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M A Karmali

Publications and source records attributed to M A Karmali.

At least 19 recordsLinked to original sources
PubMed1992

Tumor necrosis factor and interleukin-1 induce expression of the verocytotoxin receptor globotriaosylceramide on human endothelial cells: implications for the pathogenesis of the hemolytic uremic syndrome.

The epidemic form of the hemolytic uremic syndrome (HUS), beginning with an acute gastroenteritis, has been associated with a verocytotoxin-producing Escherichia coli infection. The endothelial cell is believed to play an important role in the pathogenesis of HUS. Endothelial cell damage by verocytotoxin-1 (VT-1) in vitro is potentiated by the additional exposure of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha). Preincubation of human umbilical vein endothelial cells (HUVEC) with TNF-alpha resulted in a 10- to 100-fold increase of specific binding sites for 125I-VT-1. Furthermore, interleukin-1 (IL-1), lymphotoxin (TNF-beta), and lipopolysaccharide (LPS) also markedly increase VT-1 binding. Several hours' exposure to TNF-alpha was enough to enhance the number of VT-1 receptors on the endothelial cells for 2 days. The TNF-alpha-induced increase in VT-1 binding could be inhibited by simultaneous addition of the protein synthesis inhibitor cycloheximide. Glycolipid extracts of TNF-alpha-treated cells tested on thin-layer chromatography demonstrated an increase of globotriaosylceramide (GbOse3cer), a functional receptor for VT-1, which suggests that preincubation of human endothelial cells with TNF-alpha leads to an increase in GbOse3cer synthesis in these cells. We conclude from this study that TNF-alpha and IL-1 induce one (or more) enzyme(s) that is (are) rate-limiting in the synthesis of the glycolipid VT-1 receptor, GbOse3cer. These in vitro studies suggest that, in addition to VT-1, inflammatory mediators play an important role in the pathogenesis of HUS.

Bacterial Toxins↗
PubMed1992

Experimental verocytotoxemia in rabbits.

The clinicopathologic effects of intravenously administered purified verocytotoxin 1 (VT1; Shiga-like toxin 1) in 2-kg male rabbits was studied. The 50% lethal dose was 0.2 micrograms of protein per kg of body weight (2 x 10(4) 50% cytotoxic doses per kg). The clinical features included nonbloody diarrhea and a progressive flaccid paresis, usually culminating in death. The histopathology was characterized by edema and hemorrhage in the mucosa and submucosa of the cecum and edema, hemorrhage, and neuronal necrosis in the brain and gray matter of the spinal cord. Thrombotic microangiopathy, the characteristic histopathologic renal lesion in the hemolytic-uremic syndrome, was also found to be the underlying lesion in verocytotoxemic rabbits. To determine the specific distribution of VT1 in rabbit tissues, purified 125I-labelled VT1 was administered intravenously to 20 rabbits (both immunologically naive and VT1-immune rabbits). The highest specific uptake of 125I-VT1 was in the spinal cord, brain, cecum, colon, and small bowel in unimmunized animals but in the liver, spleen, and lungs in immune animals. Immunofluorescent staining of cecal and spinal cord tissues after intravenous administration of VT1 showed evidence of specific vascular endothelial cell binding of the toxin. The striking correlation of the central nervous system and gastrointestinal localization of 125I-VT1 with the sites of known histopathology is consistent with direct toxin-mediated injury to these tissues, initiated by the specific binding of VT1 to the vascular endothelium. We conclude that the vascular damage induced by VT1 in affected rabbit tissues is similar to that seen in the kidneys and other tissues in patients with verocytotoxin-producing Escherichia coli-associated hemolytic-uremic syndrome. This suggests that although the rabbit model fails to replicate human hemolytic-uremic syndrome, it is useful for studying the pathogenesis of the vascular lesions in verocytotoxin-producing E. coli-associated diseases.

