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Biomedical subjects

C L Sears

Publications and source records attributed to C L Sears.

At least 19 recordsLinked to original sources
PubMed2001

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.

Animals↗
PubMed2001

A longitudinal study of Giardia lamblia infection in north-east Brazilian children.

OBJECTIVE: To evaluate the epidemiology of Giardia lamblia infection, investigate factors which might be associated with clinical manifestations and recurrence, and examine the role of copathogens in disease course. METHODS: Prospective 4-year cohort study of children born in an urban slum in north-eastern Brazil. RESULTS: Of 157 children followed for > or = 3 months, 43 (27.4%) were infected with Giardia. The organism was identified in 8.8% of all stool specimens, and although found with similar frequency in non-diarrhoeal (7.4%) and diarrhoeal stools (9.7%), was more common in children with persistent (20.6%) than acute diarrhoea (7.6%, P=0.002). Recurrent or relapsing infections were common (46%). Children with symptomatic infections had significantly lower weight-for-age and height-for-age than asymptomatic children. Copathogens were not associated with disease course. CONCLUSION With its protean clinical manifestations, Giardia may be associated with substantial morbidity amongst children in Brazil.

Animals↗
PubMed2000

Persistent diarrhea signals a critical period of increased diarrhea burdens and nutritional shortfalls: a prospective cohort study among children in northeastern Brazil.

Persistent diarrhea (PD; duration >/=14 days) is a growing part of the global burden of diarrheal diseases. A 45-month prospective cohort study (with illness, nutritional, and microbiologic surveillance) was conducted in a shantytown in northeastern Brazil, to elucidate the epidemiology, nutritional impact, and causes of PD in early childhood (0-3 years of age). A nested case-control design was used to examine children's diarrhea burden and nutritional status before and after a first PD illness. PD illnesses accounted for 8% of episodes and 34% of days of diarrhea. First PD illnesses were preceded by a doubling of acute diarrhea burdens, were followed by further 2.6-3.5-fold increased diarrhea burdens for 18 months, and were associated with acute weight shortfalls. Exclusively breast-fed children had 8-fold lower diarrhea rates than did weaned children. PD-associated etiologic agents included Cryptosporidium, Giardia, enteric adenoviruses, and enterotoxigenic Escherichia coli. PD signals growth shortfalls and increased diarrhea burdens; children with PD merit extended support, and the illness warrants further study to elucidate its prevention, treatment, and impact.

Bacterial Infections↗
PubMed2000

Vibrio cholerae ACE stimulates Ca(2+)-dependent Cl(-)/HCO(3)(-) secretion in T84 cells in vitro.

ACE, accessory cholera enterotoxin, the third enterotoxin in Vibrio cholerae, has been reported to increase short-circuit current (I(sc)) in rabbit ileum and to cause fluid secretion in ligated rabbit ileal loops. We studied the ACE-induced change in I(sc) and potential difference (PD) in T84 monolayers mounted in modified Ussing chambers, an in vitro model of a Cl(-) secretory cell. ACE added to the apical surface alone stimulated a rapid increase in I(sc) and PD that was concentration dependent and immediately reversed when the toxin was removed. Ion replacement studies established that the current was dependent on Cl(-) and HCO(3)(-). ACE acted synergistically with the Ca(2+)-dependent acetylcholine analog, carbachol, to stimulate secretion in T84 monolayers. In contrast, the secretory response to cAMP or cGMP agonists was not enhanced by ACE. The ACE-stimulated secretion was dependent on extracellular and intracellular Ca(2+) but was not associated with an increase in intracellular cyclic nucleotides. We conclude that the mechanism of secretion by ACE involves Ca(2+) as a second messenger and that this toxin stimulates a novel Ca(2+)-dependent synergy.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

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.

Animals↗
PubMed1999

Longitudinal study of Cryptosporidium infection in children in northeastern Brazil.

