC showed upregulation of Transcriptional profiling of B-cell compartments has identified groups of genes involved in critical molecular and cellular events that affect proliferation, survival migration, and differentiation of the cells. The gene expression study of normal B-cell compartments may additionally contribute to our understanding of the molecular abnormalities of the corresponding lymphoid tumors. Appropriate T- and B-cell migration and timely interaction with antigen presenting cells (APC) are essential for the development of humoral immune responses ,2. Specihigh, CD27- [high, IgDlow and CD27+ phenotype, suggesting that this zone contains mainly memory B-cells [In follicles with developing GCs, the resting B-cells that are not the part of the GC response are pushed outward to form the mantle zone (MNZ) or corona around the GC B-cells. The mantle cell is a pre-GC, immunologically naïve B-cell that is also the putative cell of origin of mantle cell lymphoma . These Bh, CD27-  and are h, CD27- . In the h, CD27- . Unlike  B-cells .Many previous studies ,16 have We used the Lymphochip cDNA microarray  to inves+ CD27-, similar to FACS-sorted naïve B-cells. The GC was easily recognizable and generally contained a higher percentage of non B-cells, including T-cells, macrophages and follicular dendritic cells (FDC). The MGZ was obtained from a spleen with a morphologically clearly defined MGZ containing mostly IgM+ CD27+ B-cells, corresponding to the phenotype of FACS-sorted memory B-cells [GC and MNZ could be readily dissected from tonsillar frozen sections, but MGZ could only be reliably obtained from the spleen Figure . Immunos B-cells . The MGZ B-cells ,21. The Fifteen data sets corresponding to the five sample groups were generated. Different hybridizations were correlated through a correlation matrix plot, and replicated hybridizations were shown to be closely related (R ≥ 0.85). The plots allowed us to check reproducibility of the microarray assay among different samples of each tissue Figure . The numARK2 in GC, CCL20 in MNZ and CMRF-35H in MGZ. Other transcripts were expressed in all compartments with a relatively high differential expression in one, such as SET and FAIM in GC, Cyclin G2 in MNZ, and NM23-H1 and CARD11 in MGZ . The results corresponded well with both microarray and semi-quantitative RT-PCR.Some of the results of the semi-quantitative RT-PCR were further validated by the SYBRCCNB1, PCNA, Ki67), kinetochore association (e.g., CENPF, BUB1 and BUB3), functional components of mitotic checkpoints (e.g., CENPE and TTK) and regulators of cell-cycle related events, including centrosome separation/segregation and cytokinesis (e.g. KNSL5, ARK2), as expected from the known high proliferation rate of centroblasts. GC also highly expressed genes involved in DNA repair (e.g., RAD54, BRCA2, RAD51, ERCC5 and MSH2), as expected from the frequent physiological double-strand DNA breaks associated with somatic hypermutation and isotype switching. The GC profile also showed increased levels of transcripts involved in DNA replication (e.g., DNAJ, DNA2L, DNMT1, TOP, RFC4 and RPA1) and in transcription and translation (e.g., EIF2, TAF, TCF, UHRF1, UBD and UBE2).Comparing the gene expression profiles of LCM GC and FACS-sorted GC B-cells  revealedCCNA, CCNB1and CCNF and of CDC2  is consistent with the resting state of the MNZ and MGZ B-cells. Characteristically CCNA expression is very low in Go phase and begins to increase in early G1. For the cell to enter the G2/M phase, an association with CDC2 is required [Cyclin G2 (CCNG2) was highly expressed in MNZ cells as compared to either GC or MGZ. The function of CCNG2 differs from the conventional cyclins in negatively regulating the cell cycle [CARD11 in MNZ may also be part of the program in maintaining the quiescent state. CARD11 has been shown to be critical for immune receptor signaling of both T and B-cells through the activation of JNK and NF-κB [CMRF-35H [CBL-B [GAS2 [The low expression of the cyclins required . The trarequired . Howeverll cycle . Studiesnd NF-κB . The incnd NF-κB . InteresCMRF-35H , CBL-B [H [CBL-B , and GAS-B [GAS2 , which mBCL2 expression in GC B-cells makes them vulnerable to undergo apoptosis unless rescued by survival signals [BIK, FASL and PDCD8 (AIF) suggests a further increase in susceptibility to apoptosis in the GC. However, FAIM is overexpressed in the GC and may represent a protective mechanism in GC B-cells that have appropriate BCR signaling and CD40L stimulation with resultant upregulation of FAIM and increased resistance to FASL-mediated apoptosis [BCL2, but have different profiles of other apoptosis/survival genes that may represent specific adaptation of these cells to their unique physiological states and microenvironment. The expression of BNIP3, encoding a proapoptotic protein of the BCL2 family[TCL1 was upregulated in MNZ only and may have an antiapoptotic role in that population.The markedly decreased  signals . An incrpoptosis . Presumapoptosis  showed ipoptosis . This suL2 family, is markSuppressor of Death Domains (SODD) in MGZ cells, suggesting a complex regulation of signaling through the TNFR superfamily. SODD is