Cholera.

Despite more than a century of study, cholera still presents challenges and surprises to us. Throughout most of the 20th century, cholera was caused by Vibrio cholerae of the O1 serogroup and the disease was largely confined to Asia and Africa. However, the last decade of the 20th century has witnessed two major developments in the history of this disease. In 1991, a massive outbreak of cholera started in South America, the one continent previously untouched by cholera in this century. In 1992, an apparently new pandemic caused by a previously unknown serogroup of V. cholerae (O139) began in India and Bangladesh. The O139 epidemic has been occurring in populations assumed to be largely immune to V. cholerae O1 and has rapidly spread to many countries including the United States. In this review, we discuss all aspects of cholera, including the clinical microbiology, epidemiology, pathogenesis, and clinical features of the disease. Special attention will be paid to the extraordinary advances that have been made in recent years in unravelling the molecular pathogenesis of this infection and in the development of new generations of vaccines to prevent it.     

Human submandibular-sublingual saliva promotes adhesion of Candida albicans to polymethylmethacrylate.

The purpose of this study was to identify components of saliva that interact with Candida albicans in solution and that may modulate adhesion to dental acrylic (polymethylmethacrylate [PMMA]) surfaces. Saliva-derived pellicles extracted from C. albicans blastoconidia and hyphal-form cells mixed with fresh human submandibular-sublingual saliva (HSMSL) contained predominantly high- and low-molecular-weight mucins (MG1 and MG2, respectively). In contrast, few components from fresh human parotid saliva were adsorbed to yeast cells. Coating PMMA beads with HSMSL significantly enhanced (10-fold) adhesion of both growth forms of C. albicans compared with human parotid saliva (2-fold), suggesting a role for mucins in adhesion. HSMSL-enhanced adhesion was completely abolished by preadsorbing HSMSL with either blastoconidia or hyphal-form cells prior to coating PMMA. However, coating PMMA with purified salivary mucins or the addition of mucin to preadsorbed saliva did not enhance or restore adhesion to levels found with fresh HSMSL. Adhesion assays employing guanidine-treated fresh HSMSL showed a complete lack of Candida binding, suggesting that subjecting HSMSL to dissociating conditions may alter a property of salivary mucins crucial for C. albicans adhesion. Protease and glycosidase treatment of yeast cells significantly reduced adhesion to HSMSL-coated PMMA. In addition, preincubation of C. albicans with mannose and galactose inhibited adhesion to HSMSL-coated PMMA. These results suggest that mucins may play a role in C. albicans adhesion to saliva-coated PMMA and that a glycoprotein on the yeast surface may be involved in these events.     

Usefulness of an enzyme-linked immunosorbent assay for detection of Giardia antigen in feces.

The usefulness of a recently developed enzyme-linked immunosorbent assay which detects Giardia lamblia antigen in feces was determined in experimentally infected humans. Giardia antigen was determined in serially collected fecal specimens from humans inoculated with two Giardia isolates, GS/M and Isr. A total of 277 stools from 18 volunteers were tested, 74 from Isr-inoculated volunteers and 203 from GS/M-inoculated volunteers. None of the five Isr-inoculated volunteers became infected, and none of their stools contained Giardia antigen. In contrast, all of the 13 GS/M-inoculated volunteers became infected, and Giardia antigen was present one or more times in the stools of each. Of 203 stools from GS/M-inoculated volunteers, 73 contained Giardia cysts, and 69 of these (94.5%) had detectable antigen. In contrast, 108 of the 203 specimens were positive for Giardia antigen and only 73 had cysts. Most antigen-positive but cyst-negative specimens occurred during treatment, but during patency 71 stools contained antigen and 65 had cysts. The enzyme-linked immunosorbent assay is infections and is easier to perform.     

