Biomedical subjects

S K Ahuja

Publications and source records attributed to S K Ahuja.

At least 19 recordsLinked to original sources

PubMed2004

Application of Plackett-Burman design and response surface methodology to achieve exponential growth for aggregated shipworm bacterium.

Here we report the successful implementation of the Plackett-Burman multifactorial design to screen the limiting components for growth and subsequent use of the response surface methodology (RSM) to design a medium that supported exponential growth of the aggregated morphology of the shipworm bacterium, Teredinobacter turnirae. The results obtained with the help of Plackett-Burman design indicated limitations of three components in the growth medium, MnCl2.4H2O, Na2CO3, and K2HPO4. The concentrations of these three components were further optimized using RSM. By increasing the concentrations of the above-mentioned components by 4-fold, 12-fold, and 12-fold, respectively, it became possible to achieve exponential growth of the culture.

Algorithms↗

PubMed2004

Production of an endoglucanase by the shipworm bacterium, Teredinobacter turnirae.

The nutritional behavior of a cellulolytic nitrogen-fixing shipworm bacterium, Teredinobacter turnirae, is described, with respect to various carbon and nitrogen sources, in terms of endoglucanase production. Also, the effects of various surfactants on enzyme production are reported. Among the carbon sources, sucrose results in the maximum enzyme production, followed by cellulose. Ammonium phosphate proves to be the best nitrogen source for endoglucanase production. Various surfactants enhance the enzyme titers, with Triton X-100 yielding the best results. A combination of the above-mentioned components improves the enzyme production by 3.6-fold.

Bacteria↗

PubMed2001

Extensive repertoire of membrane-bound and soluble dendritic cell-specific ICAM-3-grabbing nonintegrin 1 (DC-SIGN1) and DC-SIGN2 isoforms. Inter-individual variation in expression of DC-SIGN transcripts.

Expression in dendritic cells (DCs) of DC-SIGN, a type II membrane protein with a C-type lectin ectodomain, is thought to play an important role in establishing the initial contact between DCs and resting T cells. DC-SIGN is also a unique type of human immunodeficiency virus-1 (HIV-1) attachment factor and promotes efficient infection in trans of cells that express CD4 and chemokine receptors. We have identified another gene, designated here as DC-SIGN2, that exhibits high sequence homology with DC-SIGN. Here we demonstrate that alternative splicing of DC-SIGN1 (original version) and DC-SIGN2 pre-mRNA generates a large repertoire of DC-SIGN-like transcripts that are predicted to encode membrane-associated and soluble isoforms. The range of DC-SIGN1 mRNA expression was significantly broader than previously reported and included THP-1 monocytic cells, placenta, and peripheral blood mononuclear cells (PBMCs), and there was cell maturation/activation-induced differences in mRNA expression levels. Immunostaining of term placenta with a DC-SIGN1-specific antiserum showed that DC-SIGN1 is expressed on endothelial cells and CC chemokine receptor 5 (CCR5)-positive macrophage-like cells in the villi. DC-SIGN2 mRNA expression was high in the placenta and not detectable in PBMCs. In DCs, the expression of DC-SIGN2 transcripts was significantly lower than that of DC-SIGN1. Notably, there was significant inter-individual heterogeneity in the repertoire of DC-SIGN1 and DC-SIGN2 transcripts expressed. The genes for DC-SIGN1, DC-SIGN2, and CD23, another Type II lectin, colocalize to an approximately 85 kilobase pair region on chromosome 19p13.3, forming a cluster of related genes that undergo highly complex alternative splicing events. The molecular diversity of DC-SIGN-1 and -2 is reminiscent of that observed for certain other adhesive cell surface proteins involved in cell-cell connectivity. The generation of this large collection of polymorphic cell surface and soluble variants that exhibit inter-individual variation in expression levels has important implications for the pathogenesis of HIV-1 infection, as well as for the molecular code required to establish complex interactions between antigen-presenting cells and T cells, i.e. the immunological synapse.

Adult↗

PubMed2001

Concordance between the CC chemokine receptor 5 genetic determinants that alter risks of transmission and disease progression in children exposed perinatally to human immunodeficiency virus.