associated with TNFR1 in vivo, maintaining the receptor in an inactive monomeric state. The release of SODD from TNFR1 permits the recruitment of proteins such as TRADD and TRAF2 to the activated TNFR1 signaling complex [SODD may be a major mechanism to dampen TNFR1 signaling in MGZ B-cells in the resting state. The higher expression of CARD11 in MGZ may have a pro-survival function, but it may also have a role in MGZ organization. It has been shown that loss of CARD11 in mice resulted in the complete loss of CD5+ peritoneal cells and reduced number of IgD high IgM low mature splenic B-cells, indicating its role in B-cell development [NM23-H1 and NM23-H2, which share an amino acid identity of 88%, were highly expressed in MGZ. NM23 H1 is a granzyme A-activated DNase (GAAD) that is inhibited by SET [NM23-H1 and the low expression of its inhibitor SET was opposite in their expression profile in the GC, suggesting that this expression may influence apoptosis in opposite directions in these two B-cell compartments.There was increased expression of  complex . It has elopment . Two clod by SET . The higCCL18, encoding a chemokine secreted by immature dendritic cells (DC), is specifically upregulated in the GC compartment. Our finding was supported by a recent report showing that CCL18 is produced by GC DC and can attract MNZ B-cells towards GC [CCL18 may be especially important during the onset of a GC reaction, the time point to recruit antigen primed pre-GC B-cells, which then interact with GC DC to initiate and maintain the GC reaction. The GC compartment showed increased expression of CXCL10 (IP-10). which has pleiotropic effects, including stimulation of monocytes and T cell migration [Chemokines attract primary B-cells and play an important role in the homing and localization of B-cell subsets at different stages of antigen-independent and dependent reaction . Our micwards GC . The higigration .MIC1, also known as PLAB) [The GC also showed increased transcriptional level of genes that may suppress or control inflammatory responses; e.g., SOCS1 limits cellular response to IFNγ, IL-2 and IL-6,43. Macras PLAB) , is onlyCCL20 was observed in MNZ cells. Human naïve and memory B-cells express the cognate receptor for CCL20, CC-chemokine receptor 6 (CCR6) [CCL20 may play a vital role in naïve B-cell migration and localization in the MNZ. The chemokine gene CXCL12  is highly expressed in MGZ cells. The receptor for CXCL12 is CXCR4, which is present on CD34+ cells, myeloid cells, CD4+ T cells, B-cells, epithelial cells, endothelial cells, and dendritic cells. In the bone marrow, stromal cells secrete CXCL12, which is involved in the emigration of hematopoietic precursors to the marrow during embryogenesis [CCL14  and CCL3  were more highly expressed in the MGZ. CCL14 can activate human monocytes via receptors that also recognize CCL3 [CXCL13 and CCL5 were upregulated in both microdissected MGZ and MNZ compared with the FACS-sorted B-cell populations. Previous studies have established an important role for CXCL13 (BLC) in the development of Peyer's patches (PP) and many peripheral lymph nodes. It also controls B-cell migration and thus the organization of B-cell areas [Increased expression of the chemokine 6 (CCR6) . The higogenesis . In periize CCL3 . CCL3 isize CCL3 . Thus, tll areas . CCL5 (Rll areas . It is pAIM2, IFNGR1, and IFNAR -1 and -2) were also preferentially expressed and may reflect the unique function of the MGZ to provide the first rapid response to particulate or T cell-independent antigens. The MGZ also showed increased expression of many members of G protein pathways consistent with more active chemotaxis, cell motility and secretory functions. In addition, MGZ cells showed higher expression of IL-7Rα, consistent with the role of the IL-7R in MGZ organization [IL-7Rα expression is also required for the recruitment of precursor cells to develop in secondary lymphoid organs and for the proper structural organization of these organs [A number of IFN-induced genes , encoding a metalloproteinase that preferentially degrades elastin and takes part in the remodeling of extracellular matrix. No collagen-specific gene was up-regulated in the MNZ.Cells function within the context of a three-dimensional (3D) extracellular matrix (ECM) that participates in regulating cellular motility, proliferation and survival. In the GC, lagen IX , and COLlagen XI , were unOsteonectin (SPARC), upregulated in LCM samples, encodes a matrix-associated protein that elicits changes in cell shape, inhibits cell-cycle progression, and influences the synthesis of extracellular matrix [MafB, and c-Maf) were also part of the stromal signature. The Maf family of genes encode bZip nuclear transcription factors and play an important role in morphogenesis and cellular differentiation [ICAM1 and VCAM1. MGZ B cells express the integrin LFA1 which binds to its ligands ICAM1 and VCAM1, and this interaction may control the localization of these B cells [VCAM1, ITGAL (LFA-1) and ITGA6 in the MNZ, suggesting a role for these adhesion molecules in mantle cell localization as well. The kruppel-like transcription factor BCL11a , which is essential for normal B-cell