Role of a large plasmid of Salmonella typhi encoding multiple drug resistance

Twenty isolates of Salmonella typhi from cases of typhoid during the 1989-1990 epidemic in Calcutta were examined. Most isolates (84% of all isolates in the epidemic) were resistant to chloramphenicol, ampicillin, tetracycline and streptomycin but were sensitive to nalidixic acid and ciprofloxacin. Plasmids of 120 kb and 14 kb were identified amongst the multi-drug resistant isolates of S. typhi. However, there was no plasmid in the antibiotic-sensitive isolates. The 120-kb plasmid was transferable and transconjugants were resistant to chloramphenicol, ampicillin, tetracycline and streptomycin. Restriction endonuclease analysis patterns after EcoRI digestion of the 120-kb antibiotic-resistance plasmids from the S. typhi isolates and transconjugants were similar.     

Analysis of immune responses against T- and B-cell epitopes from Plasmodium falciparum liver-stage antigen 1 in rodent malaria models and malaria-exposed human subjects in India

Liver-stage antigen 1 (LSA-1) is a potential vaccine candidate against preerythrocytic stages of malaria. We report here the immunogenicity of linear synthetic constructs delineated as T(H)-cell determinants from the nonrepeat regions of Plasmodium falciparum LSA-1 in murine models and human subjects from areas where malaria is endemic in Rajasthan State, India. Seven peptide constructs (LS1.1 to LS1.7) corresponding to predicted T-cell sites from both the N- and C-terminal regions and peptide LS1R from a repeat region of PfLSA-1 were synthesized to analyze the cellular immune responses. These linear peptides were also tested for humoral responses in order to determine if there were any overlapping B-cell epitopes in the predicted T-cell sites. Most peptides induced cellular responses in peptide-immunized BALB/c and C57BL/6 mice as measured by proliferation and cytokine analysis. Cross-reactive T-cell recognition of P. falciparum-based peptides in Plasmodium berghei-immune animals was evaluated, but only one peptide, LS1.2 (amino acids 1742 to 1760) triggered T-cell proliferation and interleukin-2 and gamma interferon secretion in P. berghei-immune splenocytes of BALB/c and C57BL/6 mice as well as in Thamnomys gazellae (natural host of P. berghei ANKA). In an enzyme-linked immunosorbent assay with the peptides, only one peptide, LS1.1, was recognized by anti-P. berghei liver-stage serum. Three peptides (LS1. 1, LS1.2, and LS1.3) of the eight peptides tested in this study were recognized by a relatively large percentage of P. falciparum-exposed human subjects; the reactivities ranged from approximately 45% for LS1.3 to approximately 60% for LS1.1 and LS1.2. Interestingly, all of the eight putative T-cell determinants were also recognized by the sera collected from malaria patients, although the response was variable in nature. These T(H)- and B-cell epitopes may be of potential value for preerythrocytic antigen-based malaria subunit vaccine formulations.     

A conserved peptide sequence of the Plasmodium falciparum circumsporozoite protein and antipeptide antibodies inhibit Plasmodium berghei sporozoite invasion of Hep-G2 cells and protect immunized mice against P. berghei sporozoite challenge.

Minutes after injection into the circulation, malaria sporozoites enter hepatocytes. The speed and specificity of the invasion process suggest that it is receptor mediated. The region II sequence of Plasmodium falciparum circumsporozoite (CS) protein includes a nonapeptide (WSPCSVTCG) which is highly conserved in all of the CS proteins sequenced to data, including the one from Plasmodium berghei. We have found that two peptides based on the P. falciparum region II sequence, P18 (EWSPCSVTCGNGIQVRIK) and P32 (IEQYLKKIKNS ISTEWSPCSVTCGNGIQVRIK), significantly inhibited P. berghei sporozoite invasion into Hep-G2 cells in vitro. This inhibition was enhanced if either peptide was preincubated with Hep-G2 cells prior to sporozoite invasion. We confirm that region II is a sporozoite ligand for the hepatocyte receptor; moreover, despite the few differences between P. falciparum and P. berghei region II sequences around the nonapeptide sequence (66% homology), the functional characteristics of the motif sequences are not affected. Since the conserved motifs represent a crucial sequence involved in Plasmodium sporozoite invasion of hepatocytes, antibodies to region II should inhibit sporozite invasion into hepatocytes. Indeed, we found that polyclonal antibodies generated to the P. falciparum-based peptide P32 inhibited P. berghei sporozoite invasion of Hep-G2 cells. Furthermore, inbred mice (C57BL/6) immunized with P32 were protected against a lethal challenge of P. berghei sporozoites. Our results suggest that the conserved region II of the CS protein contains crucial B- and T-cell epitopes, that such peptide sequences from the human malaria parasite P. falciparum can be screened in the P. berghei rodent model, and, finally, that region II can be considered useful as one of the components of a malaria vaccine.     

