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Vijay Sharma

Publications and source records attributed to Vijay Sharma.

At least 37 records · Page 2Linked to original sources
PubMed2005

Investigation of an outbreak of scrub typhus in the himalayan region of India.

In Indira Gandhi Medical College, Himachal Pradesh, India, during autumn of 2003 (September-November), more than 100 cases of fever of unknown origin (FUO) were reported with 15 ensuing deaths. In addition to all routine investigations and cultures, the Weil-Felix test was incorporated for the investigation of these cases. Antigen was procured from the Central Research Institute, Kasauli. Forty-six percent (45/96) of the cases demonstrated a > or =1:80 titer of agglutinins against OXK antigen. A team from the National Institute of Communicable Diseases, New Delhi, confirmed the antibodies for scrub typhus in some of the serum samples tested for leptospirosis, dengue fever, and rickettsial infections. Twelve blood samples positive for OXK antigen were sent to the Defense Research Development Establishment, Gwalior, for polymerase chain reaction studies, but none of the samples were positive, as all of the patients were already on broad-spectrum antibiotics and had reported to our hospital after 7-10 days of fever. At our institute, the Weil-Felix test has now been rountinely introduced for the investigation of cases of FUO, and the results until April 2004 (150 cases) revealed the presence of other rickettsial infections prevalent in the region. To evaluate the epidemiology and magnitude of the problem, further prospective studies are required.

Agglutination Tests↗
PubMed2004

Clinical resistance of Staphylococcus keratitis to ciprofloxacin monotherapy.

PURPOSE: To review the in vitro susceptibility and the clinical response to 0.3% ciprofloxacin in Staphylococcus aureus keratitis. METHODS: This is a non-comparative case series derived from a prospectively collected database and analysed retrospectively. One hundred and twenty one patients presenting in the cornea service of LV Prasad Eye Institute, Hyderabad, India, between January 1993 and December 2000, and identified to have S. aureus keratitis were included in the study. All patients had received slitlamp evaluation of the cornea and diagnostic microbiologic workup. They were treated with topical antimicrobial therapy based on smear and culture results. All S. aureus isolates were tested for antibiotic susceptibility by disk diffusion and those resistant to ciprofloxacin were confirmed by testing for minimum inhibitory concentration. The patients' clinical response to the antimicrobial therapy was noted. RESULTS: Twenty-five (20.6%) of 121 isolates resistant to ciprofloxacin on in vitro susceptibility testing were further analysed in this study. Fifteen of these 25 (60%) patients were initially treated with topical ciprofloxacin. Twelve of 15 (80%) patients showed no clinical improvement (3-8 days). Based on antibiotic susceptibility results, the antibiotic therapy was modified to fortified cefazolin and gentamicin in seven (58.3%), to vancomycin in one and to chloramphenicol in four cases. The corneal infiltrate resolved in 11 (73.3%) of 15 cases. Two patients required penetrating keratoplasty (PK), one required evisceration, and one patient was lost to follow up. Nine of 25 patients were initially started on fortified cefazolin and gentamicin therapy based on smear positive for gram-positive cocci; this resulted in resolution of infiltrate in 44.4% (4/9) while three required change of antibiotics (vancomycin-2, chloramphenicol-1), one required PK and one patient was lost to follow up. One of 25 patients started and continued on chloramphenicol, showed no response and required PK. CONCLUSIONS: This study shows a significant resistance of S. aureus to many antibiotics including ciprofloxacin and highlights the need for an alternative to ciprofloxacin monotherapy for the treatment of staphylococcal keratitis.

Adolescent↗
PubMed2003

Tacrolimus enhances transforming growth factor-beta1 expression and promotes tumor progression.

