Importance of antibiotic class to the development of Clostridium difficile infection (CDI) in adults: A systematic review

C. difficile causes gastrointestinal infections in humans, including severe diarrhea. It is implicated in 20%-30% of cases of antibiotic-associated diarrhea, in 50%-70% of cases of antibiotic-associated colitis, and in >90% of cases of antibiotic-associated pseudomembranous colitis. Exposure to antimicrobial agent, hospitalization and age are some of the risk factors that predispose to CDI. Virtually all hospitalized patients with nosocomially-acquired CDI have a history of treatment with antimicrobials or neoplastic agent within the previous 2 months. The development of CDI usually occurs during treatment with antibiotics or some weeks after completing the course of the antibiotics. ^ After exposure to the organism (often in a hospital), the median incubation period is less than 1 week, with a median time of onset of 2days. The difference in the time between the use of antibiotic and the development of the disease relate to the timing of exogenous acquisition of C. difficile. ^ This paper reviewed the literature for studies on different classes of antibiotics in association with the rates of primary CDI and RCDI from the year 1984 to 2012. The databases searched in this systematic review were: PubMed (National Library of Medicine) and Medline (R) (Ovid). RefWorks was used to store bibliographic data. ^ The search strategy yielded 733 studies, 692 articles from Ovid Medline (R) and 41 articles from PubMed after removing all duplicates. Only 11 studies were included as high quality studies. Out of the 11 studies reviewed, 6 studies described the development of CDI in non-CDI patients taking antibiotics for other purposes and 5 studies identified the risk factors associated with the development of recurrent CDI after exposure to antibiotics. ^ The risk of developing CDI in non-CDI patients receiving beta lactam antibiotics was 2.35%, while fluoroquinolones, clindamycin/macrolides and other antibiotics were associated with 2.64%, 2.54% and 2.35% respectively. Of those who received beta lactam antibiotic, 26.7% developed RCDI, while 36.8% of those who received any fluoroquinolone developed RCDI, 26.5% of those who received either clindamycin or macrolides developed RCDI and 29.1% of those who received other antibiotics developed RCDI. Continued use of non-C. difficile antibiotics especially fluoroquinolones was identified as an important risk factor for primary CDI and recurrent CDI. ^

Rifaximin-mediated changes to the epithelial cell proteome: 2-D gel analysis

Diarrhea remains a significant cause of worldwide morbidity and mortality. Over 4 million children die of diarrhea annually. Although antibiotics can be used as prophylaxis or for treatment of diarrhea, concern remains over antibiotic resistance. Rifaximin is a semi-synthetic rifamycin derivative that can be used to treat symptoms of infectious diarrhea, inflammatory bowel syndrome, bacterial overgrowth of the small bowel, pouchitis, and fulminant ulcerative colitis. Rifaximin is of particular interest because it is poorly adsorbed in the intestines, shows no indication of inducing bacterial resistance, and has minimal effect on intestinal flora. In order to better understand how rifaximin functions, we sought to compare the protein expression profile of cells pretreated with rifaximin, as compared to cells treated with acetone, rifamycin (control antibiotic), or media (untreated). 2-D gel electrophoresis identified 38 protein spots that were up- or down-regulated by over 2-fold in rifaximin treated cells compared to controls. 16 of these spots were down-regulated, including keratin, annexin A5, intestinal-type alkaline phosphatase, histone h4, and histone-binding protein RbbP4. 22 spots were up-regulated, including heat shock protein HSP 90 alpha, alkaline phosphatase, and fascin. Many of the identified proteins are associated with cell structure and cytoskeleton, transcription and translation, and cellular metabolism. A better understanding of the functionality of rifaximin will identify additional potential uses for rifaximin and determine for whom the drug is best suited. ^