lymphopoiesis, was upregulated in LCM cells only. Interestingly, bone marrow from BCL11a-/- mouse can induce thymic lymphoma in wild type mice. Thus, the increased expression of BCL11a in the MNZ and MGZ may be physiologically relevant to the function of lymphocytes in these regions [Microdissected compartments contained a minor component of stromal T cells, macrophages, dendritic cells and fibroblasts whereas FACS-sorted cells from lymphoid tissues comprise almost exclusively B-cells. Thus, an insight into the gene expression profile of the stromal elements can be obtained by comparing the expression profile of FACS-sorted and microdissected cells. We found a set of genes that likely represent the stromal signature. r matrix . It regur matrix . Two memntiation . These g B cells  in this  regions .BCL6, CD10, GCET1, GCET2, JAW1 and CD38. A number of genes were clearly upregulated in the MNZ or MGZ but their functional significance is largely unknown. Some of these would be interesting targets for further investigation. Among the genes encoding surface molecules, CD59 was highly expressed in the GC. CD59 antigen is a small protein that inhibits complement-mediated pore formation or lysis by preventing the formation of membrane-embedded C9 multimers [CIITA was markedly down regulated in GC cells, associated with a general low expression of MHC transcripts.Many genes know to be specifically expressed in GC B-cells are found to be upregulated: e. g., ultimers . It is lultimers -63. NotaThe gene expression profiles of the three B-cell compartments reflect distinctive functional attributes of the resident B-cell populations. They also showed different molecular microenvironments that allow the different B-cell populations to differentiate and function properly. GC B-cells have a high proliferation gene signature, whereas MNZ and MGZ cells are characterized by signals that help to maintain the quiescent state. Genes involved in the apoptosis pathway are differentially expressed in the three B-cell compartments, reflecting different adaptations for survival in different B-cell populations. Expression of different chemokines, their receptors, and stromal molecules have been detected. Many of these have been implicated in the establishment of the normal lymphoid architecture in peripheral lymphoid organs and in attracting distinct immune-cell populations to specific lymphoid areas. The expression of unique sets of genes may also reflect the functional adaptation of cells in a specific location, such as genes involved in DNA repair in the GC and genes that are active in innate immune response to infection in the MGZ. Gene expression profiling of B-cell compartments has allowed us to obtain a global survey of the molecular signals that are functionally important in B-cell subpopulations as well as the respective microenvironments. One of the major challenges is to delineate the functions of the uncharacterized genes that are unique to each of the compartments. Another challenge is to exploit these normal transcriptional profiles to further our understanding of the normal immune response and the derangements resulting in the corresponding lymphoid tumors.Tissue blocks of tonsils and spleens were snap frozen in O.C.T immediately after surgery. Four micrometer thick frozen sections of reactive tonsils or spleens on plain glass slides were fixed with 70% ethanol for 30 seconds, rinsed in DEPC water and stained with hematoxylin for 30 seconds, followed by another water rinse. The sections were then dehydrated with 70%, 95% and 100% ethanol for 10 seconds each. Finally, the slides were passed through xylene twice, each for 30 seconds. A consecutive section was immunostained for CD3 to guide the dissection. The three B-cell compartments (GC, MNZ and MGZ) were isolated using LCM with the Arcturus PixCell II system (Arcturus Engineering, Mountain View, CA). To avoid contamination, only well-defined GC, MNZ and MGZ were dissected. Cells were captured at the 15-μm laser setting on CapSure LCM Caps (Arcturus). The laser was set to pulse at 60 mW for 200 ms. The Institutional Review Board of the University of Nebraska approved the usage of tissues for this study.7 B-cells were stained with IgM-Cy-chrome, IgD-FITC and CD27-PE (BD Pharmingen, SanDiego, CA) at 4°C for 30 min. MGZ B-cells were isolated from the splenic B-cells gated on the IgMhighIgDlowCD27+ fraction, whereas MNZ B-cells were selected based on IgMlowIgDhighCD27- using the BD FACSVantage™ SE high-speed cell sorter (Becton-Dickinson, SanJose, CA)Tissue from fresh spleens or tonsils was cut into small pieces in cold RPMI-1640, and cells released by grinding with a glass tissue homogenizer. The crude cell suspension was passed through a nylon mesh (Spectrum Laboratories, Inc) to generate a single-cell suspension. B-cells were firstly isolated using the Human B-cell Isolation Kit (a cocktail of hapten-modified antibodies to CD2, IgE, CD4, CD11b, CD16 and CD36) and the Midi Macs system (Miltenyi Biotec, Auburn, CA). The highly enriched B-cell population (negative fraction) was subjected to 3-color cell sorting. Briefly, 1 × 10in vitro transcription (IVT) using the Ampliscribe™ High yield