The myeloid immune signature of enterotoxigenic Bacteroides fragilis-induced murine colon tumorigenesis.

Enterotoxigenic Bacteroides fragilis (ETBF), a human commensal and candidate pathogen in colorectal cancer (CRC), is a potent initiator of interleukin-17 (IL-17)-dependent colon tumorigenesis in MinApc+/- mice. We examined the role of IL-17 and ETBF on the differentiation of myeloid cells into myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages, which are known to promote tumorigenesis. The myeloid compartment associated with ETBF-induced colon tumorigenesis in Min mice was defined using flow cytometry and gene expression profiling. Cell-sorted immature myeloid cells were functionally assayed for inhibition of T-cell proliferation and inducible nitric oxide synthase expression to delineate MDSC populations. A comparison of ETBF infection with that of other oncogenic bacteria (Fusobacterium nucleatum or pks+Escherichia coli) revealed a specific, ETBF-associated colonic immune infiltrate. ETBF-triggered colon tumorigenesis is associated with an IL-17-driven myeloid signature characterized by subversion of steady-state myelopoiesis in favor of the generation of protumoral monocytic-MDSCs (MO-MDSCs). Combined action of the B. fragilis enterotoxin BFT and IL-17 on colonic epithelial cells promoted the differentiation of MO-MDSCs, which selectively upregulated Arg1 and Nos2, produced NO, and suppressed T-cell proliferation. Evidence of a pathogenic inflammatory signature in humans colonized with ETBF may allow for the identification of populations at risk for developing colon cancer.

Serum mannose-binding lectin deficiency is associated with cryptosporidiosis in young Haitian children.

BACKGROUND: Mannose-binding lectin (MBL) is a component of the innate immune response and binds microbial surfaces through carbohydrate recognition domains. MBL deficiency may contribute to susceptibility to a variety of infectious diseases, particularly in young children. MBL binds to the Cryptosporidium sporozoite and may be important in resistance to cryptosporidiosis. METHODS: We studied the association of serum MBL levels and cryptosporidiosis in a case-control study of young Haitian children with cryptosporidiosis versus children who were control subjects. RESULTS: Ninety-nine children were enrolled, as follows: 49 children with cryptosporidiosis, 41 healthy controls, and 9 children with diarrhea from other causes. Case children were more malnourished than controls, and 49% had persistent or chronic diarrhea. At enrollment, mean serum MBL levels were markedly lower in children with cryptosporidiosis (P = .002), as was the number of children with an MBL deficiency of < or = 70 ng/mL (P = .005). In multivariate analysis, the association of cryptosporidiosis and MBL deficiency persisted (P = .002; adjusted odds ratio, 22.4), as did the association of cryptosporidiosis with general malnutrition. The subset of children with cryptosporidiosis and MBL deficiency were more likely to be male (P = .025). CONCLUSIONS: MBL may be an important component of innate immune protection against Cryptosporidium infection in young children. Additional studies are necessary to determine whether MBL intestinal losses, deficient epithelial expression, and/or genetic polymorphisms in the MBL gene contribute to MBL deficiency in cryptosporidiosis and other enteric infections in young children.

Microbiome changes associated with sustained eradication of Clostridium difficile after single faecal microbiota transplantation in children with and without inflammatory bowel disease.

BACKGROUND: Little data are available regarding the effectiveness and associated microbiome changes of faecal microbiota transplantation (FMT) for Clostridium difficile infection (CDI) in children, especially in those with inflammatory bowel disease (IBD) with presumed underlying dysbiosis. AIM: To investigate C. difficile eradication and microbiome changes with FMT in children with and without IBD. METHODS: Children with a history of recurrent CDI (≥3 recurrences) underwent FMT via colonoscopy. Stool samples were collected pre-FMT and post-FMT at 2-10 weeks, 10-20 weeks and 6 months. The v4 hypervariable region of the 16S rRNA gene was sequenced. C. difficile toxin B gene polymerase chain reaction was performed. RESULTS: Eight children underwent FMT for CDI; five had IBD. All had resolution of CDI symptoms. All tested had eradication of C. difficile at 10-20 weeks and 6 months post-FMT. Pre-FMT patient samples had significantly decreased bacterial richness compared with donors (P = 0.01), in those with IBD (P = 0.02) and without IBD (P = 0.01). Post-FMT, bacterial diversity in patients increased. Six months post-FMT, there was no significant difference between bacterial diversity of donors and patients without IBD; however, bacterial diversity in those with IBD returned to pre-FMT baseline. Microbiome composition at 6 months in IBD-negative patients more closely approximated donor composition compared to IBD-positive patients. CONCLUSIONS: FMT gives sustained C. difficile eradication in children with and without IBD. FMT-restored diversity is sustained in children without IBD. In those with IBD, bacterial diversity returns to pre-FMT baseline by 6 months, suggesting IBD host-related mechanisms modify faecal microbiome diversity.