BACKGROUND: Immunosuppressive therapy is a risk factor for the increased incidence and metastatic progression of malignancies in organ graft recipients. Transforming growth factor (TGF)-beta(1) has been associated with tumor invasion and metastasis, and we have implicated cyclosporine-associated TGF-beta(1) hyperexpression in tumor progression in mice. METHODS: BALB/c mice or severe combined immunodeficient-beige mice were treated with 2 or 4 mg/kg of tacrolimus, and the effect of treatment on mouse renal cancer cell pulmonary metastasis was investigated. We also determined whether tacrolimus induces TGF-beta(1) expression. Spleens from tacrolimus-treated mice were analyzed for level of expression of TGF-beta(1) mRNA with the use of competitive-quantitative polymerase chain reaction assay, and circulating levels of TGF-beta(1) protein were measured with the use of an enzyme-linked immunosorbent assay. RESULTS: Treatment with tacrolimus resulted in a dose-dependent increase in the number of pulmonary metastases in the BALB/c mice (197+/-16 in untreated mice, 281+/-26 in mice treated with 2 mg/kg of tacrolimus, and 339+/-25 in mice treated with 4 mg/kg of tacrolimus; no treatment vs. 4 mg/kg tacrolimus, Bonferroni's P<0.001) and in the severe combined immunodeficient-beige mice (117+/-18 in untreated mice, 137+/-19 in mice treated with 2 mg/kg of tacrolimus, and 216+/-29 in mice treated with 4 mg/kg of tacrolimus; no treatment vs. 4 mg/kg tacrolimus, P<0.05). Treatment with 4 mg/kg but not 2 mg/kg of tacrolimus resulted in a significant increase in the levels of expression of TGF-beta(1) mRNA and circulating levels of TGF-beta(1) protein. CONCLUSIONS: Tacrolimus has a dose-dependent effect on tumor progression and TGF-beta(1) expression, and tacrolimus-induced TGF-beta(1) overexpression may be a pathogenetic mechanism in tumor progression.

Animals↗
PubMed2003

Molecular imaging of protein-protein interactions: controlled expression of p53 and large T-antigen fusion proteins in vivo.

Protein-protein interactions control essential steps in signal transduction pathways and other intracellular processes, and assembly of protein complexes modulates and responds to the regulatory events that exist in living animals. We have used microPET and fluorescence imaging to detect interactions between p53 tumor suppressor and large T antigen (TAg) of SV40 virus in a tetracycline-inducible two-hybrid system. To additionally validate this molecular imaging technique, we investigated whether expression of the reporter gene, comprised of a mutant thymidine kinase from herpes simplex virus 1 fused to green fluorescent protein could quantify relative differences in amounts of interacting hybrid proteins. In HeLa cells stably transfected with the reporter gene and interacting (p53-TAg) or noninteracting (p53 and polyoma virus coat protein) pairs of proteins, treatment with doxycycline produced time- and dose-dependent increases in expression of hybrid proteins. Proportional increases in amounts of reporter gene were produced only in cells expressing p53 and TAg. In mice bearing xenografts of these stably transfected HeLa cells, amounts of hybrid proteins were regulated with doxycycline. Both microPET imaging and biodistribution studies showed time- and dose-dependent increases in accumulation of the reporter substrate 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine only in p53-TAg tumors. Fluorescence microscopy of excised tumors also showed corresponding changes in expression of the fusion reporter gene in response to binding of p53 and TAg. These data demonstrate that the imaging two-hybrid system responds in a proportional fashion to increasing amounts of interacting proteins in vivo.

Acyclovir↗
PubMed2003

Metalloantimalarials: targeting of P. falciparum strains with novel iron(III) and gallium(III) complexes of an amine phenol ligand.

