Henry F. Gomez

Concentration of milk secretory immunoglobulin A against Shigella virulence plasmid-associated antigens as a predictor of symptom status in Shigella-infected breast-fed infants

We conducted a prospective, community-based study of healthy breast-fed Mexican infants to determine the protective effects of anti-Shigella secretory IgA antibodies in milk. Milk samples were collected monthly, and stool culture specimens were obtained weekly and at the time of episodes of diarrhea. Nineteen breast-fed infants were found to have Shigella flexneri, Shigella boydii, or Shigella sonnei in stool samples. Ages of the 10 infants with symptomatic infection and the nine with asymptomatic infection did not differ significantly. Milk samples collected up to 12 weeks before infection were evaluated by enzyme-linked immunosorbent assay for secretory IgA antibodies against lipopolysaccharides of S. flexneri, S. boydii serotype 2, S. sonnei, and virulence plasmid-associated antigens. The geometric mean titers of anti-Shigella antibodies to virulence plasmid-associated antigens in milk received before infection were eightfold higher in infants who remained well than in those in whom diarrhea developed. The significance of milk secretory IgA directed against lipopolysaccharide was less clear. We conclude that human milk protects infants against symptomatic shigella infection when it contains high concentrations of secretory IgA against virulence plasmid-associated antigens.

Incomplete hemolytic-uremic syndrome in Argentinean children with bloody diarrhea☆☆☆★★★

Argentina has an exceptionally high frequency of hemolytic-uremic syndrome (HUS). We sought to define prospectively the role of verocytotoxins (Shiga-like toxins [SLTs]) in 254 Argentinean children with grossly bloody diarrhea during spring and summer. Free fecal SLTs (I/II) and/or DNA probe-positive isolates were found in 99 (39%) of the children. During the follow-up period, HUS developed in 6 patients (4 with evidence of recent SLT infection based on stool studies); another 14 patients had some, but not all, of the abnormalities seen in typical HUS. The development of HUS or incomplete HUS in these children was significantly associated with recent SLT-Escherichia coli infection (p = 0.024). The high incidence of SLT-associated bloody diarrhea in Argentina explains, at least partially, the unusually high frequency of HUS. Our data indicate that incomplete forms of HUS may be common in patients with SLT-associated bloody diarrhea.

Protective Role of Human Lactoferrin Against Invasion of Shigella Flexneri M90t

Lactoferrin is an iron-binding protein found in human mucosal secretions such as milk. A variety of functions have been ascribed to this protein, it appears to contribute to antimicrobial host defense. Still its overall physiological role remains to be defined. We sought to study the role of recombinant human lactoferrin (rhLf) in Shigella infection. Invasion of epithelial cells is essential to the development of bacillary dysentery. Shigella flexneri 5 M90T, a virulent strain, was evaluated in the classic HeLa cell invasion model, in immunoblots, and by transmission electron microscopy, immunofluorescence, and deconvolved microscopy Bacteria not exposed to rhLf were used as controls. We found that rhLf decreased significantly the invasiveness of S. flexneri 5 M90T in a HeLa cell model. The immunoblot data showed that invasion plasmid antigen B (IpaB) was released from the bacteria during incubation with rhLf. Lactoferrin treatment did not directly dissociate the complex of IpaB and IpaC (IpaBC) once the complex had been formed. Furthermore, ferric iron had no effect on release of IpaB. Electron microscopy of rhLf-treated bacteria suggested a reduction in vacuolization of the HeLa cell cytoplasm and decreased number of bacteria within HeLa cells. At 40,000 x magnification the few rhLf-treated Shigella that invaded exhibited a dense ring completely surrounding them. Immunofluorescence and deconvolved microscopy suggested that rhLf-treated bacteria were completely surrounded by a thick layer of actin. The fact that two cell surface functions (invasion and actin-mediated movement) were deranged suggests that rhLf disrupts the integrity of the bacterial outer membrane in which virulence proteins are anchored. The mechanism by which rhLf impairs Shigella invasiveness may be relevant to other enteropathogens that share similar virulence strategies.

Human Milk Lipids Bind Shiga Toxin

Hemolytic uremic syndrome, a serious complication of Shiga toxin-associated diarrhea, is rare before 6 months of age. Immunologic and nonimmunologic factors present in human milk may partially explain this observation. In prior studies, we have demonstrated that human milk contains Gb3, the receptor for the B subunit of Shiga toxin, and also contains secretory IgA (sIgA) against the toxin. We therefore sought to determine the relative importance of milk glycolipid and toxin-specific sIgA in toxin binding. We studied two populations that differed in their frequency of exposure to Shiga toxin. Human milk samples obtained from healthy donors from Boston and Buenos Aires were separated by centrifugation into aqueous (antibody enriched) and cream (glycosphingolipid enriched) fractions. An emulsion of equal volumes of aqueous phase or cream layer of each sample and purified Shiga toxin was incubated, and the amount of free toxin present in each was determined by enzyme immunoassay. The cream layers bound 85%+/-2 (mean +/- SE) (Argentina milk samples) and 86%+/-1 (Boston milk samples) of Shiga toxin. In contrast, the soluble fraction in samples from Buenos Aires, a population expected to frequently have antibodies to Shiga toxin, bound more toxin (48%+/-2) than did this fraction in samples from Boston, an area where toxin exposure is infrequent (30%+/-3) (P < 0.0001). Toxin-binding lipids present in human milk are biologically active and may contribute to the putative protective effect of human milk. In a population frequently exposed to Shiga toxins (Argentina), protection may be due to both immune (sIgA), and nonimmune (lipid) factors present in human milk. In a population infrequently exposed to Shiga toxins, cream fraction-associated glycolipids represent the major toxin binding activity in human milk.