A clinicopathologic analysis of AIDS-related cryptosporidiosis.

OBJECTIVE: To characterize the histology of AIDS-associated cryptosporidiosis and identify features that explain the clinical variability. DESIGN: A retrospective analysis of HIV-positive individuals with cryptosporidiosis who underwent endoscopy at the Johns Hopkins Hospital between 1985 and 1996. METHODS: The histologic features (intensity of Cryptosporidium infection, inflammation, mucosal damage, copathogens) of gastrointestinal biopsies from 37 HIV-positive individuals with cryptosporidiosis were systematically graded. These histologic features were correlated with the severity of the diarrheal illness obtained from a patient chart review. RESULTS: Histologic features associated with Cryptosporidium infection include a neutrophilic infiltrate in the stomach, villus blunting in the duodenum, cryptitis and epithelial apoptosis in the colon, and reactive epithelial changes in the stomach and duodenum. The nature and intensity of the inflammatory response varied widely; however, duodenal biopsies from a subset of patients (37%) revealed marked acute inflammation that was associated with concomitant cytomegalovirus infection. Although duodenal infection was common (93% of individuals), infection of other sites was variable (gastric cryptosporidiosis in 40% and colonic cryptosporidiosis in 74%). Widespread infection of the intestinal tract, which included both the large and small intestine, was associated with the most severe diarrheal illness. CONCLUSIONS: Cryptosporidium infection produces histologic evidence of gastrointestinal mucosal injury. The inflammatory response to the infection is variable, and may be modified by copathogens such as cytomegalovirus. The clinical manifestations are influenced, in part, by the anatomic distribution of the infection, with extensive infections involving both small and large intestines producing the most severe illness.

The effectiveness of a consultation. Compliance with initial recommendations.

To identify the attributes of an effective consultation, 202 general medicine consultations were analyzed to assess the extent of compliance with the consultant's initial recommendations. The overall compliance rate was 77 percent. Compliance decreased as the number of recommendations increased. The consultant made more recommendations among patients who had more complex and more severe illnesses. Although compliance did increase significantly in severely ill patients (p less than 0.01), with each severity level, compliance was higher when five or fewer recommendations were made. In fact, compliance decreased from 96 percent in severely ill patients with small consultation lists to 79 percent in those with large lists. Compliance was greatest with recommendations involving medications and least with those requiring direct physician and nursing action. Multivariate analysis confirmed that clinical severity of the patients' illnesses and the type and number of recommendations were all predictors of compliance. To promote overall compliance, consultants should limit the total number of recommendations in their initial consultation to five or fewer, focusing on issues central to current patient care. This is especially true in severely ill patients. Since recommendations that must be implemented by physicians or nurses have a lower compliance rate, consultants must carefully follow up those requests.

General medicine consultation. Lessons from a clinical service.

The 564 consultations performed by a general medicine consultation service during its first year were analyzed in order to provide a concrete definition of this new academic domain. Of the consultations, 52 percent were for patients on the surgical service. Among these patients, the most common reason for consultation was the preoperative management of chronic illness, specifically, hypertension, diabetes, and angina; 47 percent of such patients had two or more chronic illnesses. The service recommended cancellation of planned surgery in 2 percent and postponement in 9 percent of the 210 patients seen preoperatively. Patients on the psychiatric service accounted for 47 percent of the consultations. In this group, diagnostic issues were the most common reasons for consultation, that is, abdominal pain, dementia, and the suspicion of thyroid disease. Only 12 percent of the patients were seen for prognostic reasons, usually related to the planned use of electroconvulsive therapy or tricyclic antidepressants. The service was evaluated by the referring physicians who rated the service favorably on its "mechanics," as well as on its qualitative performance. However, complaints of triviality were voiced when the average length of the list of recommendations seemed disproportionate to the complexity of the problems. The service was also evaluated by the residents who had provided consultations. From their perspective, the service was more successful in teaching the "art" of consultation than the "science." This experience provides an operational definition of the work facing a general medicine consultation service as well as data useful in focusing future educational programs and research efforts.

Environmental sources of Cryptosporidium in an urban slum in northeastern Brazil.