If CC chemokine receptor 5 (CCR5)-dependent mechanisms at the time of initial virus exposure are important determinants of virus entry and disease outcome, then the polymorphisms in CCR5 that influence risk of transmission and disease progression should be similar; this hypothesis was tested in a cohort of 649 Argentinean children exposed perinatally to human immunodeficiency virus type 1 (HIV-1). Two lines of evidence support this hypothesis. First, CCR5 haplotype pairs associated with enhanced risk of transmission were the chief predictors of a faster disease course. Second, some of the haplotype pairs associated with altered rates of transmission and disease progression in children were similar to those that we previously found influenced outcome in European American adults. This concordance suggests that CCR5 haplotypes may serve as genetic rheostats that influence events occurring shortly after initial virus exposure, dictating not only virus entry but, by extension, also the extent of early viral replication.

Acquired Immunodeficiency Syndrome↗

PubMed2001

Global survey of genetic variation in CCR5, RANTES, and MIP-1alpha: impact on the epidemiology of the HIV-1 pandemic.

Expression of CC chemokine receptor 5 (CCR5), the major coreceptor for HIV-1 cell entry, and its ligands (e.g., RANTES and MIP-1alpha) is widely regarded as central to the pathogenesis of HIV-1 infection. By surveying nearly 3,000 HIV+ and HIV- individuals from worldwide populations for polymorphisms in the genes encoding RANTES, MIP-1alpha, and CCR5, we show that the evolutionary histories of human populations have had a significant impact on the distribution of variation in these genes, and that this may be responsible, in part, for the heterogeneous nature of the epidemiology of the HIV-1 pandemic. The varied distribution of RANTES haplotypes (AC, GC, and AG) associated with population-specific HIV-1 transmission- and disease-modifying effects is a striking example. Homozygosity for the AC haplotype was associated with an increased risk of acquiring HIV-1 as well as accelerated disease progression in European Americans, but not in African Americans. Yet, the prevalence of the ancestral AC haplotype is high in individuals of African origin, but substantially lower in non-Africans. In a Japanese cohort, AG-containing RANTES haplotype pairs were associated with a delay in disease progression; however, we now show that their contribution to HIV-1 pathogenesis and epidemiology in other parts of the world is negligible because the AG haplotype is infrequent in non-Far East Asians. Thus, the varied distribution of RANTES, MIP-1alpha, and CCR5 haplotype pairs and their population-specific phenotypic effects on HIV-1 susceptibility and disease progression results in a complex pattern of biological determinants of HIV-1 epidemiology. These findings have important implications for the design, assessment, and implementation of effective HIV-1 intervention and prevention strategies.

Africa↗

PubMed2001

Pleomorphism of the marine bacterium Teredinobacter turnirae.

AIMS: A morphology transition for the marine bacterium, Teredinobacter turnirae is reported. METHODS AND RESULTS: When grown in the rod-shaped morphology, the cells require high concentrations of NaCl (0.3 mol x l(-1)) and secrete extracellular protease and endoglucanase activity. When this bacterium is grown in a medium containing casein as a sole carbon and nitrogen source, a major change in morphology to a stable aggregated form is obtained. CONCLUSION: In the aggregated morphology, much higher protease production rates (170 Units x ml(-1) x d-1 for aggregates vs. 15 Units x ml(-1) x d(-1) for rods, for the same initial biomass) and negligible endoglucanase titres are obtained. In addition, the aggregated morphology does not require sodium chloride for growth. SIGNIFICANCE AND IMPACT OF THE STUDY: The phenomenon reported here describes a novel relationship between the cell morphology and the biochemical characteristics of the bacterium.

Bacterial Proteins↗

PubMed2000

Preformed membrane-associated stores of interleukin (IL)-12 are a previously unrecognized source of bioactive IL-12 that is mobilized within minutes of contact with an intracellular parasite.

The prevailing paradigm is that production of the interleukin (IL)-12 p70 heterodimer, a critical T helper cell type 1 (Th1)-inducing cytokine, depends on the induced transcription of the p40 subunit. Concordant with this paradigm, we found that dendritic cells (DCs) produced IL-12 p70 only after at least 2-4 h of stimulation with lipopolysaccharide plus interferon gamma. However, using several complementary experimental approaches, including electron and confocal microscopy, we now show that resting murine and human myeloid cells, including macrophages/DCs and DC-rich tissues, contain a novel source of bioactive IL-12 that is preformed and membrane associated. These preformed, membrane-associated IL-12 p70 stores are released within minutes after in vitro or in vivo contact with Leishmania donovani, an intracellular pathogen. Our findings highlight a novel source of bioactive IL-12 that is readily available for the rapid initiation of Th1 host responses to pathogens such as Leishmania species.

