CGVD workshop: should Haemophilus influenzae type B conjugate vaccines be introduced into the EPI?
Explore the source record for details and available documents.
Bacterial Vaccines↗
Biomedical subjects
G T Keusch
Publications and source records attributed to G T Keusch.
Interleukin-1 beta (IL-1 beta), IL-1 receptor antagonist, and TNF alpha production in whole blood.
The ability of an individual to mount defense responses to infection depend in part on the capacity to produce cytokines such as interleukin 1 (IL-1) and tumor necrosis factor (TNF). The specialized equipment, labor intensity, and sterile practice required for the standard in vitro evaluation of cytokine production can make such evaluation impractical in some clinical situations. We report a method for stimulating whole blood to produce cytokines that can be implemented in laboratories without tissue culture facilities and requires minimal sample preparation. IL-1 beta and TNF alpha production in whole blood samples was stimulated with endotoxin and/or phytohemagglutinin in standard EDTA-containing vacuum collection tubes. After incubation, plasma was removed and frozen for later assay. Comparison of this whole blood method with isolated mononuclear cell cultures indicated a significant correlation for IL-1 beta production (r = 0.746, P = 0.005). This technique also produced the newly described cytokine, IL-1 receptor antagonist. We conclude that the whole blood method is an acceptable alternative to isolated cell culture methods for measuring IL-1 beta in situations that preclude the standard in vitro approach.
Adult↗
Shiga toxin: biochemistry, genetics, mode of action, and role in pathogenesis.
Explore the source record for details and available documents.
Bacterial Toxins↗
Persistent diarrhea associated with AIDS.
Chronic diarrhea and wasting are very common manifestations of AIDS in adults in developing countries. Etiologic studies show that protozoa (including Cryptosporidium parvum, Isospora belli, and Enterocytozoon bieniusi) and Mycobacterium avium-intracellulara are the most frequently identified pathogens. Limited data in children suggest that common enteric pathogens are equally as likely in HIV+ and HIV- babies. Preliminary analysis of an ongoing longitudinal study of 469 babies born to mothers with known HIV serostatus in Kinshasa, Zaire, reveals progression of acute to persistent diarrhea is six times greater in HIV+ compared to HIV- babies, and 3.5 times greater in HIV- babies born of HIV+ mothers in comparison to HIV- babies with HIV- mothers. HIV+ babies were also at greater risk than HIV- babies to have recurrent episodes of diarrhea (RR = 2.3). Fifty percent of the deaths were due to acute or persistent diarrhea, and were strongly associated with HIV infection. Efforts to improve child survival in AIDS infected populations will need to address HIV infections in both mothers and infants.
Acquired Immunodeficiency Syndrome↗
Identification and partial purification of a lectin on the surface of the sporozoite of Cryptosporidium parvum.
A human-derived isolate of Cryptosporidium parvum from a symptomatic patient with the acquired immunodeficiency syndrome was expanded in vivo by infecting a neonatal calf with 10(8) oocysts. Sporozoites were isolated from 4 x 10(10) oocysts harvested from this single infection, and the characteristics of mixed hemagglutination (HA) with rabbit erythrocytes were determined. Sporozoite HA was inhibited by bovine submaxillary mucin (BSM), hog gastric mucin, and orosomucoid, but not by simple sugars, including sialic acid. Carbohydrate-inhibitable HA (lectin) activity increased with sporozoite lysis and was associated with the sporozoite membrane fractions. The ability of intact sporozoites to form rosettes around erythrocytes indicates that the HA (lectin) is, at least in part, present on the parasite surface. Hemagglutination (lectin) activity was partially purified from sporozoite lysates by affinity chromatography with BSM coupled to Sepharose-4B. Best elution was obtained with ethylene glycol and NaCl, which resulted in enrichment of 6 bands compared to the crude starting lysate (Mr = 60, 24, 22, 20, and 15 kDa and a 40-kDa doublet). Our results indicate that an HA (lectin) activity is present on the surface of intact sporozoites where it could play a role in cell-to-cell interactions with eukaryotic targets.