Emergence of chloroquine (CQ)-resistant Plasmodium falciparum strains necessitates discovery of potent and inexpensive antimalarial drugs. The high cost of new drugs negatively impacts their access and distribution in the regions of the world with scarce economic resources. While exploring structure-activity relationships, using gallium(III) as a surrogate marker for iron(III), we found cationic, and moderately hydrophobic, compounds, [[1,12-bis(2-hydroxy-3-ethyl-benzyl)-1,5,8,12-tetraazadodecane]metal(III)](+) (metal = Fe(III) and Ga(III); [Fe-3-Eadd](+), 3; [Ga-3-Eadd](+), 4), that possessed antimalarial activity. Crystal structure analyses revealed octahedral geometry for these complexes. The RP-HPLC analysis, after incubation in PBS or HEPES buffer (pH 7.4) at 37 degrees C for 3 days, detected only parental compounds thereby providing evidence for stability under physiological conditions. Both 3 and 4 demonstrated promising half-maximum inhibitory concentration (IC(50)) values of approximately 80 and 86 nM in the CQ-sensitive HB-3 line, respectively. However, both 3 and 4 were found to possess elevated IC(50) values of 2.5 and 0.8 microM, respectively, in the CQ-resistant Dd2 line, thus displaying preferential cytotoxicity toward the CQ-sensitive HB3 line. In cultured parasites, 3 and 4 targeted hemozoin formation. Thus, these compounds acted similarly to chloroquine with regard to action and resistance, despite the lack of structural similarity to quinolines. Finally, similarity in coordination chemistry, stability, and antimalarial cytotoxicity profiles indicated that gallium(III) ion can serve as a template for iron(III) in structure elucidation of active molecules in solution.

Amines↗
PubMed2003

Synthesis, characterization, and molecular structure of a gallium(III) complex of an amine-phenol ligand with activity against chloroquine-sensitive Plasmodium falciparum strains.

Emergence of chloroquine-resistant Plasmodium falciparum strains necessitates discovery of novel antimalarial drugs, especially if the agents can be synthesized from commercially available, inexpensive precursors via short synthetic routes. While exploring structure-activity relationships, we found a gallium(III) complex, [(1,12-bis(2-hydroxy-5-methoxybenzyl)-1,5,8,12-tetraazadodecane)-gallium(III)](+) [Ga-5-Madd](+), 1, that possessed antimalarial efficacy. Like previously reported complexes, the crystal structure of 1 revealed gallium(III) in a symmetrical octahedral environment surrounded by four secondary amine nitrogen atoms in equatorial plane and two axial oxygen atoms. In contrast to a previously reported complex, [Ga-3-Madd](+), this novel metallo-antimalarial 1 possessed modest efficacy against chloroquine-sensitive HB3 Plasmodium lines. Thus, slight variation in the positions of methoxy functionalities on the aromatic rings of the organic scaffold dramatically altered specificity thereby suggesting a targeted (e.g., transporter- or receptor-mediated) rather than non-specific (e.g., pH or other gradient-mediated) mechanism of action for these agents.

Amines↗
PubMed2003

Selective loss of c-Rel compromises dendritic cell activation of T lymphocytes.

Dendritic cells initiate the immune response by presenting antigen in the context of varying levels of costimulation. The maturation state of the dendritic cell determines the quantity and quality (Th1, Th2) of the subsequent T cell response. Members of the NF-kappaB family of transcription factors have previously been implicated in dendritic cell development. Here, we used a mouse with a homozygous c-Rel deletion to investigate the role of c-Rel in the function of bone marrow derived dendritic cells. When direct presentation was evaluated, we found c-Rel(-/-) dendritic cells induce less allogeneic T cell stimulation than c-Rel(+/+) dendritic cells. In addition, T cell encounters with c-Rel(-/-) dendritic cells generate less IFN-gamma and IL-4 when compared to those with c-Rel(+/+) DCs. A similar degree of functional compromise was observed in antigen-specific T cells that were stimulated by c-Rel(-/-) dendritic cells. Functional deficits were not linked to differences in the ability to undergo maturation per se, as LPS exposure induced similar morphologic and cell surface changes in both c-Rel(+/+) and cRel(-/-) DCs. Although LPS induced a compensatory increase in the nuclear activity of fellow NF-kappaB family members, RelB and p65, LPS exposure was unable to negate the deficiencies in autologous T cell proliferation and cytokine production associated with the loss of c-Rel in dendritic cells. Taken together, our study supports a unique and non-redundant role for c-Rel in dendritic cell costimulatory capacity.

Animals↗
PubMed2003

Visualizing protein-protein interactions in living animals.