Animals↗

PubMed2000

CC chemokine receptor (CCR)2 is required for langerhans cell migration and localization of T helper cell type 1 (Th1)-inducing dendritic cells. Absence of CCR2 shifts the Leishmania major-resistant phenotype to a susceptible state dominated by Th2 cytokines, b cell outgrowth, and sustained neutrophilic inflammation.

There is growing evidence that chemokines and their receptors regulate the movement and interaction of antigen-presenting cells such as dendritic cells (DCs) and T cells. We tested the hypothesis that the CC chemokine receptor (CCR)2 and CCR5 and the chemokine macrophage inflammatory protein (MIP)-1alpha, a ligand for CCR5, influence DC migration and localization. We found that deficiency of CCR2 but not CCR5 or MIP-1alpha led to distinct defects in DC biology. Langerhans cell (skin DC) density in CCR2-null mice was normal, and their ability to migrate into the dermis was intact; however, their migration to the draining lymph nodes was markedly impaired. CCR2-null mice had lower numbers of DCs in the spleen, and this was primarily due to a reduction in the CD8alpha(1) T helper cell type 1 (Th1)-inducing subset of DCs. Additionally, there was a block in the Leishmania major infection-induced relocalization of splenic DCs from the marginal zone to the T cell areas. We propose that these DC defects, in conjunction with increased expression of B lymphocyte chemoattractant, a B cell-specific chemokine, may collectively contribute to the striking B cell outgrowth and Th2 cytokine-biased nonhealing phenotype that we observed in CCR2-deficient mice infected with L. major. This disease phenotype in mice with an L. major-resistant genetic background but lacking CCR2 is strikingly reminiscent of that observed typically in mice with an L. major-susceptible genetic background. Thus, CCR2 is an important determinant of not only DC migration and localization but also the development of protective cell-mediated immune responses to L. major.

Animals↗

PubMed2000

Evolution of human and non-human primate CC chemokine receptor 5 gene and mRNA. Potential roles for haplotype and mRNA diversity, differential haplotype-specific transcriptional activity, and altered transcription factor binding to polymorphic nucleotides in the pathogenesis of HIV-1 and simian immunodeficiency virus.

Polymorphisms in CC chemokine receptor 5 (CCR5), the major coreceptor of human immunodeficiency virus 1 (HIV-1) and simian immunodeficiency virus (SIV), have a major influence on HIV-1 transmission and disease progression. The effects of these polymorphisms may, in part, account for the differential pathogenesis of HIV-1 (immunosuppression) and SIV (natural resistance) in humans and non-human primates, respectively. Thus, understanding the genetic basis underlying species-specific responses to HIV-1 and SIV could reveal new anti-HIV-1 therapeutic strategies for humans. To this end, we compared CCR5 structure/evolution and regulation among humans, apes, Old World Monkeys, and New World Monkeys. The evolution of the CCR5 cis-regulatory region versus the open reading frame as well as among different domains of the open reading frame differed from one another. CCR5 cis-regulatory region sequence variation in humans was substantially higher than anticipated. Based on this variation, CCR5 haplotypes could be organized into seven evolutionarily distinct human haplogroups (HH) that we designated HHA, -B, -C, -D, -E, -F, and -G. HHA haplotypes were defined as ancestral to all other haplotypes by comparison to the CCR5 haplotypes of non-human primates. Different human and non-human primate CCR5 haplotypes were associated with differential transcriptional regulation, and various polymorphisms resulted in modified DNA-nuclear protein interactions, including altered binding of members of the NF-kappaB family of transcription factors. We identified novel CCR5 untranslated mRNA sequences that were conserved in human and non-human primates. In some primates, mutations at exon-intron boundaries caused loss of expression of selected CCR5 mRNA isoforms or production of novel mRNA isoforms. Collectively, these findings suggest that the response to HIV-1 and SIV infection in primates may have been driven, in part, by evolution of the elements controlling CCR5 transcription and translation.