Cryptosporidium is an important cause of diarrheal disease in children worldwide. To elucidate the environmental sources of this parasite, we selected an urban slum in Fortaleza, Brazil, a community with a known high incidence of cryptosporidiosis, and examined both stool smears from household animals (n = 127) and filtrates from local water sources (n = 18) for Cryptosporidium oocysts. Because previous work in this community has demonstrated the seasonal nature of human infection with Cryptosporidium, collections were made separately for the dry and rainy seasons. Of the 64 stools collected during the dry season (September-December 1990), four (6.3%) were positive by acid-fast staining for Cryptosporidium. Of the 63 rainy season samples (March-May 1991), nine (14.3%) were positive. Overall, oocysts were detected in 13 (10.2%) of 127 animal stool samples. Freshwater samples were obtained from a variety of sources including open and closed wells, and running city water and then processed. Four of 18 samples (22.2%), including a sample from city water were positive by at least one of two staining techniques (acid-fast and immunofluorescence). In summary, animals may serve as a reservoir of Cryptosporidium, with potential for the contamination of immediate household water sources. These findings may help to explain the high incidence of cryptosporidiosis among infants in this impoverished community.

Seroepidemiology of Entamoeba histolytica in a slum in northeastern Brazil.

Infection with the human pathogenic parasite Entamoeba histolytica has not been well-characterized in northeastern Brazil. In this study, the prevalence of E. histolytica infection in a slum in northeastern Brazil was assayed using an enzyme-linked immunosorbent assay (ELISA) for antibodies against the galactose/N-acetyl-D-galactosamine (Gal/GalNAc)-inhibitable adherence lectin of E. histolytica. Sera from a total of 335 individuals were examined for anti-Gal/GalNAc lectin antibodies. The overall seropositivity was 24.7%; 29.4% of females and 19.4% of males were positive. Among different age groups there was a peak of 40% positivity in the 6-14-year-old age group. There was also familial clustering of seropositivity. To examine colonization, stool samples from 155 people were examined microscopically for the presence of the parasite. Fourteen of 155 stools (9.0%) were identified as containing E. histolytica or nonpathogenic E. dispar. These 14 positive stools were analyzed with an ELISA that detects Gal/GalNAc lectin antigen and can distinguish between E. histolytica and E. dispar. Four stools (29%) were positive for E. histolytica and the remaining 10 were identified as E. dispar-positive. Although the overall colonization rate by microscopy was only 9%, with a third identified as E. histolytica, up to 40% of older children develop serologic evidence of having experienced pathogenic E. histolytica infection. The results of this study demonstrate that this community in northeastern Brazil is highly endemic for E. histolytica with infection rates similar to other developing nations.

The toxins of Bacteroides fragilis.

Bacteroides fragilis are both key commensals and important human pathogens. Particular strains of B. fragilis, termed enterotoxigenic B. fragilis (ETBF), are recently identified enteric pathogens of children and adults. These strains are distinguished by secretion of a 20kDa metalloprotease toxin (B. fragilis toxin or BFT), the first recognized and only established toxin to date for B. fragilis. Three isotypes of BFT are encoded by distinct bft loci contained within a 6kb chromosomal region unique to ETBF strains termed the B. fragilis pathogenicity island (BfPAI). Experimental studies have suggested that the cellular target for BFT is E-cadherin, the primary protein of the zonula adherens. It is postulated that BFT cleavage of E-cadherin is critical in precipitating the intracellular events culminating in the two established activities for BFT; namely, stimulation of secretion in ligated intestinal segments in several animal species and alteration of cellular morphology only in epithelial cells that retain the ability to polarize and form a tight junctional complex. Future studies will be directed to characterizing in greater detail both the molecular genetics of the BFT toxin and the precise steps in its cellular mechanism of action.

Molecular physiology and pathophysiology of tight junctions V. assault of the tight junction by enteric pathogens.

Studies of the impact of enteric pathogens and their virulence factors on the proteins comprising the tight junction and zonula adherens offer a novel approach to dissection of tight junctional complex regulation. Most studies to date provide only tantalizing clues that select pathogens may indeed assault the tight junctional complex. Information on critical human pathogens such as Campylobacter jejuni and Shigella and Salmonella subspecies is lacking. Mechanistic studies are currently sparse, but available results on pathogenic Escherichia coli and specific virulence factors such as the Rho-modifying and protease bacterial toxins indicate four major mechanisms by which these pathogens may act: 1) direct cleavage of tight junctional structural proteins; 2) modification of the actin cytoskeleton; 3) activation of cellular signal transduction; and 4) triggering transmigration of polymorphonuclear cells across the epithelial cell barrier. New therapeutics may evolve from detailed studies of these pathogens and the cellular processes and proteins they disrupt.

Detection of toxin production by Bacteroides fragilis: assay development and screening of extraintestinal clinical isolates.