A prospective, 4-year cohort study of children born in an urban slum in northeastern Brazil was undertaken to elucidate the epidemiology of Cryptosporidium infection in an endemic setting, describe factors associated with Cryptosporidium-associated persistent diarrhea, and clarify the importance of copathogens in symptomatic cryptosporidiosis. A total of 1476 episodes of diarrhea, accounting for 7581 days of illness (5.25 episodes/child-year), were recorded: of these, 102 episodes (6.9%) were persistent. Cryptosporidium oocysts were identified in 7.4% of all stools, and they were found more frequently in children with persistent diarrhea (16.5%) than in those with acute (8.4%) or no (4.0%) diarrhea (P<.001). Low-birth-weight children and those living in densely crowded subdivisions were at greater risk for symptomatic infection. Disease course was highly variable and was not associated with the presence of copathogens. Recurrent Cryptosporidium infection and relapsing diarrhea associated with it were moderately common. In light of these data, the applicability of the current World Health Organization diarrheal definitions to Cryptosporidium-associated diarrheal episodes may need to be reconsidered.

Analysis of Variance↗
PubMed1999

Identification of a third metalloprotease toxin gene in extraintestinal isolates of Bacteroides fragilis.

To further understand the epidemiology of enterotoxigenic Bacteroides fragilis (ETBF), 89 extraintestinal B. fragilis strains from Seoul, Korea, were examined for secretion of B. fragilis toxin (BFT) by the HT29/C1 biologic assay and for the B. fragilis toxin gene (bft) by colony blot hybridization and PCR. Complete agreement between the three techniques was found. Overall, 34 B. fragilis strains (38%) were identified as ETBF. Eleven of the 34 ETBF strains (32%) expressed a new isoform of BFT (Korea-BFT). This new isoform is more related to BFT-2 than to BFT-1. Like BFT-1 and BFT-2, Korea-BFT cleaves E-cadherin, the zonula adherens protein.

Amino Acid Sequence↗
PubMed1999

Molecular evolution of the pathogenicity island of enterotoxigenic Bacteroides fragilis strains.

Enterotoxigenic Bacteroides fragilis (ETBF) strains, which produce a 20-kDa zinc metalloprotease toxin (BFT), have been associated with diarrheal disease in animals and young children. Studying a collection of ETBF and nontoxigenic B. fragilis (NTBF) strains, we found that bft and a second metalloprotease gene (mpII) are contained in an approximately 6-kb pathogenicity island (termed B. fragilis pathogenicity island or BfPAI) which is present exclusively in all 113 ETBF strains tested (pattern I). Of 191 NTBF strains, 100 (52%) lack both the BfPAI and at least a 12-kb region flanking BfPAI (pattern II), and 82 of 191 NTBF strains (43%) lack the BfPAI but contain the flanking region (pattern III). The nucleotide sequence flanking the left end of the BfPAI revealed a region with the same organization as the mobilization region of the 5-nitroimidazole resistance plasmid pIP417 and the clindamycin resistance plasmid pBFTM10, that is, two mobilization genes (bfmA and bfmB) organized in one operon and a putative origin of transfer (oriT) located in a small, compact region. The region flanking the right end of the BfPAI contains a gene (bfmC) whose predicted protein shares significant identity to the TraD mobilization proteins encoded by plasmids F and R100 from Escherichia coli. Nucleotide sequence analysis of one NTBF pattern III strain (strain I-1345) revealed that bfmB and bfmC are adjacent to each other and separated by a 16-bp GC-rich sequence. Comparison of this sequence with the appropriate sequence of ETBF strain 86-5443-2-2 showed that in this ETBF strain the 16-bp sequence is replaced by the BfPAI. This result defined the BfPAI as being 6,036 bp in length and its precise integration site as being between the bfmB and bfmC stop codons. The G+C content of the BfPAI (35%) and the flanking DNA (47 to 50%) differ greatly from that reported for the B. fragilis chromosome (42%), suggesting that the BfPAI and its flanking region are two distinct genetic elements originating from very different organisms. ETBF strains may have evolved by horizontal transfer of these two genetic elements into a pattern II NTBF strain.

Amino Acid Sequence↗
PubMed1998

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.

AIDS-Related Opportunistic Infections↗
PubMed1998

Bacteroides fragilis enterotoxin cleaves the zonula adherens protein, E-cadherin.