A variety of techniques have been developed to analyze protein-protein interactions in vitro and in cultured cells. However, these methods do not determine how protein interactions affect and are regulated by physiologic and pathophysiologic conditions in living animals. This article describes methodology for detecting and quantifying protein interactions in living mice, using an inducible two-hybrid system developed for positron emission tomography (PET) imaging. We discuss the methods to establish stably transfected cells with components of the imaging system, create tumor xenografts, synthesize PET radiopharmaceuticals used to visualize the imaging reporter, perform microPET imaging, and analyze data from imaging studies. Development and application of technologies for molecular imaging of protein-protein interactions in vivo should enable researchers to investigate intrinsic binding specificities of proteins during normal development and disease progression as well as aid drug development through direct interrogation of molecular targets within intact animals.

Animals↗
PubMed2003

Synthesis and characterization of a Gd-DOTA-D-permeation peptide for magnetic resonance relaxation enhancement of intracellular targets.

Many MR contrast agents have been developed and proven effective for extracellular nontargeted applications, but exploitation of intracellular MR contrast agents has been elusive due to the permeability barrier of the plasma membrane. Peptide transduction domains can circumvent this permeability barrier and deliver cargo molecules to the cell interior. Based upon enhanced cellular uptake of permeation peptides with D-amino acid residues, an all-D Tat basic domain peptide was conjugated to DOTA and chelated to gadolinium. Gd-DOTA-D-Tat peptide in serum at room temperature showed a relaxivity of 7.94 +/- 0.11 mM(-1) sec(-1) at 4.7 T. The peptide complex displayed no significant binding to serum proteins, was efficiently internalized by human Jurkat leukemia cells resulting in intracellular T1 relaxation enhancement, and in preliminary T1-weighted MRI experiments, significantly enhanced liver, kidney, and mesenteric signals.

Animals↗
PubMed2003

AgNOR counts in cervical lesions.

Present study was undertaken with a view to assess the utility of AgNOR counts in differentiating cervical lesions. One hundred cases of cervical lesions including normal, chronic cervicitis, CIN (I, II, III) and carcinoma were taken up for study during the period December 1995 to November 1996. Three micron thick sections of paraffin were subjected to AgNOR staining as described by Crocker and Smith with a little modification of 0.01% safranin counterstain. Statistical counts of AgNORs were done. The mean AgNOR counts in cervical epithelium showed a progressive and statistically significant increase from normal to chronic cervicitis to CIN, I, II and III (P<0.001). Scores in carcinoma also exceeded that of CIN (P<0.05). This can prove to be a useful adjunct to routine histopathology to evaluate cervical lesions.

Cervix Uteri↗
PubMed2002

Imaging pulmonary gene expression with positron emission tomography.

We evaluated positron emission tomographic imaging of pulmonary transgene expression, using an enhanced mutant herpes simplex virus-1 thymidine kinase as the reporter gene, in the lungs of normal rats. Sixteen rats were studied 3 days after an intratracheal administration of 5 x 10(9) to 1 x 10(11) viral particles of a replication-incompetent adenovirus containing a fusion gene of the mutant kinase and green fluorescent protein. Three rats infected with adenovirus containing no insert (null vector) served as control subjects. Images were obtained 1 hour after an intravenous injection of 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine, an imaging substrate for the viral kinase. After euthanasia, tissue radioactivity was determined in a gamma counter, and thymidine kinase activity and green fluorescent protein levels were measured in lung tissue samples. Imaging and gamma counting radioactivity measurements were strongly and linearly correlated (r2 = 0.96, p < 0.001). Imaging detected thymidine kinase expression above background (null vector) in 15 of 16 rats, even at low viral doses that produced little to no measurable green fluorescent protein expression. Lung 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine uptake (as assessed by imaging) correlated with in vitro assays of both kinase activity (r(2) = 0.48, p < 0.001) and fluorescent protein (r(2) = 0.46, p < 0.001). We conclude that positron emission tomographic imaging is a sensitive and quantitative method for detecting pulmonary reporter gene expression noninvasively.

Analysis of Variance↗
PubMed2002

Evidence for a plasma membrane-mediated permeability barrier to Tat basic domain in well-differentiated epithelial cells: lack of correlation with heparan sulfate.

