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.