Molecular Analysis of Non-O1, Non-O139 Vibrio cholerae Associated with an Unusual Upsurge in the Incidence of Cholera-Like Disease in Calcutta, India

There was an inexplicable upsurge in the incidence of non-O1, non-O139 Vibrio cholerae among hospitalized patients admitted to the Infectious Diseases Hospital, Calcutta, India, between February and March 1996. Of the 18 strains of V. cholerae isolated during this period, 15 belonged to the non-O1, non-O139 serogroups (4 belonged to O144, 3 belonged to O11, 1 each belonged to O6, O8, O12, O19, O39, and O58, and 2 strains could not be typed), 2 belonged to the O139 serogroup, and 1 belonged to the O1 serogroup. Cell-free culture supernatants of 13 representative non-O1, non-O139 V. cholerae strains evoked a distinct cytotoxic effect on CHO and HeLa cells, and the strains examined produced the nonmembrane-damaging cytotoxin. By several PCR assays, it was determined that none of the non-O1, non-O139 strains were positive for the ctxA, zot, ace, and tcpA genes and for the genes representing the heat-labile toxin, heat-stable toxin, and verotoxin of Escherichia coli and the various variants of these genes. Studies on the clonality of non-O1, non-O139 V. cholerae strains by restriction fragment length polymorphism (RFLP) analysis of rRNA genes and of other genes (hlyA, hlyU, hlx, toxR, and attRS1) and by pulsed-field gel electrophoresis (PFGE) collectively indicate that the upsurge which occurred in February and March 1996 was caused by strains belonging to different clones. Overall, there was an excellent correlation between the results of ribotyping, RFLP analysis of various genes, and PFGE, with strains belonging to a particular serogroup showing nearly identical restriction patterns and PFGE profiles. It is clear from this study that some serogroups of V. cholerae can cause diarrhea by a mechanism quite different from that of toxigenic V. cholerae O1 and O139, and we have proposed the nomenclature of enteropathogenic V. cholerae to include these serogroups.     

Scavenger receptor-mediated delivery of muramyl dipeptide activates antitumor efficacy of macrophages by enhanced secretion of tumor-suppressive cytokines

We showed that muramyl dipeptide (MDP) conjugated to maleylated bovine serum albumin (MBSA) was internalized by macrophages (Mphi) through scavenger receptor (SCR)-mediated endocytosis, which leads to 50-fold higher cytotoxic activity against non-Mphi tumor cells compared with that elicited by free MDP-treated Mphi. The enhanced cytotoxic effect of MBSA-MDP was found to be a result of higher secretion of interleukin (IL)-1, IL-6, tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO) because the addition of antibodies directed against IL-1, IL-6, or TNF-alpha in combination with Mphi cultures totally abrogated the tumoricidal activity of MBSA-MDP. It is interesting to note that MBSA-MDP triggers the secretion of IL-12, whereas IL-10, a Mphi suppressor cytokine, could be detected only on free MDP treatment. The cytotoxic activity of MBSA-MDP was inhibited by indomethacin, indicating a regulatory role for prostaglandin E2 (PGE2). Efficient SCR-mediated intracellular delivery of MDP leading to elimination of cancer cells suggests the immunotherapeutic potential of this approach for treatment of neoplasia.     

Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-kappa B pathway

Dendritic cells (DC) are key components of innate and adaptive immune responses. The identity of endogenous signals that activate DC is a crucial and unresolved question. We report here that heat shock proteins (HSP), the most abundant and conserved mammalian molecules, constitute such an internal signal. Necrotic but not apoptotic cell death leads to release of HSP gp96, calreticulin, hsp90 and hsp70. HSP stimulate macrophages to secrete cytokines, and induce expression of antigen-presenting and co-stimulatory molecules on the DC. The HSP gp96 and hsp70 act differentially, and each induces some but not all molecules. HSP interact with these antigen-presenting cells through the highly conserved NF-kappa B pathway. As HSP are intracellular, abundant and soluble, their presence in the extra-cellular milieu and the consequent activation of antigen-presenting cells (APC) constitutes an excellent mechanism for response to cell death. As HSP are conserved from bacteria to mammals, the ability of HSP to activate APC provides a unified mechanism for response to internal and external stimuli.     

Atropine-antagonized miosis induced in the goat by intravenous infusions of prostaglandin E (PGE1 and PGE2)

Effects of 30 min intravenous infusions of prostaglandin (PG) E1 (total dosage 0.07 mg kg-1), E2 and F2 alpha (total dosage 0.12 mg kg-1), respectively, were studied concomitant with measurements of renal excretion of PGF metabolites in hyperhydrated goats. None of the PGs induced any rise in rectal temperature. However, the PGEs elicited pronounced, atropine-antagonized miosis and an inhibition of the water diuresis accompanied by some increase in renal excretion of arginine vasopressin (AVP). These effects were not obtained in response to PGF2 alpha. The PGF2 alpha rapidly induced a conspicuous and long-lasting increase in the renal excretion of PGF metabolites. The corresponding effect of the PGEs was more delayed, and less than 10% of the increase obtained in response to PGF2 alpha. Nevertheless, the excretion of these metabolites in response to the PGEs was of the same magnitude as that previously observed during endotoxin fever in the goat. It is concluded that endotoxin-induced miosis and stimulation of AVP secretion previously demonstrated in the goat might well have been secondary to systemic PGE production. However, this does not seem to hold true for endotoxin-induced fever.     