Membrane permeation peptides, such as Tat basic domain, have emerged as useful membrane transduction agents with potential utility in therapeutic delivery and diagnostic imaging. While generally thought to universally permeate all cells by a nonselective process, the mechanism of membrane transduction remains poorly characterized. To examine vectorial transport properties of Tat basic domain in well-differentiated epithelial cells possessing tight junctions, L and D stereoisomers of Tat(48-57) peptide conjugates labeled with (99m)Tc were quantitatively analyzed in confluent monolayers of MDCK renal epithelial and CaCo-2 colonic carcinoma cells grown in transwell configurations. In both cell lines, vectorial transepithelial apparent permeability coefficients (P(app)) for L- and D-[(99m)Tc]Tat-peptides ranged from 30 to 70 nm/s, comparable to values for the macromolecular impermeant marker inulin in both apical-to-basolateral and basolateral-to-apical directions, but 100-fold less than the P(app) values for propranolol, a highly permeable control compound. Upon direct instillation of [(99m)Tc]Tat-peptide into the urinary bladder of living rats in vivo, no transepithelial permeation into other tissues was identified. Furthermore, MDCK and CaCo-2 cells showed a complete lack of intracellular accumulation of fluorescein conjugated Tat-peptide. However, translocation into cells was induced by treatment with plasma membrane permeabilizing agents such as digitonin and acetone/methanol, while cholesterol depletion with beta-methyl-cyclodextrin and metabolic inhibition with CCCP or 4 degrees C showed no effect. By contrast, in Hela and KB 3-1 cells, epithelial lines that do not form tight junctions in monolayer culture, baseline cytoplasmic and nucleolar accumulation was readily observed. Because all four cell lines expressed heparan sulfate proteoglycans, putative receptors for Tat basic peptides, we found no correlation between heparan sulfate and the permeation barrier observed in MDCK and CaCo-2 cells. The unanticipated presence of a permeation barrier to Tat-peptides in well-differentiated epithelial cells suggests the existence of cell-specific mechanisms for mediated translocation of these permeation peptides.

Administration, Intravesical↗
PubMed2002

Noninvasive imaging of protein-protein interactions in living animals.

Protein-protein interactions control transcription, cell division, and cell proliferation as well as mediate signal transduction, oncogenic transformation, and regulation of cell death. Although a variety of methods have been used to investigate protein interactions in vitro and in cultured cells, none can analyze these interactions in intact, living animals. To enable noninvasive molecular imaging of protein-protein interactions in vivo by positron-emission tomography and fluorescence imaging, we engineered a fusion reporter gene comprising a mutant herpes simplex virus 1 thymidine kinase and green fluorescent protein for readout of a tetracycline-inducible, two-hybrid system in vivo. By using micro-positron-emission tomography, interactions between p53 tumor suppressor and the large T antigen of simian virus 40 were visualized in tumor xenografts of HeLa cells stably transfected with the imaging constructs. Imaging protein-binding partners in vivo will enable functional proteomics in whole animals and provide a tool for screening compounds targeted to specific protein-protein interactions in living animals.

Animals↗
PubMed2002

Molecular imaging of gene expression and protein function in vivo with PET and SPECT.

Molecular imaging is broadly defined as the characterization and measurement of biological processes in living animals, model systems, and humans at the cellular and molecular level using remote imaging detectors. One underlying premise of molecular imaging is that this emerging field is not defined by the imaging technologies that underpin acquisition of the final image per se, but rather is driven by the underlying biological questions. In practice, the choice of imaging modality and probe is usually reduced to choosing between high spatial resolution and high sensitivity to address a given biological system. Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) inherently use image-enhancing agents (radiopharmaceuticals) that are synthesized at sufficiently high specific activity to enable use of tracer concentrations of the compound (picomolar to nanomolar) for detecting molecular signals while providing the desired levels of image contrast. The tracer technologies strategically provide high sensitivity for imaging small-capacity molecular systems in vivo (receptors, enzymes, transporters) at a cost of lower spatial resolution than other technologies. We review several significant PET and SPECT advances in imaging receptors (somatostatin receptor subtypes, neurotensin receptor subtypes, alpha(v)beta(3) integrin), enzymes (hexokinase, thymidine kinase), transporters (MDR1 P-glycoprotein, sodium-iodide symporter), and permeation peptides (human immunodeficiency virus type 1 (HIV-1) Tat conjugates), as well as innovative reporter gene constructs (herpes simplex virus 1 thymidine kinase, somatostatin receptor subtype 2, cytosine deaminase) for imaging gene promoter activation and repression, signal transduction pathways, and protein-protein interactions in vivo.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2002

Vanadium increases GLUT4 in diabetic rat skeletal muscle.

The effect of vanadium in lowering blood glucose in diabetic animals is well established; however, the exact mechanism of action of vanadium still eludes us. There are several reports from in vitro studies indicating that vanadium increases enzyme activity in insulin signalling pathways; however, these findings have not been duplicated in vivo. Glucose transporters (GLUT) have a major role to play in any glucoregulatory effects. Insulin dependent GLUT4 is a major glucose transporter present in skeletal muscle, adipocytes and heart. In the present study we found that the plasma glucose in streptozotocin (STZ) diabetic animals was restored to normal following treatment with a single dose of BMOV, an organic vanadium compound, given by oral gavage (0.6 mmol/kg), similar to the response with chronic BMOV treatment. The response to BMOV by oral gavage was rapid and the animals were normoglycemic within 24 h of treatment and still demonstrated a significant effect even after 72 h. Using a specific antibody against GLUT4 we found an overall reduction in the GLUT4 in the total membrane fraction in skeletal muscle of diabetic animals. However, with a single dose of BMOV the GLUT4 level was restored to normal. This is the first report that establishes a direct effect of vanadium on the regulation of GLUT4 expression in diabetic animals in vivo, and may at least partially explain the glucoregulatory effects of vanadium.

Animals↗
PubMedDate not supplied

Characterization of novel histidine-tagged Tat-peptide complexes dual-labeled with (99m)Tc-tricarbonyl and fluorescein for scintigraphy and fluorescence microscopy.

To enable concurrent whole body scintigraphy and direct imaging of subcellular localization of permeation peptides, dual-labeled Tat-peptides useful for both radiometric analysis and fluorescence microscopy are desired for molecular imaging applications. Thus, novel dual-labeled D-Tat-peptides comprising Tat-basic domain (hgrkkrrqrrrgc), C-terminus conjugated with fluorescein-5-maleimide (FM) and N-terminus chelated with [(99m)Tc(CO)(3)] via histidine coordination, were synthesized and characterized. In human Jurkat cells, radiotracer uptake and washout studies revealed concentration-dependent accumulation of the dual-labeled Tat-peptide within cells. Subcellular localization of Tat-peptide was confirmed by fluorescence microscopy using an analogous [Re(CO)(3)] dual-labeled Tat-peptide. As seen with C-terminus single-labeled Tat-peptides, localization to the nucleoli was observed with the dual-labeled Tat-peptide, suggesting that the mechanism of Tat-peptide uptake and localization was not dependent on free peptide termini at either end. In Balb/c mice, biodistribution studies performed with the dual-labeled Tat-peptide showed fluorescence intensity by microscopic analysis that visually confirmed and correlated directly with scintigraphic and radiometric data. Of note, following intravenous administration, little brain penetration of these permeation sequences was observed in vivo. His[(99m)Tc(CO)(3)]-, DTPA[(99m)Tc(CO)(3)]-, and epsilon-lys-gly-cys[(99m)Tc(O)]-labeled Tat-peptides showed significant pharmacokinetic differences in liver and kidney depending on labeling strategy, indicating that Tat-peptide biodistribution can be impacted by the chelation moiety coordinated with (99m)Tc. Thus, we have shown that dual-labeled (99m)Tc-tricarbonyl Tat-peptide-FM conjugates can be conveniently synthesized and enable direct comparison of quantitative radiometric and qualitative fluorescence data both in vitro as well as in vivo.

Animals↗