Acquired Immunodeficiency Syndrome↗
Crystallization of the B chain of Shiga-like toxin I from Escherichia coli.
Shiga-like toxin I (SLT-I) is produced by several pathogenic strains of Escherichia coli associated with diarrheal disease. The toxin consists of an A chain, which attacks eukaryotic ribosomes, inhibiting protein synthesis, and multiple copies of a 69 amino acid B chain. The B subunit mediates cell binding and uptake through its interactions with cell surface carbohydrate moieties. Here we report that the B chain has been crystallized in a form suitable for high-resolution X-ray analysis. The space group is P2(1)2(1)2(1), with a = 56.2 A, b = 59.9 A and c = 102.5 A. A rotation function using three-dimensional diffraction data suggests that the asymmetric unit is a tetramer.
Bacterial Toxins↗
In vitro encystation of Giardia lamblia: large-scale production of in vitro cysts and strain and clone differences in encystation efficiency.
A method for obtaining large numbers of Giardia lamblia cysts in vitro was developed based on modification of earlier methods of in vitro encystation. Maximal numbers of cysts were obtained by growing trophozoites to confluence in TYI-S-33 growth medium containing 0.5 mg/ml of bovine bile, followed by incubation in medium containing 10 mg/ml of bovine bile, at pH 7.8 for 96 h at 37 C. Up to 4 x 10(5) cysts were obtained per milliliter of encystation medium. Cysts thus obtained were similar in structure to those in vivo, were resistant to hypotonic lysis, and reacted with a cyst-specific monoclonal antibody. Further modification of this method by returning the trophozoites to growth medium after 24 hr of exposure to encystation medium resulted in production of cysts that were shown to be viable by fluorogenic dye staining and ability to excyst. This method was scaled up using roller bottles, which resulted in production of up to 1.6 x 10(8) cysts per roller bottle. In addition, of 4 strains tested, the LT strain yielded the highest number of cysts. Of 4 clones of the WB strain, clone A consistently produced the largest number of cysts.
Animals↗
Vitamin A supplements--too good not to be true.
Explore the source record for details and available documents.
Child↗
Induction of a phosphomannosyl binding lectin activity in Giardia.
Giardia lamblia, a protozoan parasite that causes widespread diarrheal disease, expresses a surface membrane associated lectin, taglin, which is specifically activated by limited proteolysis with trypsin, a protease that is present in abundance at the site of infection. When activated, taglin agglutinates enterocytes which are the cells to which the parasite adheres in vivo, and in addition, binds to isolated brush border membranes of these cells. These findings suggest that this lectin may be involved in the host-parasite interaction. Taglin is most specific for terminal phosphomannosyl residues and its binding to red cells is mediated by cell surface phosphate residues. Hemagglutinating activity induced by taglin is most active at pH 6.5 and is dependent on divalent cations. A monoclonal antibody to taglin reacts with the surface membrane of live trophozoites and recognizes a protein of 28/30 kDa in lysates of Giardia trophozoites, by immunoblotting. This finding is confirmed by direct demonstration of lectin activity by erythrocyte binding to proteins electroblotted to nitrocellulose, which revealed specific red cell binding to giardial protein bands in the same molecular weight range as those recognized by the monoclonal antibody.
Animals↗
A comparison of HEp-2 cell invasion by enteropathogenic and enteroinvasive Escherichia coli.
In order to further characterize cellular invasion by enteropathogenic Escherichia coli (EPEC), we compared invasion of HEp-2 cells by EPEC and enteroinvasive E. coli (EIEC). We used a gentamicin HEp-2 cell assay and measured bacterial recovery under conditions of varying incubation time and temperature, and in the presence or absence of inhibitors of cellular microfilaments and microtubules. We found that, unlike EIEC, EPEC did not rapidly multiply within HEp-2 cell but invaded well at 32 degrees C. While microfilament inhibitors reduced invasion by both EIEC and EPEC, microtubule inhibitors reduced invasion by EPEC only. These results suggest that EPEC and EIEC differ in their mechanisms of epithelial cell invasion.