Transcription Kit (Epicentre Technologies, Madison, WI) containing 1000 units AmpliScribe T7 enzyme at 37°C for 8–12 hours, as per the manufacture's instruction. Second-round amplification and IVT were performed as described previously [Total RNA was extracted from each sample of microdissected cells with Trizol™ (Gibco BRL, Carlsbad, CA) and further purified with the RNeasy Mini Kit . RNA amplification was performed using a modified Eberwine protocol . Brieflyeviously . The qua. The aminoallyl group on the dUTP reacts with the ester group on the cyanine dyes. Cy3 dye was used to label the standard cDNA and Cy5 dye the test probe, and hybridization was performed as previously described [Analysis of gene expression was performed using the Lymphochip cDNA microarray, which contained 15,132 cDNA clones representing 7399 known or uncharacterized genes . Labeledescribed .© (Mathworks, Inc, Natick, MA), which provided an overview of the similarity of expression profiles between multiple samples. Only genes with at least two values out of the triplicate experiments showing similar behavior were included for analysis. The expression data for each gene from an individual compartment was median/mean centered with weighted variance across the two or three replicates showing similar behavior. The initial data reduction was performed using the two-tailed student t-test to compare the differences in gene expression levels between individual compartments. Genes differentially expressed between the two compartments with a p-value of less than 0.05 were selected for further analysis using the Significance Analysis of Microarrays (SAM) approach, as described previously [ or ) and viewed by TreeView.Each tissue type was independently isolated, amplified and profiled in three separate experiments to enhance the reliability of the gene expression data. Images of hybridized microarrays were obtained and processed using GenePix 4000B microarray scanner (Axon Instruments, Union City, CA). Spots or areas of an array with obvious blemishes were flagged and excluded from subsequent analyses. Fluorescence ratios were normalized for each array by applying a single scaling factor to all fluorescent ratios from the array . The coreviously . SAM ass- reverse transcriptase as per the manufacturer's instructions (Invitrogen, Carlsbad, CA). Five-fold serially diluted cDNAs from GC, MNB and MZB were amplified with gene-specific primers for 30 cycles with the following cycling conditions: A denaturation step at 94°C for 2.5 minutes and then each PCR cycle at 94°C for15 sec, 52°C for 30 sec, and 72°C for 30 sec followed by a final extension at 72°C for 5 min. The human HPRT transcript was used as the comparative standard. The products were analyzed by electrophoresis in 2% agarose gel. The primers were designed to amplify the cDNA close to the 3' end of the transcript, and all the PCR products were less than 200 bp in length.To confirm the differential mRNA expression of the genes identified by the Lymphochip in different B-cell compartments, a semi-quantitative RT-PCR was employed. In brief, 200 ng aRNA was reversely transcribed into cDNA with 200 ng random primer using MMLV-RNase H® Green qPCR Kit (MJ Research, Reno, NV) on DNA Engine OPTICON2 (MJ Research, Reno, NV) as per the manufacturer's instructions. The PCR protocol used an initial denaturation of 95°C for 15 minutes followed by 35 cycles (95°C for 10 sec, 52°C for 15 sec and 70°C for 20 sec). The plate was read at 70°C according to the melting point of the amplicon. Serial dilutions of cDNA from the lymphoid standard [FAIM, CCL3, SODD, NM23-H1, CARD11, Cyclin G2 and CIITA-8) and the endogenous reference genes (HPRT). For each unknown sample, the relative amounts of target cDNAs and reference cDNAs applied to the PCR reaction system were calculated using linear regression analysis from the corresponding standard curves [Some of the results from the semi-quantitative RT-PCR were also validated by the real-time quantitative PCR with DyNAmo™ HS SYBRstandard  were used curves . Then thin vitro RNA amplification and semi-quantitative PCR. JI participated in the design of the study, microarray procedure, data analysis, and drafted the manuscript. LX, RL and SS participated in data analysis. JE provided the microarray facility and various technical assistance and advice. LS and AR provided the reference standard, the Lymphochip and helpful discussions. KD, GZ, TM participated in helpful discussions and interpretation of the data. WCC conceived, organized and supervised the study, and participated in the analysis and interpretation of the data. All authors have read and approved the final manuscript.YS carried out the LCM,