Optimization of plasmid maintenance in the attenuated live vector vaccine strain Salmonella typhi CVD 908-htrA

The broad objective of the research presented here is to develop a noncatalytic plasmid maintenance system for the stabilization of multicopy expression plasmids encoding foreign antigens in a Salmonella typhi live-vector vaccine strain such as CVD 908-htrA. We have enhanced the maintenance of expression plasmids at two independent levels. First, we removed dependence upon balanced-lethal maintenance systems that involve catalytic enzymes expressed from multicopy plasmids; we accomplished this through incorporation into expression plasmids of a postsegregational killing system based on the noncatalytic hok-sok plasmid addiction system from the antibiotic resistance factor pR1. We also included at least one naturally occurring plasmid partition function in our expression plasmids, which eliminates random segregation of these plasmids, thereby enhancing their inheritance and stability; to accomplish this, we incorporated either the par locus from pSC101, the parA locus from pR1, or both. We monitored the stability of optimized expression plasmids within CVD 908-htrA by quantitating expression of a variant of green fluorescent protein (GFPuv) by using flow cytometry. In this report, we demonstrate the utility of this novel plasmid maintenance system in enhancing the stability of our expression plasmids and go on to show that as the copy number of stabilized plasmids increases, the toxicity of GFPuv synthesis also increases. The implications of these observations for the rational design of immunogenic and protective bacterial live vector vaccines are discussed.     

Cellular dysfunction in the diabetic fibroblast: impairment in migration,vascular endothelial growth factor production,and response to hypoxia

Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P < 0.001) and was not significantly stimulated by hypoxia (1% O(2)), whereas wild-type fibroblast migration was up-regulated nearly twofold in hypoxic conditions (P < 0.05). Diabetic fibroblasts produced twice the amount of pro-matrix metalloproteinase-9 as normal fibroblasts, as measured by both gelatin zymography and enzyme-linked immunosorbent assay (P < 0.05). Adult diabetic fibroblasts exhibited a sevenfold impairment in vascular endothelial growth factor (VEGF) production (4.5 +/- 1.3 pg/ml versus 34.8 +/- 3.3 pg/ml, P < 0.001) compared to wild-type fibroblasts. Moreover, wild-type fibroblast production of VEGF increased threefold in response to hypoxia, whereas diabetic fibroblast production of VEGF was not up-regulated in hypoxic conditions (P < 0.001). To address the question whether these differences resulted from chronic hyperglycemia or absence of the leptin receptor, fibroblasts were harvested from newborn db/db mice before the onset of diabetes (4 to 5 weeks old). These fibroblasts showed no impairments in VEGF production under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show selective impairments in discrete cellular processes critical for tissue repair including cellular migration, VEGF production, and the response to hypoxia. The VEGF abnormalities developed concurrently with the onset of hyperglycemia and were not seen in normoglycemic, leptin receptor-deficient db/db mice. These observations support a role for fibroblast dysfunction in the impaired wound healing observed in human diabetics, and also suggest a mechanism for the poor clinical outcomes that occur after ischemic injury in diabetic patients.     

Interleukin-1 dysregulation is an intrinsic defect in macrophages from MRL autoimmune-prone mice

Macrophages (M?) from pre-diseased autoimmune-prone MRL mice (both MRL/+ and MRL/1pr) dramatically underproduce the cytokine interleukin-1 (IL-1) in comparison to M? from a number of normal strains. In this study we show that IL-1 dysregulation by MRL M? is fully expressed at birth, and that this defect does not change with time or the development of disease. We also constructed adult irradiation chimeras (consisting of A/J → MRL and MRL → A/J mice), and show that M? isolated from these chimeras display a pattern of IL-1 production indistinguishable from that of the donor strain controls. Moreover, when we constructed a mixed chimera (A/J + MRL → A/J), the A/J and MRL M? coexisting within the same animal retained their individual patterns of IL-1 production when isolated by negative selection. Taken together, these results provide the first substantive evidence for an intrinsic defect (IL-1 dysregulation) in M? from MRL autoimmune-prone mice.     

Aberrant chromatin at genes encoding stem cell regulators in human mixed-lineage leukemia

Mixed-lineage leukemia (MLL) fusion proteins are potent inducers of leukemia, but how these proteins generate aberrant gene expression programs is poorly understood. Here we show that the MLL-AF4 fusion protein occupies developmental regulatory genes important for hematopoietic stem cell identity and self-renewal in human leukemia cells. These MLL-AF4-bound regions have grossly altered chromatin structure, with histone modifications catalyzed by trithorax group proteins and DOT1 extending across large domains. Our results define direct targets of the MLL fusion protein, reveal the global role of epigenetic misregulation in leukemia, and identify new targets for therapeutic intervention in cancer.     