Nonlinear dimension reduction in pathway-based genome-wide association analysis

Pathway based genome wide association study evolves from pathway analysis for microarray gene expression and is under rapid development as a complementary for single-SNP based genome wide association study. However, it faces new challenges, such as the summarization of SNP statistics to pathway statistics. The current study applies the ridge regularized Kernel Sliced Inverse Regression (KSIR) to achieve dimension reduction and compared this method to the other two widely used methods, the minimal-p-value (minP) approach of assigning the best test statistics of all SNPs in each pathway as the statistics of the pathway and the principal component analysis (PCA) method of utilizing PCA to calculate the principal components of each pathway. Comparison of the three methods using simulated datasets consisting of 500 cases, 500 controls and100 SNPs demonstrated that KSIR method outperformed the other two methods in terms of causal pathway ranking and the statistical power. PCA method showed similar performance as the minP method. KSIR method also showed a better performance over the other two methods in analyzing a real dataset, the WTCCC Ulcerative Colitis dataset consisting of 1762 cases, 3773 controls as the discovery cohort and 591 cases, 1639 controls as the replication cohort. Several immune and non-immune pathways relevant to ulcerative colitis were identified by these methods. Results from the current study provided a reference for further methodology development and identified novel pathways that may be of importance to the development of ulcerative colitis.^

Papel de la interleuquina 23 en la diferenciación hacia Th17

The incidence of inflammatory and autoimmune diseases has increased among developed countries in the past 30 years, creating a demand for the development of effective and economic therapies for these diseases. Interleukin 23 (IL-23) is a pro-inflammatory cytokine whose increased production has been shown to play a key role in the establishment and maintenance of inflammatory and autoimmune diseases in different murine models such as inflammatory bowel disease, psoriasis and experimental autoimmune encephalomyelitis. More importantly, increased levels of IL-23 have been found in biopsies from patients with Crohn’s disease and ulcerative colitis, and psoriasis. The pathological consequences of excessive IL-23 signalling have been linked to its ability to promote the production of interleukin 17 (IL-17), particularly in the subpopulation of CD4 T cells Th17. However, the precise molecular mechanisms by which IL-23 sustains the Th17 response and induces pathogenic effector functions in these cells remain largely unknown. The global objective of the experiments carried out in this work was to determine the effect of IL-23 on the proliferation, survival and IL-17 and interferon gamma (IFN-ɣ) production in Th17 cells. These experiments have shown that IL-23 does not promote proliferation or survival of in vitro generated Th17 cells, and that there is no difference in the production of IL -17 in the absence or presence of IL -23. The IL-23 receptor, like other cytokine receptors, lacks intrinsic enzymatic activity. Instead, IL-23 receptor associates with members of the Janus tyrosine kinase family (Jaks). Cytokine binding to a Jak-associated receptor triggers the activation of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors. Previous work indicated that the IL-23 receptor complex is associated with the tyrosine kinases Jak2 and Tyk2 that promote STAT3 phosphorylation. Subsequent studies showed that IL23 activation of STAT3 induces the expression of the transcription factor RORγt, which is crucial for IL-17 production. This work has explored the IL-23 signalling cascade, determining the optimal conditions for STAT3 activation and demonstrating the activation of other transcription factors such as STAT4, STAT5 and STAT1 that contribute to IL-23-mediated signalling pathways.

Identification of novel susceptibility loci for inflammatory bowel disease on chromosomes 1p, 3q, and 4q: Evidence for epistasis between 1p and IBD1