Animals↗

PubMed2000

Identification of vaccine candidates for experimental visceral leishmaniasis by immunization with sequential fractions of a cDNA expression library.

Visceral leishmaniasis caused by the intracellular parasite Leishmania donovani is a significant public health problem in many regions of the world. Because of its large genome and complex biology, developing a vaccine for this pathogen has proved to be a challenging task and, to date, protective recombinant vaccine candidates have not been identified. To tackle this difficult problem, we adopted a reductionist approach with the intention of identifying cDNA sequences in an L. donovani amastigote cDNA library that collectively or singly conferred protection against parasite challenge in a murine model of visceral leishmaniasis. We immunized BALB/c mice with plasmid DNA isolated and pooled from 15 cDNA sublibraries ( approximately 2,000 cDNAs/sublibrary). Following systemic challenge with L. donovani, mice immunized with 6 of these 15 sublibraries showed a significantly reduced (35- to 1,000-fold) hepatic parasite burden. Because of the complexity and magnitude of the sequential fractionation-immunization-challenge approach, we restricted our attention to the two sublibraries that conferred the greatest in vivo protection. From one of these two sublibraries, we identified several groups of cDNAs that afforded protection, including a set of nine novel cDNAs and, surprisingly, a group of five cDNAs that encoded L. donovani histone proteins. At each fractionation step, the cDNA sublibraries or the smaller DNA fractions that afforded in vivo protection against the parasite also induced in vitro parasite-specific T helper 1 immune responses. Our studies demonstrate that immunization with sequential fractions of a cDNA library is a powerful strategy for identifying anti-infective vaccine candidates.

Animals↗

PubMed1999

Defects in the generation of IFN-gamma are overcome to control infection with Leishmania donovani in CC chemokine receptor (CCR) 5-, macrophage inflammatory protein-1 alpha-, or CCR2-deficient mice.

We investigated the immune responses in mice lacking CCR2, CCR5, or macrophage inflammatory protein-1 alpha (MIP-1 alpha), a ligand for CCR5, in two situations: following T cell stimulation or after challenge with Leishmania donovani, an intracellular microbe whose control is dependent on a Th1 immune response. Mice deficient in CCR5, MIP-1 alpha, or CCR2 had reduced IFN-gamma responses following ligation of the TCR. Reduced IFN-gamma responses following PMA and ionomycin were also observed in CD8+ T cells of CCR5-/- and CCR2-/- mice. During the early phases of infection, all three knockout mice had low Ag-specific IFN-gamma responses. However, this reduced IFN-gamma response was overcome during a state of persistent Ag stimulation (chronic infection), and was not associated with an adverse parasitologic outcome in any of the gene-targeted mouse strains. To the contrary, during the late phase of infection, an exaggerated Ag-specific IFN-gamma response was evident in CCR5-/- and MIP-1 alpha-/- mice, and this correlated with an enhanced control of parasite replication. Although granuloma formation was abnormal in each of the knockout mice, there was no correlation between the number or architecture of the granulomas and parasite burden. Collectively, these findings indicate an important role for CCR5, MIP-1 alpha, and CCR2 in granulomatous inflammation, and that CCR5 and MIP-1 alpha, possibly acting through CCR5, might play a deleterious role in the outcome of chronic L. donovani infection. Our data also suggest that there might be cross-talk between TCR and chemokine receptor signaling pathways.

Animals↗

PubMed1999

Race-specific HIV-1 disease-modifying effects associated with CCR5 haplotypes.

Genetic variation in CC chemokine receptor 5 (CCR5), the major HIV-1 coreceptor, has been shown to influence HIV-1 transmission and disease progression. However, it is generally assumed that the same CCR5 genotype (or haplotype) has similar phenotypic effects in different populations. To test this assumption, we used an evolutionary-based classification of CCR5 haplotypes to determine their associated HIV-1 disease-modifying effects in a large well-characterized racially mixed cohort of HIV-1-seropositive individuals. We demonstrate that the spectrum of CCR5 haplotypes associated with disease acceleration or retardation differs between African Americans and Caucasians. Also, we show that there is a strong interactive effect between CCR5 haplotypes with different evolutionary histories. The striking population-specific phenotypic effects associated with CCR5 haplotypes emphasize the importance of understanding the evolutionary context in which disease susceptibility genes are expressed.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Dendritic cell (DC)-based anti-infective strategies: DCs engineered to secrete IL-12 are a potent vaccine in a murine model of an intracellular infection.