Development of isotopic and non-isotopic microwell based immunoassays for hCG using 125I and biotin labeled hCG

Isotopic and non-isotopic immunoassays of hCG, based on the principle of competitive inhibition, using micro-well as solid support and 125I and biotin as labels for hCG, have been developed. In both the assays, rabbit polyclonal antibody was immobilized onto micro-wells. In the non-isotopic assay, to the hCG antibody coated micro-wells, 50 microL of standard or samples along with 100 microL of biotinylated hCG were incubated for 1 hour at 37 degrees C. After incubation, wells were washed and 100 microL of streptavidin-HRP conjugate was added to each well and incubated again for a half hour at 37 degrees C. Bound enzyme activity was measured using tertramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. In the isotopic assay, to the hCG antibody coated micro-wells, 50 microL of standard or samples along with 100 microL of 125I-hCG were incubated for 1 hour at 37 degrees C. The bound radioactivity was measured using a gamma counter. The sensitivities of the non-isotopic and isotopic assays were 0.12 IU/mL and 0.1 IU/mL, respectively. The intra- and inter-assay CVs for both the assays were less than 12.3%. There was a good correlation between the developed non-isotopic and isotopic immunoassays (r = 0.97, n = 20).     

Deletion mapping of chromosome 1 in early onset and late onset breast tumors--a comparative study in eastern India

Younger women exhibit more aggressive pathologic features of breast cancer (BC) compared to their older counterparts. Young age has been shown to be an independent predictor of adverse prognosis. These findings have raised the question of whether these differences are also present at the genetic level. Twenty-five early onset (age < or = 40 years) tumors including 4 bilateral tumors, and 26 late onset (>40 years) breast tumors, including 2 bilateral tumors, were examined for loss of heterozygosity (LOH) at chromosome 1 using 11 polymorphic microsatellite markers. A comparative study revealed high frequencies of LOH in chr. 1p36 (61%), 1p31.3 (40%), 1p21.3 (50%) and 1q22-23.2 (56%) in a younger group, and chr. 1p36 (46%), 1p34.2 (48%), and 1q22-23.2 (52%) in an older group. These differences in LOH frequency in these two age groups were significant for chr. 1p21.3 (p = 0.025) only. These data suggest that the deletion pattern in early onset breast tumors is not fully identical to late onset breast tumors. Similar differential deletion patterns of LOH in the 5 highly deleted regions were seen in premenopausal and postmenopausal groups. An association was seen between LOH at chr. 1p34.2 and chr. 1q22-23.2 and higher grade of the tumors in older women. Among the highly deleted regions, the deletion at chr. 1p36 was found to occur early in both groups because of common allelic loss in the bilateral tumors.     

Internal herniation of wandering spleen--a rare cause of recurrent abdominal pain

A rare case of wandering spleen herniating through a defect in transverse mesocolon, which was managed electively by splenopexy using polyglycolic acid mesh is reported. An enlarged wandering spleen is at constant risk of trauma, torsion and infarction. Internal herniation seems to increase the above risks. Early recognition and elective splenopexy should be the treatment of choice.     

Micronuclei as biomarkers of carcinogen exposure in populations exposed to arsenic through drinking water in West Bengal, India: a comparative study in three cell types.

Contamination of groundwater by arsenic, a paradoxical human carcinogen, has become a cause of global public health concern. In West Bengal, India, the groundwater in 9 of 18 districts is heavily contaminated with arsenic. Various adverse health effects including cancer have been reported from these districts and are associated with prolonged arsenic exposure. A cross-sectional biomarker study was conducted to evaluate and compare the frequencies of micronuclei in peripheral blood lymphocytes, oral mucosa cells, and urothelial cells from the inhabitants of North 24 Parganas, one of the arsenic-affected districts. The three cell types were collected from 163 residents exposed to high levels of arsenic in drinking water (214.7213 +/- 9.0273 microg/l) and from 154 unexposed subjects residing in the unaffected East Midnapur district with very little or no exposure to arsenic through drinking water (9.2017 +/- 0.3157 microg/l). Our analysis revealed that micronuclei frequencies in the exposed group were significantly elevated to 5.33-fold over unexposed levels for lymphocytes, 4.63-fold for oral mucosa cells, and 4.71-fold for urothelial cells (increases in micronuclei frequencies significant at P < 0.01). The results indicate that chronic ingestion of arsenic in drinking water by the exposed subjects is linked to the enhanced incidence of micronuclei in all the three cell types, slightly higher level of micronuclei being observed in lymphocytes compared with oral mucosa and urothelial cells.