Animals↗
PubMed1992

Binding of Pseudomonas cepacia to normal human intestinal mucin and respiratory mucin from patients with cystic fibrosis.

Although not as prevalent as Pseudomonas aeruginosa, Pseudomonas cepacia is another opportunistic pathogen which colonizes the lungs of at least some patients with cystic fibrosis. A subgroup of these patients exhibits the "cepacia syndrome", i.e., a rapid clinical deterioration and death within one year. To investigate potential early sites of bacterial attachment, we have measured the specific binding of P. cepacia isolates from cystic fibrosis (CF) sputa to both CF and non-CF mucins purified from respiratory and intestinal secretions, respectively. As shown in microtiter binding assays, clinical isolates from 19/22 patients were found to bind to both mucins, with the highest specific binding exhibited by isolates from eight patients, seven of whom later died with the cepacia syndrome. No differences were observed in the binding capacity of the two (CF versus non-CF) mucins. Binding was specific, saturable, and not influenced by tetramethylurea, a disruptor of hydrophobic associations. Individual sugars were ineffective as hapten inhibitors, as were several lectins. Mucins treated by reduction/alkylation or chloroform/methanol extraction showed enhanced bacterial binding, findings which were attributed to exposure of underlying binding sites. Deglycosylation procedures indicated that mucin receptors for P. cepacia include N-acetylglucosamine and N-acetylgalactosamine, probably linked together as part of core oligosaccharide structures. P. cepacia isolates also bound to buccal epithelial cells, and mucin partially inhibited the binding of those isolates of P. cepacia that also had the ability to bind to mucin. We speculate that specific binding of P. cepacia to secreted mucins may be an early step in the pathogenesis of the cepacia syndrome.

Bacterial Adhesion↗
PubMed1991

Preparation of VT1 and VT2 hybrid toxins from their purified dissociated subunits. Evidence for B subunit modulation of a subunit function.

Verocytotoxins comprise a family of closely related subunit proteins. Two members of this group, VT1 and the immunologically distinct VT2, have been found to share similar physical properties, and yet several differences in their biological activities have been noted. The subunits of these toxins were separated using urea and isolated by high performance liquid chromatography gel filtration. Reconstituted VT1 and VT2 as well as VT1-A:VT2-B and VT2-A:VT1-B hybrid toxins were then prepared. The B subunit was found to determine cell culture specificity, cytotoxic titer, and antibody neutralizability as determined on Vero and MRC-5 cells. Cross-linking isolated B chains revealed 5 species upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis for both VT1-B and VT2-B. Using an in vitro translation system, both toxin A subunits inhibited protein synthesis at concentrations as low as 4 pM. In glycolipid binding assays, VT1 and VT1-B subunits competed equally on a molar basis with 125I-VT1 for the receptor, globotriaosylceramide, however, a 1000-fold excess of VT2 was required. Ligand analysis of direct VT1 and VT2 receptor binding assays revealed a difference in binding affinity constants (Kd of VT1 = 4.6 x 10(-8) M; VT2 = 3.7 x 10(-7) M).

Animals↗
PubMed1991

Development of verotoxin 2- and verotoxin 2 variant (VT2v)-specific oligonucleotide probes on the basis of the nucleotide sequence of the B cistron of VT2v from Escherichia coli E32511 and B2F1.

We and others have noted that there are serological differences between verotoxin 2 (VT2) (also known as Shiga-like toxin II) produced by Escherichia coli C600(933W) and the VT2 variant (VT2v) produced by strain E32511. Recent reports have described nucleotide sequence differences between the VT2v B subunit cistron of E32511 and B2F1 and that of VT2. We have confirmed the sequence differences and have used them to design oligonucleotide probes which differentiate the B subunit cistron of VT2v from that of VT2. Isolates of VT-producing E. coli obtained from human as well as food and veterinary sources were classified according to the toxin phenotype by using a toxin neutralization assay with VT2-specific monoclonal antibody and VT2v-specific polyclonal antisera. Using the oligonucleotide probes in colony hybridization, we detected 35 of 35 VT2 producers and 16 of 16 VT2v producers. One VT2 producer was falsely identified as containing the VT2v gene. The E32511 strain in our collection hybridized only with the VT2-specific probe. Southern hybridization of radiolabeled oligonucleotide probes showed that strains carried zero to one copy of the VT2 gene and zero to two copies of the VT2v gene. We conclude that colony hybridization with the VT2- and VT2-specific probes is highly predictive of the toxin phenotypes in the clinical isolates described in this study.