Strains of Bacteroides fragilis associated with diarrheal disease (enterotoxigenic B. fragilis) produce a 20-kDa zinc-dependent metalloprotease toxin (B. fragilis enterotoxin; BFT) that reversibly stimulates chloride secretion and alters tight junctional function in polarized intestinal epithelial cells. BFT alters cellular morphology and physiology most potently and rapidly when placed on the basolateral membrane of epithelial cells, suggesting that the cellular substrate for BFT may be present on this membrane. Herein, we demonstrate that BFT specifically cleaves within 1 min the extracellular domain of the zonula adherens protein, E-cadherin. Cleavage of E-cadherin by BFT is ATP-independent and essential to the morphologic and physiologic activity of BFT. However, the morphologic changes occurring in response to BFT are dependent on target-cell ATP. E-cadherin is shown here to be a cellular substrate for a bacterial toxin and represents the identification of a mechanism of action, cell-surface proteolytic activity, for a bacterial toxin.

Bacteroides fragilis↗
PubMed1998

Cryptosporidiosis in northeastern Brazilian children: association with increased diarrhea morbidity.

To evaluate the impact of Cryptosporidium infection on diarrheal disease burden and nutrition status, a nested case-control study was done among children who were followed from birth in Fortaleza, Brazil. The diarrhea history and growth records of 43 children with a symptomatic diarrhea episode of cryptosporidiosis (case-children) were compared with those of 43 age-matched controls with no history of cryptosporidiosis. After Cryptosporidium infection, case-children < or = 1 year old experienced an excessive and protracted (nearly 2 years) diarrheal disease burden. Case-children < or = 1 year old with no history of diarrhea prior to their Cryptosporidium infection also experienced a subsequent increased diarrheal disease burden with an associated decline in growth. Control subjects experienced no change in their diarrhea burden over time. This study suggests that an episode of symptomatic Cryptosporidium infection in children < or = 1 year of age is a marker for increased diarrhea morbidity.

Age Factors↗
PubMed1998

Cryptosporidiosis in patients with AIDS: correlates of disease and survival.

Although 10%-15% of patients with AIDS in the United States may acquire cryptosporidium infection, little data exist on clinical or histological characteristics that differentiate clinical outcomes. A case-control study of 83 HIV-positive adult patients with cryptosporidiosis was conducted, as was a histopathologic review of data on gastrointestinal biopsy specimens from 30 patients. Four clinical syndromes were identified: chronic diarrhea (36% of patients), choleralike disease (33%), transient diarrhea (15%), and relapsing illness (15%). A multivariate analysis of data for cases and controls revealed that acquiring cryptosporidiosis was associated with the presence of candidal esophagitis (odds ratio [OR], 2.53; P < .002) and Caucasian race (OR, 6.71; P = .0001) but not with sexual orientation. Cases had a significantly shorter duration of survival from the time of diagnosis than did controls (240 vs. 666 days, respectively; P = .0004), which was independent of sex, race, or or injection drug use. Antiretroviral use was protective against disease (OR, 0.072; P = .0001). All four clinical syndromes were represented among the histological data. There was no statistically significant correlation between histological intensity of infection and clinical severity of illness.

AIDS-Related Opportunistic Infections↗
PubMed1997

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.

Actins↗
PubMed1997

Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene.

Strains of Bacteroides fragilis that produce a ca. 20-kDa heat-labile protein toxin (termed B. fragilis toxin [BFT]) have been associated with diarrheal disease of animals and humans. BFT alters the morphology of intestinal epithelial cells both in vitro and in vivo and stimulates secretion in ligated intestinal segments of rats, rabbits, and lambs. Previous genetic and biochemical data indicated that BFT was a metalloprotease which hydrolyzed G (monomeric) actin, gelatin, and azocoll in vitro. In this paper, the cloning and sequencing of the entire B. fragilis toxin gene (bft) from enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 is reported. The bft gene from this ETBF strain consists of one open reading frame of 1,191 nucleotides encoding a predicted 397-residue holotoxin with a calculated molecular weight of 44,493. Comparison of the predicted BFT protein sequence with the N-terminal amino acid sequence of purified BFT indicates that BFT is most probably synthesized by ETBF strains as a preproprotein. These data predict that BFT is processed to yield a biologically active toxin of 186 residues with a molecular mass of 20.7 kDa which is secreted into the culture supernatant. Analysis of the holotoxin sequence predicts a 20-residue amphipathic region at the carboxy terminus of BFT. Thus, in addition to the metalloprotease activity of BFT, the prediction of an amphipathic domain suggests that oligomerization of BFT may permit membrane insertion of the toxin with creation of a transmembrane pore. Comparison of the sequences available for the bft genes from ETBF 86-5443-2-2 and VPI 13784 revealed two regions of reduced homology. Hybridization of oligonucleotide probes specific for each bft to toxigenic B.fragilis strains revealed that 51 and 49% of toxigenic strains contained the 86-5433-2-2 and VPI 13784 bft genes, respectively. No toxigenic strain hybridized with both probes. We propose that these two subtypes of bft be termed bft-1 (VPI 13784) and bft-2 (86-5433-2-2).