Infections with intracellular pathogens such as Leishmania donovani and Mycobacterium tuberculosis pose serious health problems worldwide. Effective vaccines for these pathogens are not available. Furthermore, despite optimal therapy, disease progression is often seen with several intracellular infections. For these reasons, we initiated studies to develop novel anti-infective vaccine and treatment strategies that couple the potent Ag-presenting capacity of dendritic cells (DC) with paracrine delivery of potent anti-infective cytokines such as IL-12 to local immune response sites. We tested this strategy in a murine model of visceral leishmaniasis. Adoptive transfer of DCs pulsed ex vivo with soluble L. donovani Ags (SLDA) to naive mice induced the Ag-specific production of IFN-gamma, and increased the percentage of activation markers on spleen lymphocytes. SLDA-pulsed DCs engineered by retroviral gene transfer techniques to secrete high levels of biologically active murine IL-12 augmented this immune response further. In several different vaccination and immunotherapy protocols, compared with sham-treated mice, animals receiving SLDA-pulsed DCs either before or following infection had 1-3 log lower parasite burdens, and this protection was associated with a pronounced enhancement in the parasite-specific IFN-gamma response. The augmentation of this protection by IL-12-engineered DCs was striking. First, live parasites were not detected in the liver of mice vaccinated with IL-12-transduced, SLDA-pulsed DCs. Second, this parasitological response was associated with a nearly normal liver histology. In contrast, parasites and granulomas were found in mice vaccinated with SLDA-pulsed, nontransduced DCs. Collectively, these studies provide the rationale for the development of potent DC-based immunotherapies.

3T3 Cells↗

PubMed1999

Stimulation of collagen production in an in vitro model for Peyronie's disease.

AIMS OF THIS STUDY: This study evaluated whether human cavernosal myofibroblasts in cell culture is a viable model for the study of the role of oxygen free radicals in the production of collagen types I and III, as observed in Peyronie's disease. METHOD: Human cavernosal cells in primary culture were incubated with 3H-proline in the absence or presence of (i) glyceraldehyde; (ii) alpha-tocopherol (vitamin E); (iii) a combination of the two; or (iv) gamma interferon alone or in combination with glyceraldehyde. Collagen production was monitored after precipitation by specific monoclonal antibody and quantitated using a scintillation counter. RESULTS: Collagen type III was stimulated to higher than baseline values after doses of 10 and 100 microM glyceraldehyde was added and showed suppression of stimulation with incorporation of alpha-tocopherol. There was a 40% increase in collagen type III production as compared to baseline values in glyceraldehyde-treated cells. Collagen type I showed no consistent stimulation or suppression. In glyceraldehyde-stimulated transformed caveronsal cells, alpha-tocopherol treatment caused a 10-60% decrease in collagen type I and III production. With the addition of 100,000 IU/ml gamma interferon, a significant reduction of both collagen types I and III was observed. CONCLUSIONS: The generation of oxygen radicals is associated with the stimulation of collagen production in cavernosal cells. Transformed fibroblasts from cavernosal cells in culture can be utilized to explore possible etiologies of Peyronie's disease and to further evaluate potential medical therapies for this pathological condition.

Cells, Cultured↗

PubMed1999

Disseminated zygomycosis due to Rhizopus schipperae after heatstroke.

A 21-year-old woman suffered heatstroke and developed diarrhea while trekking across south Texas. The heatstroke was complicated by seizures, rhabdomyolysis, pneumonia, renal failure, and disseminated intravascular coagulation. The patient's stool and blood cultures grew Campylobacter jejuni. The patient subsequently developed paranasal and gastrointestinal zygomycosis and required surgical debridement and a prolonged course of amphotericin B. The zygomycete cultured was Rhizopus schipperae. This is only the second isolate of R. schipperae that has been described. R. schipperae is characterized by the production of clusters of up to 10 sporangiophores arising from simple but well-developed rhizoids. These asexual reproductive propagules are produced on Czapek Dox agar but are absent on routine mycology media, where only chlamydospores are observed. Despite multiorgan failure, bacteremia, and disseminated zygomycosis, the patient survived and had a good neurological outcome. Heatstroke has not been previously described as a risk factor for the development of disseminated zygomycosis.