Volunteer studies of deletion mutants of Vibrio cholerae O1 prepared by recombinant techniques

Vibrio cholerae O1 A-B- vaccine strain JBK 70 and A-B+ CVD 101 prepared by recombinant DNA techniques from pathogenic EI Tor Inaba N16961 and classical Ogawa 395, respectively, were fed to 38 volunteers in single doses of 10(4) to 10(10). Although severe diarrhea did not occur in any vaccine, more than one-half developed mild diarrhea. These attenuated strains colonized well and elicited prominent vibriocidal and antitoxic (CVD 101) antibody responses. Recipients of a single dose of JBK 70 were significantly protected when challenged with 10(6) wild-type N16961. Diarrhea occurred in 7 of 8 controls but in only 1 of 10 vaccines (P less than 0.003, 89% vaccine efficacy), demonstrating the potency of immune mechanisms that do not involve cholera antitoxin. Further derivatives were prepared to explore the pathogenesis of the residual diarrhea, considering that either intestinal colonization by the vaccine itself or accessory toxins might be responsible. CVD 102, an auxotrophic mutant of CVD 101, did not cause diarrhea but colonized poorly and elicited feeble immune responses. Derivatives of JBK 70 and CVD 101 (CVD 104 and 105) deleted of genes encoding the EI Tor hemolysin still caused mild diarrhea. Genetically engineered strains can be colonizing, highly immunogenic, and protective single-dose oral vaccines, but they must be further attenuated before they can be considered for use as public health tools.     

Increased Escherichia coli enterotoxin detection after concentrating culture supernatants: possible new enterotoxin detectable in dogs but not in infant mice.

The heat-stable enterotoxin (ST) of Escherichia coli can be detected by infant mouse or dog intestinal loop tests. These tests differ in that the dog assay uses concentrated culture supernatants and is based on measurements of net intestinal absorption, whereas the mouse test uses unconcentrated supernatants and depends on gross fluid accumulation. To compare the relative sensitivities of these assays, culture supernatants of randomly selected E. coli isolates from 34 Bangalee diarrhea patients were tested for ST in dog loops and infant mice. Supernatants were also tested for heat-labile enterotoxin (LT) in dog loops, Y-1 adrenal cells, and Chinese hamster ovary cells. E. coli supernatants that produced positive responses for both ST and LT in the dog loop assay (ST+/LT+) also produced positive responses when tested for ST in infant mice and for LT in cell lines. Supernatants of strains negative for ST and LT in dog loop (ST-/LT) were also negative in other assays. Of 10 strains positive for just ST in the dog loop test (ST+/LT-), only 5 were ST positive in the standard infant mouse test. Supernatants of the other five strains (dog loop positive, mouse test negative) were then concentrated 100-fold and retested in mice. Three of these five gave consistently positive results after concentration, and two were only intermittently positive. Concentrated supernatants of negative control strains (ST-/LT-) were all negative in mice. The dog assay detects more strains producing ST than the infant mouse test. The infant mouse test, which detects only gross fluid accumulation, failed to detect approximately half of the 10 strains which produced ST alone (ST+/LT-; P = 0.025). Concentrating supernatants for the mouse assay increases sensitivity for detection of ST, but certain E. coli strains produce a variety of ST to which infant mice do not respond.     

The capsule and O antigen in Vibrio cholerae O139 Bengal are associated with a genetic region not present in Vibrio cholerae O1.

Vibrio cholerae O139 Bengal, although closely related to V. cholerae O1 El Tor, produces a polysaccharide capsule and has a distinct O antigen. We have identified a chromosomal region of at least 11 kb, as defined by three TnphoA mutations, that is required for the expression of both polysaccharides. Electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis show that these TnphoA mutants have lost the abilities both to express capsule and to produce lipopolysaccharide beyond the core oligosaccharide. Reactivity with O139 typing serum and resistance to serum are also lost in the mutants. DNA probes for this region do not hybridize with O1 V. cholerae but do react with other vibrios, implying that the region was recently acquired.