Strains of Bacteroides fragilis that produce an extracellular 20-kD heat-labile toxin have been epidemiologically associated with diarrheal disease. To standardize detection of the B. fragilis toxin (BFT), a detailed description of the methods for identifying BFT production by B. fragilis strains is reported. To further study the role of toxigenic strains of B. fragilis in clinical disease, extraintestinal clinical isolates that were recovered at an urban east-cost hospital were evaluated. Four (6.2%) of 65 isolates produced BFT. Three cases in which BFT-producing B. fragilis were isolated from extraintestinal sites are reported. In contrast to studies from Japan that have reported BFT production by 23.4% of extraintestinal B. fragilis isolates, we conclude that BFT-producing strains of B. fragilis are infrequently associated with extraintestinal B. fragilis disease at our institution. Additional study will be necessary to better define the importance of BFT production in human disease due to B. fragilis.

The pathogenesis of cryptosporidiosis.

Human infection with the protozoan parasite Cryptosporidium parvum has recently emerged as a global public health problem. Although infection is unrelenting in patients classically regarded as immunocompromised, a tantalizing observation is that infection with this parasite results in both acute self-limited as well as chronic diarrhea in young children. Recent data have begun to elucidate multiple potential mechanisms by which parasitism of the intestinal epithelium may yield an intestinal secretory response. However, a central issue for future studies is to understand how Cryptosporidium infection in young children results in such a broad spectrum of clinical presentation. An answer to this question is likely to result through a dual understanding of how systemic or enteric immunity impacts on intestinal secretory responses and how intra-cellular parasitism alters intestinal epithelial cell function and signals the submucosal intestinal compartment. The virulence factors of Cryptosporidium mediating these events need to be identified. Douglas Clark and Cynthia Sears here review the current understanding of the pathogenesis of intestinal secretion in response to Cryptosporidium infection, and discuss key questions requiring additional study.

Bacteroides fragilis toxin rapidly intoxicates human intestinal epithelial cells (HT29/C1) in vitro.

Enterotoxigenic Bacteroides fragilis strains associated with childhood diarrhea produce a 20-kDa protein toxin (BFT). Purified BFT causes striking morphologic changes in subconfluent human colonic epithelial cells (HT29/C1). In a 3-h HT29/C1 cell assay, the estimated half-maximal effective concentration of BFT was 12.5 pM, and morphologic effects were detectable as early as 30 min and nearly complete by 1.5 h. Concentrations as low as 0.5 pM could also cause intoxication, but morphologic changes were detectable only when the assay was extended to 18 h. The onset of this intoxication was concentration dependent and rapid, occurring within minutes (<7 min at 0.25 nM, <2 min at 2.5 nM). Notably, the onset of intoxication at 37 degrees C became irreversible to washing within 2 min after exposure to BFT. Morphologic changes were completely inhibited by treatment of HT29/C1 cells with BFT at 4 degrees C but could be demonstrated by subsequent warming to temperatures of 15 degrees C or higher after washing. The time required for the association of BFT with HT29/C1 cells at 4 degrees C was inversely correlated with concentration. Inhibitors of endosomal and Golgi trafficking (NH4Cl and brefeldin A) prevented the intoxication of HT29/C1 cells by Clostridium difficile toxin A and cholera toxin, respectively, but not by BFT. Agents altering microtubule structure did not affect the cellular activity of BFT. These data indicate that a purified toxin from B. fragilis strains associated with diarrhea rapidly and irreversibly intoxicates human intestinal epithelial cells (HT29/C1) in a concentration- and temperature-dependent manner and that the process of intoxication may not involve internalization mechanisms utilizing microtubules or sensitive to pH or brefeldin A.

Food poisoning syndromes.

Despite our society's advances in sanitation, food preservation, and hygiene, the prevalence of foodborne disease remains high (12.6 million cases per year in the United States). Although there is a constant need for education of food handlers and consumers, there is also a need for continued vigilant monitoring of coastal waters, meat packing facilities, and imported foods. As long as antibiotics are used in poultry and cattle feeds, one can expect the incidence of antibiotic-resistant foodborne pathogens to rise. There are several promising areas of research in the field of foodborne illnesses. Molecular biologists are actively characterizing the genes that enable invasive enteric pathogens such as Salmonella and Yersinia to enter tissues, and the bacterial toxins associated with secretory diarrheas continue to be the subject of intense scrutiny. Epidemiologists are implementing new techniques such as DNA fingerprinting and multilocus enzyme electrophoresis for tracing pathogens in disease outbreaks. Similarly, the use of computers in the food industry facilitates the tracing of contaminated foods. Although the rates of foodborne illness may not decrease significantly during the next decade, we can expect more rapid identification and tracing of outbreaks as well as an improved understanding of the pathogenesis of the foodborne diseases.