Drug-resistant Salmonella, Shigella, and diarrhea-associated Escherichia coli

Summary Antimicrobial resistance is common and clearly is increasing in frequency in clinically important enteropathogens. Antibiotic resistance often is associated with greater morbidity and mortality because it leads to delay in the initiation of adequate therapy. More prudent use of antibiotics, both for diarrheal disease and other infections, would decrease the frequency of these strains. At present, if a strain of Salmonella species, Shigella species, or diarrhea-associated E coli is multidrug-resistant, it is still likely to be susceptible to third-generation cephalosporins or ciprofloxacin.

AN IDIOTYPIC NATURAL AUTOANTIBODY NETWORK IN HEMOLYTIC UREMIC SYNDROME (HUS).|[dagger]| 1020

Introduction: We hypothesized that immune-mediated events rather than direct toxin injury might be involved in the pathogenesis of HUS associated with shiga-like toxin (SLT) producing E. coli.Purposes: Our objectives were to learn whether antibodies (AB) exist to the SLT glycolipid receptor Gb3, to define the occurrence and relationship between such anti-Gb3 ABs (α-Gb3) and anti-idiotypic ABs to them (α-α-Gb3), and to evaluate children infected with E. coli O157:H7 for these ABs.Methods: ELISAs were used for detecting α-Gb3 orα-α-Gb3 in sera from normal adults [n=15] and from children with E. coli O157:H7 associated with [n=24] or without [n=38] development of HUS. Results: Normals have both α-Gb3 and α-α-Gb3; levels of these ABs are correlated [r=0.9654]. The relationship between these antibodies in E. coli O157:H7 infection is shown below: Table Conclusions:(1) Autoantibodies to Gb3 are normally found. (2) α-Gb3 exists in balance with α-α-Gb3. (3) Children with HUS during the acute phase have significantly decreased levels of these ABs. We hypothesize that these ABs are low because α-α-Gb3 binds to SLT leaving α-Gb3 available to bind to Gb3 of endothelial cells and induce vascular injury. [Supported in part by NIH-PO1 HD-13021]

Protection against Invasion of Various Enteric Pathogens in HeLa Cells by Human Lactoferrin

Protection against Invasion of Various Enteric Pathogens in HeLa Cells by Human Lactoferrin

Lactoferrin Impairs the Initial Steps of Shigella flexneri-induced Phagocytosis in HeLaCells |[diams]| 842

Introduction: Previously we have shown that human lactoferrin decreases significantly the invasiveness of S. flexneri in a HeLa cell model. A unique aspect of Shigella invasion is that the bacteria induces its own uptake by mammalian cells that are not normally phagocytic (bacterium-induced phagocytosis). The earliest step in this process is induction of pseudopods projecting from the HeLa cell surface, a process that can be seen within minutes of exposure of HeLa cells to Shigella.

PROTECTIVE ROLE OF HUMAN LACTOFERRIN AGAINST INVASION OF Shigella flexneri M90T. ♦ 707

Lactoferrin is an iron-binding protein found in human mucosal secretions such as milk. A variety of functions have been ascribed to this protein, it appears to contribute to antimicrobial host defense. Still its overall physiological role remains to be defined. We sought to study the role of recombinant human lactoferrin (rhLf) in Shigella infection. Invasion of epithelial cells is essential to the development of bacillary dysentery. Shigella flexneri 5 M90T, a virulent strain, was evaluated in the classic HeLa cell invasion model, in immunoblots, and by transmission electron microscopy, immunofluorescence, and deconvolved microscopy Bacteria not exposed to rhLf were used as controls. We found that rhLf decreased significantly the invasiveness of S. flexneri 5 M90T in a HeLa cell model. The immunoblot data showed that invasion plasmid antigen B (IpaB) was released from the bacteria during incubation with rhLf. Lactoferrin treatment did not directly dissociate the complex of IpaB and IpaC (IpaBC) once the complex had been formed. Furthermore, ferric iron had no effect on release of IpaB. Electron microscopy of rhLf-treated bacteria suggested a reduction in vacuolization of the HeLa cell cytoplasm and decreased number of bacteria within HeLa cells. At 40,000 x magnification the few rhLf-treated Shigella that invaded exhibited a dense ring completely surrounding them. Immunofluorescence and deconvolved microscopy suggested that rhLf-treated bacteria were completely surrounded by a thick layer of actin. The fact that two cell surface functions (invasion and actin-mediated movement) were deranged suggests that rhLf disrupts the integrity of the bacterial outer membrane in which virulence proteins are anchored. The mechanism by which rhLf impairs Shigella invasiveness may be relevant to other enteropathogens that share similar virulence strategies.

PURIFIED SERUM ANTIBODIES DIRECTED AGAINST Gb 3 INDUCE APOPTOSIS IN VERO AND HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC). † 2150

PURIFIED SERUM ANTIBODIES DIRECTED AGAINST Gb 3 INDUCE APOPTOSIS IN VERO AND HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC). † 2150