The idiopathic inflammatory bowel diseases, Crohn’s disease (CD) and ulcerative colitis (UC), are chronic, frequently disabling diseases of the intestines. Segregation analyses, twin concordance, and ethnic differences in familial risks have established that CD and UC are complex, non-Mendelian, related genetic disorders. We performed a genome-wide screen using 377 autosomal markers, on 297 CD, UC, or mixed relative pairs from 174 families, 37% Ashkenazim. We observed evidence for linkage at 3q for all families (multipoint logarithm of the odds score (MLod) = 2.29, P = 5.7 × 10−4), with greatest significance for non-Ashkenazim Caucasians (MLod = 3.39, P = 3.92 × 10−5), and at chromosome 1p (MLod = 2.65, P = 2.4 × 10−4) for all families. In a limited subset of mixed families (containing one member with CD and another with UC), evidence for linkage was observed on chromosome 4q (MLod = 2.76, P = 1.9 × 10−4), especially among Ashkenazim. There was confirmatory evidence for a CD locus, overlapping IBD1, in the pericentromeric region of chromosome 16 (MLod = 1.69, P = 2.6 × 10−3), particularly among Ashkenazim (MLod = 1.51, P = 7.8 × 10−3); however, positive MLod scores were observed over a very broad region of chromosome 16. Furthermore, evidence for epistasis between IBD1 and chromosome 1p was observed. Thirteen additional loci demonstrated nominal (MLod > 1.0, P < 0.016) evidence for linkage. This screen provides strong evidence that there are several major susceptibility loci contributing to the genetic risk for CD and UC.

Transcription initiation is controlled by three core promoter elements in the hgl5 gene of the protozoan parasite Entamoeba histolytica

Entamoeba histolytica is a single cell eukaryote that is the etiologic agent of amoebic colitis. Core promoter elements of E. histolytica protein encoding genes include a TATA-like sequence (GTATTTAAAG/C) at −30, a novel element designated GAAC (GAACT) that has a variable location between TATA and the site of transcription initiation, and a putative initiator (Inr) element (AAAAATTCA) overlying the site of transcription initiation. The presence of three separate conserved sequences in a eukaryotic core promoter is unprecedented and prompted examination of their roles in regulating transcription initiation. Alterations of all three regions in the hgl5 gene decreased reporter gene activity with the greatest effect seen by mutation of the GAAC element. Positional analysis of the TATA box demonstrated that transcription initiated consistently 30–31 bases downstream of the TATA region. Mutation of either the TATA or GAAC elements resulted in the appearance of new transcription start sites upstream of +1 in the promoter of the hgl5 gene. Mutation of the Inr element resulted in no change in the site of transcription initiation; however, in the presence of a mutated TATA and GAAC regions, the Inr element controlled the site of transcription initiation. We conclude that all three elements play a role in determining the site of transcription initiation. The variable position of the GAAC element relative to the site of transcription initiation, and the multiple transcription initiations that resulted from its mutation, indicate that the GAAC element has an important and apparently novel role in transcriptional control in E. histolytica.

Cattle lack vascular receptors for Escherichia coli O157:H7 Shiga toxins

Escherichia coli O157:H7 causes Shiga toxin (Stx)-mediated vascular damage, resulting in hemorrhagic colitis and the hemolytic uremic syndrome in humans. These infections are often foodborne, and healthy carrier cattle are a major reservoir of E. coli O157:H7. We were interested in knowing why cattle are tolerant to infection with E. coli O157:H7. Cattle tissues were examined for the Stx receptor globotriaosylceramide (Gb3), for receptivity to Stx binding in vitro, and for susceptibility to the enterotoxic effects of Stx in vivo. TLC was used to detect Gb3 in tissues from a newborn calf. Gb3 was detected by TLC in kidney and brain, but not in the gastrointestinal tract. Immunohistochemistry was used to define binding of Stx1 and Stx2 overlaid onto sections from cattle tissues. Stx1 and Stx2 bound to selected tubules in the cortex of the kidney of both newborn calves (n = 3) and adult cattle (n = 3). Stx did not bind to blood vessels in any of the six gastrointestinal and five extraintestinal organs examined. The lack of Gb3 and of Stx receptivity in the gastrointestinal tract raised questions about the toxicity of Stx in bovine intestine. We found that neither viable E. coli O157:H7 nor Stx-containing bacterial extracts were enterotoxic (caused fluid accumulation) in ligated ileal loops in newborn calves. The lack of vascular receptors for Stx provides insight into why cattle are tolerant reservoir hosts for E. coli O157:H7.

Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor

Clostridium difficile, a causative agent of antibiotic-associated diarrhea and its potentially lethal form, pseudomembranous colitis, produces two large protein toxins that are responsible for the cellular damage associated with the disease. The level of toxin production appears to be critical for determining the severity of the disease, but the mechanism by which toxin synthesis is regulated is unknown. The product of a gene, txeR, that lies just upstream of the tox gene cluster was shown to be needed for tox gene expression in vivo and to activate promoter-specific transcription of the tox genes in vitro in conjunction with RNA polymerases from C. difficile, Bacillus subtilis, or Escherichia coli. TxeR was shown to function as an alternative sigma factor for RNA polymerase. Because homologs of TxeR regulate synthesis of toxins and a bacteriocin in other Clostridium species, TxeR appears to be a prototype for a novel mode of regulation of toxin genes.

On the etiology of Crohn disease.

Crohn disease (CD) is a chronic, panenteric intestinal inflammatory disease. Its etiology is unknown. Analogous to the tuberculoid and lepromatous forms of leprosy, CD may have two clinical manifestations. One is aggressive and fistulizing (perforating), and the other is contained, indolent, and obstructive (nonperforating) [Gi]-berts, E. C. A. M., Greenstein, A. J., Katsel, P., Harpaz, N. & Greenstein, R. J. (1994) Proc. Natl. Acad. Sci. USA 91, 12721-127241. The etiology, if infections, may be due to Mycobacterium paratuberculosis. We employed reverse transcription PCR using M. paratuberculosis subspecies-specific primers (IS 900) on total RNA from 12 ileal mucosal specimens (CD, n = 8; controls, n = 4, 2 with ulcerative colitis and 2 with colonic cancer). As a negative control, we used Myobacterium avium DNA, originally cultured from the drinking water of a major city in the United States. cDNA sequence analysis shows that all eight cases of Crohn's disease and both samples from the patients with ulcerative colitis contained M. paratuberculosis RNA. Additionally, the M. avium control has the DNA sequence of M. paratuberculosis. We demonstrate the DNA sequence of M. paratuberculosis from mucosal specimens from humans with CD. The potable water supply may be a reservoir of infection. Although M. paratuberculosis signal in CD has been previously reported, a cause and effect relationship has not been established. In part, this is due to conflicting data from studies with empirical antimycobacterial therapy. We conclude that clinical trials with anti-M. paratuberculosis therapy are indicated in patients with CD who have been stratified into the aggressive (perforating) and contained (nonperforating) forms.

An increased risk of Crohn's disease in individuals who inherit the HLA class II DRB3*0301 allele.

The role of inflammatory T cells in Crohn's disease suggests that inherited variations in major histocompatibility complex (MHC) class II genes may be of pathogenetic importance in inflammatory bowel disease. The absence of consistent and strong associations with MHC class II genes in Caucasian patients with inflammatory bowel disease probably reflects the use of less precise typing approaches and the failure to type certain loci by any means. A PCR-sequence-specific oligonucleotide-based approach was used to type individual alleles of the HLA class II DRB1, DRB3, DRB4, and DRB5 loci in 40 patients with ulcerative colitis, 42 Crohn's disease patients, and 93 ethnically matched healthy controls. Detailed molecular typing of the above alleles has previously not been reported in patients with inflammatory bowel disease. A highly significant positive association with the HLA-DRB3*0301 allele was observed in patients with Crohn's disease (P = 0.0004) but not in patients with ulcerative colitis. The relative risk for this association was 7.04. Other less significant HLA class II associations were also noted in patients with Crohn's disease. One of these associations involved the HLA-DRB1*1302 allele, which is known to be in linkage disequilibrium with HLA-DRB3*0301. These data suggest that a single allele of an infrequently typed HLA class II locus is strongly associated with Crohn's disease and that MHC class II molecules may be important in its pathogenesis.