AIDS enteropathy.

Chronic diarrhea is one of the hallmarks of advanced human immunodeficiency virus (HIV) disease. The symptoms of this complication are troublesome, have a significant impact on the patient's quality of life, and in severe cases can lead to extreme abnormalities in fluids and electrolytes and can even cause death. The workup for AIDS-associated diarrhea is often frustrating and frequently unrewarding. However, during the last 10 years, much has been learned about the causes of diarrhea; while treatment is still often ineffective, some advances have been made. Dr. John G. Bartlett and his colleagues in the Department of Medicine at Johns Hopkins University School of Medicine have been responsible for many of these advances. In this AIDS Commentary, these experts discuss recent advances that have enhanced our understanding of chronic diarrhea in HIV-infected persons and offer their recommendations for the most efficient and effective approach to managing these patients.

Human intestinal epithelial cells swell and demonstrate actin rearrangement in response to the metalloprotease toxin of Bacteroides fragilis.

Enterotoxigenic Bacteroides fragilis (ETBF) cells produce a 20-kDa heat-labile metalloprotease toxin which is potentially important in the pathogenesis of diarrhea associated with this infection. Previous studies indicate that subconfluent HT29/C1 cells treated with the B. fragilis toxin (BFT) develop morphologic changes with dissolution of tight clusters and apparent swelling. Such alterations suggest toxin-stimulated reorganization of the cellular cytoskeleton. The purpose of the current study was to evaluate the effect of BFT on actin microfilaments (F-actin) and cell volume. As assessed by fluorescent phallicidin staining which detects F-actin, BFT treatment of HT29/C1 cells resulted in redistribution of F-actin with loss of stress fibers, a floccular staining pattern, and cellular membrane blebbing without quantitative changes in F-actin fluorescence intensity. The F-actin redistribution was time and concentration dependent. In contrast to the cell shrinkage observed in response to the F-actin-depolymerizing agents cytochalasin D and Clostridium difficile toxin A, BFT stimulated an increase in HT29/C1 cell volume of 10 to 25% (compared with control cells) over a 24-h time course. Only 10 to 30 ng of BFT per ml was necessary to stimulate a maximal increase in HT29/C1 cell volume. The effect of BFT on cell volume was persistent and dependent on the proteolytic activity of BFT. In agreement with cell viability assays indicating that BFT did not injure HT29/C1 cells, intoxicated cells exhibited regulatory volume decrease, suggesting that toxin-treated cells remain physiologically dynamic. We conclude that BFT acts on the intestinal epithelial cell cytoskeleton to alter F-actin structure and to stimulate an increase in HT29/C1 cell volume. Although these two activities of BFT appear to be linked, the precise sequence of cellular events following intoxication of HT29/C1 cells with BFT remains unclear. We hypothesize that these F-actin and cell volume changes may lead to an alteration in tight junction function in the polarized intestinal epithelium, contributing to the pathogenesis of diarrhea in ETBF infections.

Bacteroides fragilis toxin rearranges the actin cytoskeleton of HT29/C1 cells without direct proteolysis of actin or decrease in F-actin content.

Enterotoxigenic strains of B. fragilis associated with childhood diarrhea produce a 20 kD zinc metalloprotease toxin (BFT). BFT is reported to cleave G-actin in vitro and also causes dramatic rounding and rearrangement of the F-actin cytoskeleton in human intestinal epithelial cell lines (HT29) and HT29/C1). To test the hypothesis that the proteolysis of cellular actin by BFT in vivo may contribute to these alterations in morphology and cytoskeletal architecture, we assessed the F-actin content and the arrangement of the F- and G-actin cytoskeleton in BFT-treated HT29/C1 cells by spectrofluorimetry, confocal microscopy, and immunoblotting. BFT-treated cells were compared to cells treated with C. difficile toxin A (CDA) or cytochalasin D. Using spectrofluorimetric quantification, the F-actin content of BFT- and cytochalasin D-treated cells was unchanged in contrast to a significant decrease in CDA-treated cells. By confocal microscopy, the arrangement of F- and G-actin in all treated cells was markedly different than control cells. There was no change in the immunoblotting pattern of actin in the Triton-soluble or -insoluble cellular fractions of BFT-treated HT29/C1 cells. We conclude that BFT alters the F- and G-actin cytoskeletal architecture of HT29/C1 cells without direct proteolysis of actin or decrease in F-actin content.