Tumours arising from odontogenic tissues are rare and constitute a heterogenous group of interesting lesions. The aim of this study was to determine the relative frequency of odontogenic tumors (OT) among Nigerian children and adolescents 19 years or younger.The histopathology records were retrospectively reviewed for all the tumors and tumor-like lesions of the oral cavity and the jaws seen in children and adolescents ≤ 19 years seen between January 1980 and December 2003. Hematoxylin and eosin-stained sections were re-evaluated and the diagnosis in each case was confirmed or modified according to World Health Organization (WHO) classification, 1992; and were subjected to analysis of age, sex, site of tumor and histopathologic type.A total of 477 tumors and tumor-like lesions were seen in patients ≤ 19 years during the period of the study. Of these, 92 (19.3%) were odontogenic tumors. Benign odontogenic tumors constituted 98.9% of the cases seen, while only 1 case (1.1%) of malignant variety was seen during the period. The mean (SD) age of patients was 14.9 (± 3.1) years (range, 4–19 years). Male-to-female ratio was 1:1; and mandible-to-maxilla ratio was 2.7:1. OT's were most frequently seen in patients aged 16–19 years (46.7%) and the least number (2.2%) were found in patients aged 0–5 years. Among nine histologic types of OT seen, ameloblastoma (48.9%), adenomatoid odontogenic tumor (19.6%) and odontogenic myxoma (8.7%) were predominant. Multicystic/solid and unicystic variants of ameloblastoma were diagnosed in 40 (89%) and 5 (11%) cases respectively.Odontogenic tumors are relatively common in children and adolescents in Nigeria. One out of every 5 children and adolescents with tumors and tumor-like lesions of oral cavity and the jaws seen in this study had a diagnosis of odontogenic tumor. Tumors and tumor-like growths arising from the odontogenic tissues constitute a heterogenous group of particularly interesting lesions, as they display the various inductive interactions that normally occur among the embryologic components of the developing tooth germ . In humaThere are few reports especially from Africa, which have specifically reported the high frequency of odontogenic tumors (OT) in children and adolescents in the literature. In most previous African reports, odontogenic tumours in children were presented as part of orofacial  or oral The histopathology records of the Department of Oral Pathology and Biology, College of Medicine, University of Lagos, Lagos, Nigeria, were reviewed for all the tumors and tumor-like lesions of the oral cavity and the jaws seen in children and adolescent ≤ 19 years from January 1980 to December 2003. Hematoxylin and eosin-stained sections was re-evaluated and the diagnosis in each case was confirmed or modified according to World Health Organization (WHO) 1992 classification . The datA total of 477 tumors and tumor-like lesions were seen in patients ≤ 19 years during the period of the study. Of these, 92 (19.3%) were odontogenic tumors. Benign odontogenic tumors constituted 98.9% of the cases seen, while only 1 case (1.1%) of malignant variety was seen during the period. Table Table Ameloblastoma constituted almost half (48.9%) of the odontogenic tumors with female-to-male and maxilla-to-mandible ratios of 1:1.7 and 1:8 respectively. The mean (SD) age of patients in this group was 15.1 (± 3.0) years (range, 4–19 years) with most patients (49%) in age group 3. Multicystic/solid and unicystic variants were diagnosed in 40 (89%) and 5 (11%) cases respectively.Adenomatoid odontogenic tumor (AOT) was the second most common tumor in this series (Table es Table  accountiOdontogenic myxoma accounted for 8 cases (8.7%) of odontogenic tumors with a male-to-female ratio of 1:3. Only 1 case occurred in the maxilla, the rest (7) were found in the mandible. The patients were found between 10 and 19 years.Odontogenic fibroma accounted for 7 cases (7.6%) of tumors in this series. More cases were found in males and in the mandible. Patients were seen between 5 and 19 years in this group.Ameloblastic fibroma accounted for 5.4% of cases seen. The lesion occurred exclusively in the mandible with a male-to-female ratio of 1:4.Odontomas accounted for 4.3% of OT seen. They were exclusively found in the maxilla with equal sex predilection.Table We present a report of odontogenic tumours in children and adolescents aged ≤ 19 years. This report represents the largest series of odontogenic tumors in children and adolescents in Africa. We found that 19.3% of tumors and tumor-like lesions of the oral cavity and the jaws in children and adolescent in this study were odontogenic tumors. This is similar to the findings of Arotiba . Howeveret al [et al [A major problem in comparing our report with previous studies is the lack of uniformty in the age group studied in those reports. Some studies were restricted to children under the age of 14 years ,15, or 1et al . Al-Khatl [et al ,13; thesOdontogenic tumors were less frequently seen in patients aged 0–5 years in this study in agreement with most reports in the literature ,11-14,17et al [et al [et al [Odontogenic tumors in children are known to have predilection for the mandible -13. Thiset al  however l [et al ; whereasl [et al  reportedet al [Ameloblastoma was the most common tumor in this study in agreement with reports from Africa ,11. Otheet al  found odet al ,11-14. Aet al . Previouet al ,21. Unicet al ,22,23.Adenomatoid odontogenic tumor (AOT) was the second most common OT in this study and 50% of this tumor was found in patients >15 years. Asamoa  reportedet al [In children, the reported incidence of myxoma ranges from 1.2% to 39% ,6,9,13. et