Amino Acid Sequence↗
PubMed1990

Clostridium difficile invasion and toxin circulation in fatal pediatric pseudomembranous colitis.

The direct involvement of Clostridium difficile in the lesional tissue of pseudomembranous colitis has not been demonstrated; the organism's effects have been assumed to be strictly toxin mediated. Because C. difficile cytotoxin may be found incidentally in the intestinal lumina of asymptomatic infants, the role of the organism in a variety of pediatric intestinal diseases is uncertain. The authors studied seven cases of fatal pediatric pseudomembranous colitis in which the presence of C. difficile was uniformly demonstrable in lesional tissues with the use of both an intestinal spore stain and a specific immunostain. The patients had either underlying Hirschsprung's disease or hematologic malignancy; the striking pathologic features peculiar to these patients were altered mucosal mucin and immunologic barriers in the former group and neutropenia in the latter. Two patients had demonstrable circulating cytotoxin in serum or ascitic fluid, and C. difficile was identified invading colonic mucosa or submucosa. Such phenomena did not occur in control pediatric patients with multiple other intestinal lesions. Altered host factors may be responsible for the intestinal invasion of C. difficile and its systemic toxin circulation in cases of fatal pediatric pseudomembranous colitis.

Adolescent↗
PubMed1989

Infection by verocytotoxin-producing Escherichia coli.

Verocytotoxin (VT)-producing Escherichia coli (VTEC) are a newly recognized group of enteric pathogens which are increasingly being recognized as common causes of diarrhea in some geographic settings. Outbreak studies indicate that most patients with VTEC infection develop mild uncomplicated diarrhea. However, a significant risk of two serious and potentially life-threatening complications, hemorrhagic colitis and the hemolytic uremic syndrome, makes VTEC infection a public health problem of serious concern. The main reservoirs of VTEC appear to be the intestinal tracts of animals, and foods of animal (especially bovine) origin are probably the principal sources for human infection. The term VT refers to a family of subunit exotoxins with high biological activity. Individual VTEC strains elaborate one or both of at least two serologically distinct, bacteriophage-mediated VTs (VT1 and VT2) which are closely related to Shiga toxin and are thus also referred to as Shiga-like toxins. The holotoxins bind to cells, via their B subunits, to a specific receptor which is probably the glycolipid, globotriosyl ceramide (Gb3). Binding is followed by internalization of the A subunit, which, after it is proteolytically nicked and reduced to the A1 fragment, inhibits protein synthesis in mammalian cells by inactivating 60S ribosomal subunits through selective structural modification of 28S ribosomal ribonucleic acid. The mechanism of VTEC diarrhea is still controversial, and the relative roles of locally acting VT and "attaching and effacing adherence" of VTEC to the mucosa have yet to be resolved. There is increasing evidence that hemolytic uremic syndrome and possibly hemorrhagic colitis result from the systemic action of VT on vascular endothelial cells. The role of antitoxic immunity in preventing the systemic complications of VTEC infection is being explored. Antibiotics appear to be contraindicated in the treatment of VTEC infection. The most common VTEC serotype associated with human disease is O157:H7, but over 50 different VT-positive O:H serotypes have now been identified. The best strategies for diagnosing human VTEC infection include testing for the presence of free VT in fecal filtrates and examining fecal cultures for VTEC by means of deoxyribonucleic acid probes that specify genes encoding VT1 and VT2. Both methods are currently confined to specialized laboratories and await commercial development for wider use. In the meantime, most laboratories should continue to screen for the most common human VTEC serotype, O157:H7, using a sorbitol-containing MacConkey medium.