Actin Cytoskeleton↗
Enzyme-linked immunosorbent assay for Shiga toxin and Shiga-like toxin II using P1 glycoprotein from hydatid cysts.
Shiga toxin from Shigella dysenteriae type 1 strains and Shiga-like toxins (SLT) I and II from Escherichia coli bind to terminal alpha-D-Galp-(1----4)-D-Galp containing glycolipids. Hydatid cyst fluid isolated from sheep infected with Echinococcus granulosus contains a glycoprotein (P1gp) with a terminal alpha-D-Galp-(1----4)-D-Galp disaccharide. Preparations of P1gp were shown to interact directly with Shiga toxin and to inhibit the binding and cytotoxicity of Shiga toxin to HeLa cells. A sandwich ELISA was developed using preparations of P1gp as the toxin capture molecule, which, with an appropriate polyclonal antibody, was capable of detecting as little as 80 pg/well Shiga toxin and 132 pg/well SLT-II. Thus, the P1gp-toxin interaction forms the basis for a simple antigen-capture ELISA that may be useful clinically for the rapid detection and quantitation of Shiga and Shiga-like toxins.
Animals↗
Identification of developmentally regulated Giardia lamblia cyst antigens using GCSA-1, a cyst-specific monoclonal antibody.
GCSA-1, a monoclonal antibody raised against cysts generated in vitro was shown to be Giardia cyst-specific by immunoblot analysis and immunofluorescence. GCSA-1 recognized four polypeptides ranging from 29-45 kD present in the cyst wall. These antigens appeared within eight hours of exposure of trophozoites to encystation medium and were shown to be synthesized by encysting parasites by means of metabolic labelling with [35S]-cysteine. Trophozoites were not stained by the antibody. GCSA-1 also reacted with in vivo cysts obtained from faeces of infected humans, gerbils and mice. These data demonstrate that the determinants recognized by GCSA-1 are early cyst antigens which are developmentally regulated and conserved components of the cyst wall. The actual role of the antigens detected by GCSA-1 in encystation are unknown, but they represent a potential target for strategies directed at inhibiting this process.
Animals↗
Micronutrients and susceptibility to infection.
Iron deficiency and vitamin A deficiency are both reported to predispose to infection morbidity and to mortality. In both situations, however, the data are insufficient to draw firm conclusions, primarily owing to flaws in the design of the studies. To be sure, these are difficult studies to carry out, and the investigators whose reports have been reviewed should be praised rather than adversely criticized for their efforts. In the case of iron deficiency, there is a further complication in interpretation, that is the suggestion that iron deficiency states may be protective and that conditions of iron overload may predispose to infection. These concepts appear to pertain most convincingly to malaria and Yersinia infections, and to situations in which iron dextran is given parenterally to young children in the first few months of life. There are still two few data to suggest that oral iron is harmful and there is no reason at present that it should not be employed for the correction of iron deficiency anemia.
Disease Susceptibility↗
Construction and analysis of TnphoA mutants of enteropathogenic Escherichia coli unable to invade HEp-2 cells.
Enteropathogenic Escherichia coli (EPEC) strains have recently been shown to invade tissue culture cells. We describe a set of 22 Tn5 IS50L::phoA (TnphoA) insertion mutants of EPEC strain E2348-69 that are unable to invade HEp-2 cells. Each mutant was tested for the ability to adhere to and to induce the polymerization of actin in HEp-2 cells. Southern hybridization of plasmid and total DNA of each strain was performed to determine the location of each TnphoA insert, and each TnphoA insert along with flanking EPEC sequences was cloned. These studies resulted in the grouping of the mutants into five main categories. These include strains with plasmid and chromosomal insertions that alter adherence, chromosomal insertions that alter the ability to induce actin polymerization, and chromosomal insertions that do not affect adherence or actin polymerization. These studies indicate that genes affecting EPEC adherence may be located on both the plasmid and chromosome, that several genes are involved in the induction of actin polymerization in epithelial cells, and that EPEC invasion is a complex process involving multiple genetic loci.