al  reportedet al ,26,27. Tet al ,27 reporet al [et al [Odontogenic fibroma was reported to be rare in children with incidence ranging from 0% to 1.3% of OT ,9,11,13.et al . Arotibaet al  reportedl [et al ; whereasl [et al  have repl [et al ,26 have et al [et al [et al [Odontomas are often regarded as dental hamartomas, rather than odontogenic neoplasms ,28,29. Oet al , Tanaka l [et al  and Satol [et al  that repl [et al . This mal [et al ,17, othel [et al ,14 and sl [et al ,25. OdonMalignant odontogenic tumors are rare, most especially in children ,12,15,30Odontogenic tumors are relatively common in children and adolescents in Nigeria. One out of every 5 children and adolescents with tumor and tumor-like lesions of oral cavity and the jaws seen in this study had a diagnosis of odontogenic tumor.The author(s) declare that they have no competing interests.OFA conceived the study, coordinated the write-up and submission of the article, and also reviewed the slides.ALL, WLA and MOO did the literature search and participated in the writing of the manuscript.All the authors read and approved the final manuscript.None declared


A conditional-lethal recombinant virus was constructed in which the expression of the vaccinia virus I7L gene is under the control of the tetracycline operator/repressor system. In the absence of I7L expression, processing of the major VV core proteins is inhibited and electron microscopy reveals defects in virion morphogenesis subsequent to the formation of immature virion particles but prior to core condensation. Plasmid-borne I7L is capable of rescuing the growth of this virus and rescue is optimal when the I7L gene is expressed using the authentic I7L promoter. Taken together, these data suggest that correct temporal expression of the VV I7L cysteine proteinase is required for core protein maturation, virion assembly and production of infectious progeny. The cysteine proteinase encoded by the VV I7L gene, was originally identified based on a sequence comparison with the African Swine Fever virus proteinase and an ubiquitin-like proteinase in yeast ,2. We haresidues ,4. To delac operator and driven off of the T7 promoter, was responsible for cleaving the A17L membrane protein as well as the L4R core protein precursor. In this work, we show that I7L proteinase, in a different inducible mutant virus, this one regulated by the tetracycline (TET) operator/repressor system and driven off of the I7L native promoter, is responsible for cleaving the other core protein precursors (p4a and p4b). We also demonstrate that expression of the I7L gene from its native promoter appears to be important for optimal viral assembly and replication.While this work was in progress, Ansarah-Sobrinho and Moss  publisheTo investigate the role of the I7L proteinase in the viral life cycle, an inducible mutant virus was constructed in which the expression of the I7L gene could be regulated by the presence or absence of TET using the components of the bacterial tetracycline operon ,7. This 40 cells [40 cells. At an MOI of 0.1 or 0.5 there was an average reduction of 99.1% of infectious virus particles  and infected with vtetOI7L at an MOI of 0.2 plaque-forming units per cell in the absence of TET. Cells were harvested 24 hours post infection (hpi) and the titer determined on BSCene Fig. . This waene Fig. , suggest35S-met and harvested after 24 h. Extracts were immunoprecipitated with I7L antisera and protein detected by autoradiography. With wild type virus, I7L protein was expressed at each TET concentration (data not shown). However, in the mutant virus, expression of I7L enzyme was repressed in the absence of TET and increased with the addition of TET.We have previously shown through transient expression assays that the I7L proteinase is capable of cleaving the p4b, p4a, and p25k core protein precursors ,4 which To determine the effect of TET concentration on p4b core protein precursor processing, cells were infected in the presence of 0 to 5 μg/ml TET, harvested 24 hpi, and the extracts immunoblotted with anti-4b antisera. With wild type virus p4b was processed at each TET concentration as expected, however with the mutant virus, p4b processing was repressed in the absence of TET (data not shown). The slight processing in the absence of TET is likely due to slight leak-through of I7L gene expression in this system. The same results were seen for the processing of p4a, with processing in each of the wild type virus lanes, repressed processing with the mutant in the absence of TET and increased processing in the presence of TET (data not shown). Kane and Shuman  have preet al [The morphogenesis of vtetOI7L under nonpermissive conditions was analyzed via electron microscopy. TREx-293 cells were infected with vtetOI7L at an MOI of 1 in the presence or absence of TET and harvested 24 h later. In the presence of TET, cells contained a variety of both immature and mature forms of the virus  declare that there are no competing interests.CMB conducted all the experiments and wrote the manuscript. DEH conceived the study, coordinated the research efforts and edited the paper. Both authors read and approved the final manuscript.