Bacterial Toxins↗
PubMed1989

Direct isolation of atypical thermophilic Campylobacter species from human feces on selective agar medium.

Campylobacter upsaliensis is the name which has been proposed for a new group of thermophilic campylobacter strains which differ from C. jejuni and C. coli in having a negative or weak catalase reaction. Primary isolation of these strains from human feces has been achieved only by use of filtration techniques. We report here direct isolation of strains corresponding to C. upsaliensis from stools of six children. The strains were isolated on a newly described campylobacter-selective medium. The strains were oxidase positive, hippurate negative, nitrate positive, negative for H2S in triple sugar iron, and susceptible to cephalothin (30-micrograms disk) and nalidixic acid (30-micrograms disk), and they grew at 37 and 43 degrees C, but not at 25 degrees C. The selective medium used was a blood-free, charcoal-based medium consisting of Columbia agar base, activated charcoal, cefoperazone (32 micrograms/ml), vancomycin (20 micrograms/ml), and cycloheximide (100 micrograms/ml). The medium supported the growth of the weakly reacting or catalase-negative strains, with colony counts equivalent to those obtained on antibiotic-free horse blood agar. These strains could not be isolated directly from stool on Skirrow medium, and colony counts confirmed that this medium could not support a low inoculum of these organisms. The clinical significance of these strains is unknown. We conclude that C. upsaliensis can be isolated directly from stool by using a selective medium, without the need for filtration.

Agar↗
PubMed1979

Campylobacter enteritis.

Campylobacter jejuni/coli has recently become recognized as a common bacterial cause of diarrhea. Infection can occur at any age. The usual incubation period of campylobacter enteritis is 2 to 5 days. Fever, diarrhea and abdominal pain are the most common clinical features. The stools frequently contain mucus and, a few days after the onset of symptoms, frank blood. Significant vomiting and dehydration are uncommon. A rapid presumptive laboratory diagnosis may be made during the acute phase of the illness by direct phase-contrast microscopy of stools. Isolation of the organism from stools requires culture in a selective medium containing antibiotics and incubation under reduced oxygen tension at 42 degrees C. The organism persists in the stools of untreated patients for up to 7 weeks following the onset of symptoms. Erythromycin may produce a rapid clinical and bacteriologic cure, and should be used to treat moderately to severely ill patients as well as patients with compromised host defences. The emergence of erythromycin-resistant strains requires close monitoring. The epidemiologic aspects of campylobacter enteritis will be fully understood only when methods become available for differentiating strains of C. jejuni/coli. The historical background and current knowledge of campylobacter enteritis are reviewed in this paper.

Animals↗
PubMed1979

Campylobacter enteritis in children.

In 37 children with Campylobacter enteritis seen over a 6-month period, ages ranged from 2 weeks to 15 years. The sex ratio (male:female) was three:two. Fever, diarrhea, and bloody stools occurred in about 90% of patients. Blood appeared in the stools characteristically 2 to 4 days after onset of symptoms. Over 90% of older children developed abdominal pain. Vomiting was mild and occurred in 30% of patients. Dehydration was not a feature. Infection occurred in all social classes and was not associated with parental occupation, travel, or animal contact. The illness often presented characteristically and a rapid laboratory diagnosis could be made in patients presenting acutely by direct phase-contrast microscopy of stools. The organism persisted in the stools for up to seven weeks in untreated patients, but could no longer be cultured after 48 hours of therapy with erythromycin, to which all strains were highly sensitive. Significant serologic responses were elicited using a serum bactericidal assay. The Skirrow-type selective medium used by us could be improved by increasing the concentration of polymyxin B sulfate to 5 microgram/ml.

Adolescent↗