Adult↗

PubMed1998

Human dendritic cell (DC)-based anti-infective therapy: engineering DCs to secrete functional IFN-gamma and IL-12.

An imbalance in the Th1- and Th2-type cytokine responses may allow certain microbes to modify the host response to favor their own persistence. We now show that infection/pulsing of human CD34+ peripheral blood hemopoietic progenitor cell-derived dendritic cells (DCs) with Leishmania donovani promastigotes, Histoplasma capsulatum, and Mycobacterium kansasii impairs the constitutive production of IL-12 from these cells. Thus, strategies aimed at modulating a dysregulated Th1/Th2 response to infection would be of great interest. To both augment the host immune response and deliver potent immunomodulatory cytokines such as IL-12 and IFN-gamma, our goal is to develop a therapeutic strategy using genetically modified, microbial Ag-pulsed DCs. Toward developing such immunotherapies, we used retrovirus-mediated somatic gene transfer techniques to engineer human DCs to secrete biologically active IL-12 and IFN-gamma. DCs pulsed with microbial antigens (e.g., leishmania and histoplasma Ags) were capable of inducing proliferative responses in autologous CD4+ lymphocytes. CD4+ lymphocytes cocultured with IL-12-transduced autologous DCs had enhanced Ag-specific proliferative responses compared with CD4+ lymphocytes cocultured with nontransduced or IFN-gamma- transduced DCs. In this cell culture model system we demonstrate that IL-12 has a negative effect on IL-4 secretion that is independent of its ability to induce IFN-gamma secretion. Taken together, these results indicate that IL-12-transduced DCs may be specifically suited in inducing or down-modulating Ag-specific Th1 or Th2 responses, respectively, and thus may be useful as adjunctive therapy in those intracellular infections in which a dominant Th1 response is critical for the resolution of infection.

3T3 Cells↗

PubMed1998

Genealogy of the CCR5 locus and chemokine system gene variants associated with altered rates of HIV-1 disease progression.

Allelic variants for the HIV-1 co-receptors chemokine receptor 5 (CCR5) and CCR2, as well as the ligand for the co-receptor CXCR4, stromal-derived factor (SDF-1), have been associated with a delay in disease progression. We began this study to test whether polymorphisms in the CCR5 regulatory regions influence the course of HIV-1 disease, as well as to examine the role of the previously identified allelic variants in 1,090 HIV-1 infected individuals. Here we describe the evolutionary relationships between the phenotypically important CCR5 alleles, define precisely the CCR5 regulatory sequences that are linked to the CCR5-delta32 and CCR2-641 polymorphisms, and identify genotypes associated with altered rates of HIV-1 disease progression. The disease-retarding effects of the CCR2-641 allele were found in African Americans but not in Caucasians, and the SDF1-3'A/3'A genotype was associated with an accelerated progression to death. In contrast, the CCR5-delta32 allele and a CCR5 promoter mutation with which it is tightly linked were associated with limited disease-retarding effects. Collectively, these findings draw attention to a complex array of genetic determinants in the HIV-host interplay.

Adolescent↗

PubMed1998

Penile prostheses in the management of impotence in patients with end-stage renal disease.

Erectile dysfunction, which has multifactorial causes including uremia, diabetes mellitus, hypertension, vascular insufficiency, autonomic neuropathy, and psychogenic pathology, occurs in a majority of patients with end-stage renal disease. After evaluation in the Sexual Dysfunction Clinic to exclude reversible disorders that may cause erectile dysfunction, hormonal supplementation, vacuum erection devices, and a self-injection program are offered to patients. Due to concern about patient's immunocompromised status, penile prostheses have not been considered appropriate therapy for those on dialysis or for renal transplant recipients. We report our 8-year experience with penile prostheses in 12 ESRD/renal transplant patients. Eleven patients have maintained their prostheses. Three patients had prostheses with mechanical failures that required reimplantation, and one prosthesis became infected and was explanted. Penile prostheses can be successfully implanted without excessive risk of infection in patients with erectile dysfunction resulting from end-stage renal disease.

Erectile Dysfunction↗