Estudo do potencial imunomodulador de Dehidroepiandrosterona (DHEA) na inflamação intestinal experimental

As Doenças inflamatórias intestinais (DII) são multifatoriais e sua etiologia envolve susceptibilidade genética, fatores ambientais, disbiose e ativação exacerbada do sistema imunológico no intestino. Essas doenças também tem sido relacionadas a baixos níveis de dehidroepiandrosterona (DHEA), um hormônio precursor de diversos esteroides e relacionado à modulação das respostas imunes. Porém, os mecanismos precisos que relacionam as ações deste hormônio com a proteção ou susceptibilidade à doença de Crohn ou colite ulcerativa ainda não são totalmente conhecidos. Sendo assim, este projeto buscou entender o papel imunomodulador do DHEA exógeno in vitro e in vivo durante a inflamação intestinal experimental induzida por dextran sulfato de sódio (DSS) em camundongos C57BL/6. Inicialmente, in vitro, DHEA inibiu a proliferação de células do baço de forma dose dependente nas concentrações de 5?M, 50?M ou 100?M, com diminuição da produção de IFN-?. Este hormônio não foi tóxico para células de linhagem mieloide, embora tenha causado necrose em leucócitos nas doses mais elevada (50 ?M e 100?M), o que pode ter influenciado a diminuição das citocinas in vitro. Nos ensaios in vivo, os camundongos tratados com DHEA (40 mg/Kg) foram avaliados na fase de indução da doença (dia 6) e durante o reparo tecidual, quando os animais expostos ao DSS e ao DHEA por 9 dias foram mantidos na ausência destas drogas até o dia 15. Houve diminuição do escore pós-morte, melhora no peso e nos sinais clínicos da inflamação intestinal, com redução de monócitos no sangue periférico com 6 dias e aumento de neutrófilos circulantes na fase de reparo tecidual (15 dias). Ainda, a suplementação com DHEA levou à redução da celularidade da lâmina própria (LP) e ao restabelecimento do comprimento normal do intestino. O uso deste hormônio também diminuiu a expressão do RNAm de IL-6 e TGF-?, enquanto aumentou a expressão de IL-13 no colón dos animais durante a fase de indução da doença, o que provavelmente ajudou na atenuação da inflamação intestinal. Além disso, houve acúmulo de linfócitos CD4+ e CD8+ no baço e diminuição apenas de linfócitos CD4+ nos linfonodos mesentéricos (LNM), indicando retenção das células CD4+ no baço após uso do DHEA. O tratamento foi também capaz de aumentar a frequência de células CD4 produtoras de IL-4 e diminuir CD4+IFN-?+ no baço, além de reduzir a frequência de CD4+IL-17+ nos LNM, sugerindo efeito do DHEA no balanço das respostas Th1/Th2/Th17 relacionadas à colite. Em adição, as células de baço dos animais tratados com DHEA e expostos ao DSS se tornaram hiporresponsivas, como visto pela diminuição da proliferação após re-estímulos in vitro. Finalmente, DHEA foi capaz de atuar no metabolismo dos camundongos tratados, levando à diminuição de colesterol total e da fração LDL no soro durante a fase de indução da doença, sem gerar quaisquer disfunções hepáticas. Com isso, podemos concluir que o DHEA atua por meio do balanço das respostas imunes exacerbadas, minimizando os danos locais e sistêmicos causados pela inflamação intestinal induzida por DSS.

Doença inflamatória intestinal associada a colangite esclerosante primária : um fenótipo raro

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Tratamento da infeção por Clostridium difficile

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Colite microscópica : uma causa rara de diarreia crónica em pediatria

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Reflexiones anticolicas : experimentos medico-practicos chymico-galenicos, quinta esencia de los remedios contra la colica epidemica endemica y respuesta a la Breve reflexión, y crisis medica, que sobre el dolor colico sacò à luz ... don Vicente Boyvia .../

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