Actin Cytoskeleton↗
Nutritional aspects of AIDS.
Explore the source record for details and available documents.
Acquired Immunodeficiency Syndrome↗
Dissociation of immunoregulatory function of blood monocytes from maturational state and expression of interleukin-1 in humans chronically infected with Schistosoma mansoni.
We investigated the immunoregulatory function and properties of monocytes in 54 S. mansoni infected Egyptians (40-3,840 S. mansoni eggs excreted per gram of stool) 13-35 years of age. Adherent cell-mediated suppression was found in 11 of 36 patients. Cytochemical studies and reactivity with monoclonal antibodies directed at differentiation markers failed to show alterations in the distribution or maturation of monocytes. Peripheral blood mononuclear cells (PBMC) from these individuals produced lower levels of interleukin-1 (IL-1) in response to bacterial lipopolysaccharide and Staphylococcus albus stimulation as compared to the other S. mansoni infected individuals. Overall, IL-1 production by PBMC stimulated with LPS or Staphylococcus albus was higher in infected individuals compared to uninfected controls and correlated with intensity of infection (r = 0.41, P = 0.002 for LPS; r = 0.45, P = 0.006 for S. albus). IL-1 expression by monocytes from individuals with heavy S. mansoni infection exceeded that of subjects with light infection. The intensity-related increase in IL-1 expression did not correlate with the maturational state or the immunoregulatory properties of the monocytes.
Adolescent↗
Quantitation of the rabbit intestinal glycolipid receptor for Shiga toxin. Further evidence for the developmental regulation of globotriaosylceramide in microvillus membranes.
Shiga toxin, produced by Shigella dysenteriae 1, causes enterotoxic, cytotoxic, and neurotoxic effects, which may be mediated by a glycolipid receptor, globotriaosylceramide, Gb3. To study the relationship of this receptor and toxin effects, globotriaosylceramide was quantitated and further characterized in rabbit small intestinal microvillus membranes at various ages. Glycolipids were extracted from rabbit microvillus membranes, purified on Unisil columns, and quantitated by high-performance liquid chromatography. The major glycolipid peaks were hydroxylated fatty acid-containing glucosylceramide, lactosylceramide, and globotriaosylceramide. There was a marked increase of globotriaosylceramide levels with age, ranging from 0.02 to 16.2 pmol/micrograms microvillus membrane protein in neonates and adults, respectively. The globotriaosylceramide peak was susceptible to alpha-galactosidase treatment, which produced an elevation in the lactosylceramide peak, but markedly reduced globotriaosylceramide content in 34-day-old rabbits. Binding of iodinated Shiga toxin to globotriaosylceramide was documented on high-performance thin-layer chromatography plates by autoradiography. The glycolipid receptor for Shiga toxin in rabbit microvillus membranes is thus a hydroxylated fatty acid-containing globotriaosylceramide. This moiety is virtually absent in neonates and gradually increases with age. Quantitative differences in globotriaosylceramide may be the underlying basis for the age-specific differences in functional responsiveness of rabbit intestinal tissue to Shiga toxin.
Animals↗
Pathogenesis of Shigella diarrhea. XIV. Analysis of Shiga toxin receptors on cloned HeLa cells.
Binding kinetics of Shiga toxin to HeLa CCL-2 cells and to cell lines cloned by limiting dilutions were determined. Lines with a wide range of sensitivity to Shiga toxin were obtained. Binding data, analyzed by a computer-based Scatchard model program, revealed two classes of binding sites, one of low affinity and high capacity and one of high affinity and low capacity. The number of high affinity, but not low affinity, sites present on the clones correlated with their sensitivity to toxin. Tunicamycin-treated CCL-2 cells became resistant to Shiga toxin in parallel with a reduction in the capacity of the high-affinity site. Cell content of Gb3, the glycolipid receptor for Shiga toxin, decreased as the sensitivity of the cells diminished. These data show that a minority of Shiga toxin binding sites of HeLa cells are involved in the cytotoxic response and suggest that Gb3 is the high-affinity functional cytotoxin receptor.
Bacterial Toxins↗