The objective of this study was to investigate the relationship between perceived walkability and overall self-rated health among patients who use community-based clinics.A cross-sectional survey was distributed to a convenience sample in three community clinics. Forms were completed by 793 clinic patients. Multiple logistic regression analysis was to control for the effects of demographic variables and lifestyles.Perceiving the availability of places to walk was related to better self-rated health. The most important places were work (OR = 3.2), community center (OR = 3.12), park (OR = 2.45) and day care (OR = 2.05). Respondents who said they had zero (OR = .27) or one (OR = .49) place to walk were significantly less healthy than persons who said they had five or more places to walk.Persons who perceived that they had no place to walk were significantly less healthy than persons who thought they had at least one place to walk (OR = .39). Support for walkable neighborhoods and education of patients about options for walking may be in the best interests of community medicine patients. Much recent interest and research has been directed at the relationship between one's health and where one lives . A numbeNeighborhoods where walking is convenient might encourage their inhabitants to exercise. Indeed, having convenient places to walk in the neighborhood has been related to the proportion of persons in the neighborhood who met current activity recommendations  and withThe hypothesis that perceived walkability is directly related to an individual's overall self-rated health has not previously been investigated. The purpose of this project was to test that hypothesis in patients attending community-based clinics. Self-rated health has been shown to accurately predict overall health as measured by other more traditional measures of health .A cross-sectional survey was used to test the hypothesis that patients with good self-rated health perceived the neighborhoods in which they live as having good walkability, and that this effect was independent of demographic characteristics and lifestyle habits that would predict good health. The sample was drawn from three clinics that primarily serve low-income populations. Clients, parents (if the patient was a child) and accompanying visitors were asked to complete surveys and drop them into a box. Participation was voluntary. A total of 1471 surveys were distributed with an overall return of 825 (56.1%) surveys of which 793 (54%) met eligibility parameters. Pregnant women and persons under age 18 were excluded. Assuming 80% power, p < .05, 20% poor health among the persons inhabiting neighborhoods with good perceived walkability and 30% poor health among persons inhabiting neighborhoods with poor perceived walkability, 626 cases were needed to test the hypothesis. Completed forms were received from a total of 793 persons.Return rates varied by clinic. Clinic 1 is a university-based family medicine clinic providing a full range of primary care services to cross-generational clients. It is staffed by family medicine physicians and residents. Census was approximately 85 clients daily, of which, less than 5 % were non-English speaking. Clinic personnel distributed 500 survey forms over an eight week period with an 80.8% return rate.Clinic 2 serves women and children, providing obstetrical, well care (including immunizations), and acute care services to a targeted high-risk, low socioeconomic sub-population. It is staffed by pediatric and OB-GYN physicians and residents. Approximately 30% of the clinic clients do not speak English. A total of 471 surveys were distributed over a period of 18 weeks with a return of 37.4%. Both the large number of obstetrical patients (ineligible for survey) and percentage of non-English speaking clients contributed to the low return rate.Clinic 3 provides primary care services to a population of indigent adults meeting residential and income screening requirements. It is staffed by internal medicine physicians and residents. A total of 500 surveys were distributed in this clinic over a ten week period with a return of 42.6%.The dependant variable for the study was self-rated health. Subjects were asked whether in general they would say their health was excellent, very good, good, fair or poor. Excellent, very good, and good responses were combined to form a category called 'good health' while fair and poor comprised 'poor health'.Leyden's scale of walkability was modified for a US population for this study by droppBoth demographic characteristics and lifestyle variables were used to adjust the associations between perceived walkability and self-rated health. Lifestyle variables were: numbers of fruits and vegetables eaten per day (zero, one, two, three, four, five or more), smoking status (not a smoker, smokes one-20 cigarettes per day, smokes more than 20 per day), days of physical activity per week that involve at least 20 minutes of exercise (zero, one, two, three, four, five or more), and obese (yes vs no). Obese was defined as body mass index (BMI) >30. BMI was computed from self-reported height and weight.Demographic variables were race/ethnicity (Hispanic, non-Hispanic white, non-Hispanic black, non-Hispanic Asian, non-Hispanic