Amino Acid Sequence↗
PubMed1997

Cloning, characterization, and chromosomal mapping of a phospholipase (lecithinase) produced by Vibrio cholerae.

Phospholipases are associated with virulence in bacterial diseases. Vibrio cholerae produces a phospholipase (lecithinase), with enzyme production visualized as a zone of clearing around colonies plated on egg yolk agar. The role of phospholipase in gut colonization or disease pathogenesis is unknown. We used the egg yolk agar assay to clone and characterize a gene encoding a phospholipase from V. cholerae El Tor strain E7946. Sequence analysis revealed a 1,254-bp open reading frame (lec) encoding a 418-amino-acid protein with a predicted molecular weight of 47,600. The predicted sequence exhibits DNA homology to other Vibrionaceae phospholipases. A potential signal sequence exists in the predicted amino acid sequence, as does a lipid binding motif found in prokaryotic and eukaryotic phospholipases and lipases. Polyacrylamide gel electrophoresis combined with an egg yolk agarose overlay demonstrated phospholipase activity migrating at a relative molecular weight of 45,000 in preparations of V. cholerae and the Escherichia coli clone. Restriction mapping and Southern blot analysis revealed that lec, hlyA (hemolysin), and hlyC (lipase) are adjacent on the V. cholerae chromosome, and chromosomal digests of several El Tor, classical, and O139 (Bengal) strains demonstrated conservation of this gene arrangement. An in-frame internal deletion of the lec gene was constructed and recombined into the chromosome of attenuated V. cholerae El Tor strain CVD 110. The resulting mutant lacked lecithinase activity on egg yolk agar but had undiminished reactivity in rabbit ligated ileal loop assays.

Amino Acid Sequence↗
PubMed1997

Bacteroides fragilis toxin exhibits polar activity on monolayers of human intestinal epithelial cells (T84 cells) in vitro.

Strains of Bacteroides fragilis associated with diarrhea in children (termed enterotoxigenic B. fragilis, or ETBF) produce a heat-labile ca. 20-kDa protein toxin (BFT). The purpose of this study was to examine the activity of BFT on polarized monolayers of human intestinal epithelial cells (T84 cells). In Ussing chambers, BFT had two effects. First, BFT applied to either the apical or basolateral surfaces of T84 monolayers diminished monolayer resistance. However, the time course, magnitude, and concentration dependency differed when BFT was applied to the apical versus basolateral membranes. Second, only basolateral BFT stimulated a concentration-dependent and short-lived increase in short circuit current (Isc; indicative of C1- secretion). Time course experiments indicated that Isc returned to baseline as resistance continued to decrease, indicating that these two electrophysiologic responses to BFT are distinct. Light microscopic studies of BFT-treated monolayers revealed only localized cellular changes after apical BFT, whereas basolateral BFT rapidly altered the morphology of nearly every cell in the monolayer. Transmission and scanning electron microscopy after basolateral BFT confirmed a striking loss of cellular microvilli and complete dissolution of some tight junctions (zonula occludens) and zonula adherens without loss of desmosomes. The F-actin structure of BFT-treated monolayers (stained with rhodamine-phalloidin) revealed diminished and flocculated staining at the apical tight junctional ring and thickening of F-actin microfilaments in focal contacts at the basolateral monolayer surface compared to those in similarly stained control monolayers. BFT did not injure T84 monolayers, as assessed by lactic dehydrogenase release and protein synthesis assays. These studies indicate that BFT is a nonlethal toxin which acts in a polar manner on T84 monolayers to stimulate C1- secretion and to diminish monolayer resistance by altering the apical F-actin structure of these cells. BFT may contribute to diarrheal disease associated with ETBF infection by altering epithelial barrier function and stimulating C1- secretion.

Actins↗