Mechanisms of nitric oxide interplay with Rho GTPase family members in modulation of actin membrane dynamics in pericytes and fibroblasts

Migration of pericytes such as hepatic stellate cells is fundamentally important for diverse biological and pathological processes including tumor invasion and fibrosis. In prototypical migratory cells such as fibroblasts, the small GTPases Rac1 and RhoA govern the assembly of lamellipodia and stress fibers, respectively, cytoskeletal structures that are integral to the cell migration process. The gaseous signaling molecule nitric oxide (NO) influences growth factor chemotactic responses, although this occurs primarily in cell-type-specific ways and through cell biological effects that are poorly characterized. In this study, we use complementary molecular and cell biological approaches to delineate important roles for Rac1, RhoA, and NO in migration of the human hepatic stellate cell line LX2 and primary rat hepatic stellate cells. Both platelet-derived growth factor (PDGF) and Rac1 overexpression drove migration through formation of actin-positive filopodia spikes in LX2 as compared to the formation of lamellipodia in fibroblasts. NO inhibited PDGF- and Rac1-driven migration in LX2 by abrogating filopodia formation and inhibited migration of fibroblasts by attenuating lamellipodial protrusions. Additionally, RhoA conferred resistance to NO inhibition of migration and restored chemotactic responses to PDGF in the absence of functional Rac1 in LX2. In conclusion, these studies identify novel crosstalk between small GTPases, cytoskeletal structures, and NO in pericyte-specific pathways, providing counterbalances in the chemotactic responses to growth factors.     

Juvenile rhesus monkeys have more colonic granulomas than adults after primary infection with<Emphasis Type="Italic"> Schistosoma mansoni</Emphasis>

Adults and children have differences in their susceptibility to schistosomiasis. Whether this age-dependent innate susceptibility influences parasite-caused granulomogenesis is difficult to assess in humans. Therefore, we exposed juvenile and adult female rhesus monkeys to primary infection with Schistosoma mansoni. Hepatic and intestinal granuloma formation was observed in both pre-pubescent and adult monkeys. Two distinct stages of granulomas were discerned, the exudative and the productive stage. In the intestine, more granulomas were generated in the colon than in the ileum. In contrast to the adult animals, the juvenile rhesus monkeys had higher numbers of colonic granulomas, these higher numbers being predominantly of the more advanced productive stage. Juvenile animals had a statistically non-significant increased worm burden. These results suggest that juvenile rhesus monkeys have a significantly more intense and advanced colonic response towards entrapped S. mansoni eggs after primary schistosome infections and, thereby, are more susceptible to parasite infection.Research protocols involving non-human primates received ethical clearance by the Institutional Animal Care and Use Committee of the Biomedical Primate Research Centre (Rijswijk, The Netherlands), according to Dutch Law.     

Comparative mutagenic and genotoxic effects of three antimalarial drugs, chloroquine, primaquine and amodiaquine

Comparative mutagenic and genotoxic effects of three antimalarialdrugs, chloroquine, primaquine and amodiaquine, were assessedin the Ames mutagenicity assay (in strains TA97a, TA100, TA102and TA104) and in vivo sister chromatid exchange (SCE) and chromosomeaberration (CA) assays in bone marrow cells of mice. These arethe most commonly used antimalarial drugs available at presentthroughout the world. The results of the bacterial mutagenicityassays showed a very weak mutagenic effect of all three drugsin Salmonella strains TA97a and TA100 both with and withoutS9 mix and in TA104 only with S9 mix. The results of the invivo SCE and CA assays indicate that these three drugs are genotoxicin bone marrow cells of mice. ³To whom correspodence should be addressed. Tel: +91 33 473 3491; Fax: +91 33 473 5197; Email: iichbio{at}giascl01.vsnl.net.in     

Cell membrane-associated MT1-MMP-dependent activation of pro-MMP-2 in A375 melanoma cells.

Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, can degrade extracellular matrix components under physiological conditions and during cancer invasion and metastasis. Among the MMPs, the 72 kDa type IV collagenase MMP-2 (gelatinase A) is activated in a membrane-associated manner by an activation complex composed of membrane type 1 matrix metalloproteinase (MT1-MMP), tissue inhibitor of matrixmetalloproteinase-2 (TIMP-2), and pro-MMP-2 in the presence of alphavbeta3 integrin receptor. The activation of pro-MMP-2 correlates with increased occurrence of metastases. Increased MMP-2 activity has been demonstrated in many human tumors. In the present communication, we studied cell surface-associated activation of MMP-2 (72 kDa collagenase type IV) in the moderately metastatic human melanoma cell line A375. RESULTS: Activation of purified 72 kDa collagenase type IV, pro-MMP-2 from cervical cancer tissue homogenate and from serum-free culture medium of HT1080 cells grown in presence of concanavalin A, by A375 cells, was shown by gelatin zymography. A375 cells activated only pro-MMP-2 from purified MMP-9/MMP-2 mixture indicating that the activation is specific for MMP-2. Activation of MMP-2 and purified collagenase type IV by A375 membrane fraction and membrane extract was also demonstrated by gelatin zymography. When A375 cells were first incubated with anti-MT1-MMP polyclonal antibody, activation of collagenase type IV was significantly decreased, indicating that membrane-associated MMP-2 activation is MT1-MMP-mediated. Immunocytochemistry showed MT1-MMP localization at focal adhesion sites. The presence of the components of activation complex-MT1-MMP and integrin alphavbeta3-were confirmed by Western blot, cell adhesion assay, and integrin subunit assay. CONCLUSION: Our experimental findings furnish another example of the unique membrane-associated MMP-2 activation mechanism in A375 melanoma cells and clearly indicate the role of MT1-MMP in MMP-2 activation. The information could help in developing new therapies designed to interfere with MMP activation and management of cancer and metastases.