other), gender, age category, marital status (married vs. not), and highest level of education achieved.Chi square tests were performed to test for any unadjusted associations between self-rated health and each categorical independent variable. Multivariate logistic regression modeling was employed to determine if associations between perceived walkability and self-rated health remained significant after adjustment for demographic and lifestyle variables. Separate logistic regression models were run for each variable. Statistical analysis was performed using EpiInfo 3.2.2.The question about self-rated overall health was answered by 793 respondents. Of these, 67 percent were classified as healthy because they said their health was excellent, very good, or good. The remainder was classified as having poor health because they said their health was fair or poor.The typical respondent was non-Hispanic white, female, had at least a high school education. The sample was evenly spread across age groups. About half were married and about half were not married Table .Unadjusted relationships between demographic variables and self-rated health are shown in Table The most common number of days of exercise per week was zero (34.9 percent). Most people said they ate at least one fruit or vegetable each day, though only less than half had three or more. The percent obese was 43.6. Over 70 percent were non-smokers . Meeting recommended physical activity guidelines is related to better self-rated health . CertainIf promotion of intensive physical activity is problematic, then more moderate activity might be a more reasonable goal. Simple walking, for example, reduces the cost of medical care for the elderly  and inteIn addition to valuing leisure-time walking, researchers and clinicians should take into account the benefits of work related activity and housework . So callOne approach to encouraging moderate physical activity is to increase walkability. The convenience of places to exercise is widely recognized to be important; adults generally support local policies that increase the availability of places to exercise -28. WalkOur purpose was to investigate the effect of perceived walkability on the health of patients attending community based clinics. The results should not be interpreted as being relevant to all persons in the community or even to all persons who live in neighborhoods they perceive to be unwalkable, but only to patients attending clinics that serve a disproportionate share of disadvantaged persons. This is important to public health because the success of strategies designed to improve in overall community health will have to focus on the disadvantaged people who bear the greatest burden of poor health. We believe that more studies of disadvantaged clinic populations such as this one are needed in the public health literature.The study reported here of low income primary care patients confirms the relationship between perceptions of convenient walking locations and self-rated health. Health status is measured by just one question, a practice which has become increasingly common in the public health literature,30. HoweAnother limitation of the study is the subjective nature of the walkability measure. Perceptions of walkability may not be accurate and thus objective assessment of walkability might have led to a different conclusion. However, since our purpose was to investigate the relationship between perceptions of walkability and self-rated health, the results are valid for this sample. If community medicine patients are incorrect in their perceptions of neighborhood walkability, then public health education campaigns and personal health education in clinics can inform them about options for walking in their neighborhoods and how they might overcome any perceived barriers.Our results also may have been influenced by the context from which subjects were drawn. Amarillo is located in the Panhandle of Texas. Most of the year the climate is mild, but during the summer season temperatures can average over 90 degrees. In some neighborhoods, crime rates are above the national average and walking after dark might be worrisome. Furthermore, cities in West Texas are designed for automobile traffic, to the detriment of pedestrians and bicyclists. Furthermore, the vast open spaces in the region make it impossible to reach many locations by foot. Accordingly the, culture of the area has not evolved to be supportive of walking. Therefore, our results may not be generalizable to communities where walking has historical been a visible aspect of the culture.Despite these limitations, the results of this analysis are intriguing and warrant further investigation using a prospective study design. The evidence presented suggests that support for walkable neighborhoods and health education about options for walking in their neighborhoods may be in the best interests of community medicine patients.The author(s) declare that they have no competing interests.JER planned the project, analyzed the data and wrote the first draft of the manuscript. JRP contributed comments and revisions to the manuscript. AD organized data collection and wrote a portion of the manuscript.The pre-publication history for this paper can be accessed here: