Biomedical subjects
Publications and source records attributed to S Roy.
The industrialization of proteomics demands reproducible, robust and high-throughput profiling technologies that current two-dimensional gel electrophoresis cannot achieve. New technologies in protein arrays, either on chips or with self-encoded elements in solution, hold much promise for interrogating the diverse and immense proteome.
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Leishmania donovani, an obligate intracellular parasite resides and multiplies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishmaniasis has not yet been clearly established. Generation of superoxide anion (O2-) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macrophages challenged with Lipophosphoglycan (LPG) deficient attenuated leishmanial parasite UR-6. The generation of O2- essentially needs the prior activation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenuated during infection. This was supported by PKC activity study, where Ca-dependent PKC activity was inhibited but, Ca-independent PKC activity was enhanced. This result was further confirmed by using isotype specific pseudosubstrate inhibitors of Ca-dependent PKC beta and Ca-independent PKC zeta. Application of beta-pseudosubstrate could not alter the Ca-dependent PKC activity but zeta-pseudosubstrate inhibited the Ca-independent PKC activity in infected macrophages. Our immunoblot analysis with specific antibody against PKC beta and PKC zeta isotypes showed down regulation of PKC beta-II expression with concomitant induction of PKC zeta. Such inhibition of Ca-dependent PKC beta was reversed in macrophages treated with UR-6. Taken together, our observations revealed that infection with L. donovani selectively attenuates both the expression and activity of Ca-dependent PKC beta.
PURPOSE: To evaluate the clinical outcome of patients who received a Baerveldt implant for refractory glaucoma and to identify factors which may influence the outcome. METHODS: Retrospective study including 51 eyes of 51 patients with medically uncontrolled glaucoma who underwent Baerveldt implant surgery between June 1994 and December 1998. Criteria for success were intraocular pressure (IOP) < or = 21 mmHg and > 6 mmHg, necessity of further antiglaucoma medications, absence of additional glaucoma surgery and no loss of light perception. RESULTS: Over a mean follow-up of 37.6 (SD: +/-18.8) months, the mean intraocular pressure decreased from 34.8 (+/-12.5) mmHg to 14.0 (+/-4.3) mmHg at month 60. Qualified success rate, achieved when IOP was below 21 mmHg and higher than 6 mmHg with medications was 25/48 (52%), complete success rate (same IOP limits without medication) was 14/48 (29%). Seven eyes had major complications or lost light perception. Postoperative visual acuity improved or remained within one Snellen line of the preoperative visual acuity in 35 patients (73%). Factors associated with a better prognosis were a preoperative visual acuity better than 20/400 and etiology of glaucoma. CONCLUSION: The Baerveldt implant is effective in lowering intraocular pressure in most patients with refractory glaucoma. Long-term results are promising with satisfactory IOP control.
AIM: to study age-related Schlemm's canal endothelial changes and evaluate consequences on the filtration function. MATERIAL AND METHODS: the inner wall endothelium of Schlemm's canal was examined in 9 non-glaucomatous subjects aged between 32 and 75 years, by a combined technique of light and electron microscopy (scanning and transmission). Quantitative analysis included counts of bulges, pores, nuclei, giant vacuoles and other protruding structures, as well as measures of pores, giant vacuoles and Schlemm's canal size parameters (diameter and inner wall width). Outflow facility calculations were realised using a modified previously described mathematical model. RESULTS: The main structures affected by ageing in Schlemm's canal appeared to be giant vacuoles. Their density but also their size is significantly reduced with increasing age. The intracellular pore population is also found to diminish with age and is correlated to that of giant vacuoles, suggesting that those pores are luminal openings of vacuoles. Outflow facility calculations revealed a global decrease of endothelial outflow facility of about 60% between the 3rd and 7th decades. The study also showed a different age-related pattern for the two subtypes of endothelial pores. CONCLUSIONS: Our study demonstrated that Schlemm's canal filtration function is significantly influenced by age, as the endothelial inner wall outflow facility is found to be widely reduced. This is partly the result of an age-related reduction in counts of giant vacuoles and intracellular pores. The second pore population (border or intercellular) doesn't follow the same evolution, but may have a more significant regulator role in transendothelial permeability.
In the present study, we examined the involvement of intracellular ceramide in host pathogen interaction of BALB/c mouse peritoneal macrophages infected with the obligate intracellular protozoan, Leishmania donovani. Our findings indicate that the level of intracellular ceramide was enhanced as a result of the in vitro infection. While the elevated ceramide was largely due to de novo synthesis, activation of the sphingomyelinases was also observed. The enhanced ceramide was responsible for the downregulation of classical PKC activity, upregulation of calcium independent atypical PKC-zeta expression and activity of calcium independent PKC. Ceramide also impaired the phosphorylation of MAPK. Evidently, ceramide suppressed the generation of nitric oxide during leishmanial infection and also facilitated the survival of leishmanial parasites in the intramacrophageal milieu. These data present newer insight to the signaling events in leishmania-infected murine macrophages, which might offer ceramide as a new therapeutic target in the future.
PURPOSE: To compare the intraocular pressure (IOP) values obtained using a Goldmann tonometer (Haag-Streit) with those obtained with the new Tonopen XL (Mentor), which has certain differences compared with first- and second-generation models. METHODS: The IOPs of 104 patients were assessed by Goldmann tonometer and Tonopen XL tonometer. Goldmann measurements was done first in 145 eyes and Tonopen measurements were done first in 53 eyes. Four observers measured the IOP. Observers A, B and C used the Goldmann tonometer first and then the Tonopen XL, while observer D used the Tonopen XL first and then the Goldmann tonometer. The results were analysed by descriptive analysis and, when the distribution of the data was normal, paired t-test and Pearson's r coefficient were used to compare and correlate IOP measurements between Goldmann and Tonopen measurements. When the distribution of the data was non-normal, the Wilcoxon matched-pair test and Spearman coefficient were used. The agreement between Goldmann and Tonopen values was also calculated. ANOVA test was used to compare the difference obtained by 'Goldmann minus Tonopen' measurements among the three different observers. RESULTS: A statistically significant difference (p < 0.0001) was found between the IOP readings obtained by Goldmann tonometer and the Tonopen XL and a significant correlation was found between the Goldmann values and Tonopen XL values (p < 0.001). When the Goldmann IOP was more than 20 mmHg the Tonopen XL measurements were lower than the Goldmann values. Also in this group this difference was statistically significant. No significant difference was found between Goldmann values and Tonopen values among the three observers, even though a significant difference was found between Goldmann values and Tonopen values for observer B. When the values obtained by first the Goldmann tonometer and then the Tonopen XL were compared with those obtained by first the Tonopen XL and then the Goldmann tonometer, no significant difference was found between the two groups. CONCLUSION: The new Tonopen XL provides similar results to the Goldmann tomometer in 62% of the cases and was slightly less accurate than the Goldmann tonometer for extreme values, just like the previous Tonopen. Nevertheless the precision is good enough for the purpose of adequate screening.
Parkinson's disease (PD) is a progressive neurodegenerative disorder of the basal ganglia, associated with the inappropriate death of dopaminergic neurons of the substantia nigra pars compacta (SNc). Here, we show that adenovirally mediated expression of neuronal apoptosis inhibitor protein (NAIP) ameliorates the loss of nigrostriatal function following intrastriatal 6-OHDA administration by attenuating the death of dopamine neurons and dopaminergic fibres in the striatum. In addition, we also addressed the role of the cysteine protease caspase-3 activity in this adult 6-OHDA model, because a role for caspases has been implicated in the loss of dopamine neurons in PD, and because NAIP is also a reputed inhibitor of caspase-3. Although caspase-3-like proteolysis was induced in the SNc dopamine neurons of juvenile rats lesioned with 6-OHDA and in adult rats following axotomy of the medial forebrain bundle, caspase-3 is not induced in the dopamine neurons of adult 6-OHDA-lesioned animals. Taken together, these results suggest that therapeutic strategies based on NAIP may have potential value for the treatment of PD.
Morphological and functional heterogeneity of the splenic macrophage (M phi) population was studied in Leishmania donovani (LD) infected BALB/c mice. On a discontinuous percoll gradient two distinct M phi populations were separated. They differed significantly in size as evident from Scanning Electron Microscopy (SEM). Morphologically, the bigger M phi (LM) showed surface projections, whereas the smaller M phi (SM) was round. As regards the antigen-presenting abilities, the LM of infected animals showed defective antigen-presenting abilities at a later stage of the disease, i.e. 6 months post infection ((6)I-LM) but not earlier, whereas the SM population remained functionally intact throughout the course of the infection. Further, the (6)I-LM showed a much enhanced Ad status as compared to their controls. Interestingly, both the (6)I-LM and the control set showed a comparable level of binding of a known A(d) restricted peptide. Despite the presence of sufficient A(d) molecules and the ability to bind the appropriate peptide, (6)I-LM were unable to stimulate peptide specific T-cell hybridoma. Further, the (6)I-LM showed an increase in membrane fluidity and distorted morphology with membrane fissure and blebs as evident from SEM. It is possible that an increase in the membrane fluidity may lead to the defective antigen-presenting ability of (6)I-LM. Thus, the LD infection functionally keep the (6)I-LM out of antigen presentation and this may contribute to the defective cell mediated immune response in leishmaniasis.
PURPOSE: The purpose of this study was to compare the aqueous humor outflow using three type of implants after deep sclerectomy. A numeric imaging system with ICG and fluorescein angiography was used. The evolution over time was recorded, while filtration and outflow facility were measured. MATERIAL AND METHODS: Numeric imaging system with ICG and fluorescein angiography were performed before and after deep sclerectomy at regular intervals after surgery. Both resorbable (collagen) and non-resorbable (hema) intrascleral devices have been tested in eight rabbits. The resorption time of the collagen implants has been monitored using UBM technique. The outflow facility was measured with the technique of anterior chamber canulation and constant pressure method. RESULTS: The resorbable collagen implants could be observed with UBM and angiography during the first six weeks post-operatively. The image of the non-resorbable Hema implant remained clearly visible over time. New drainage vessels were observed around the filtering bleb. The filtration significantly increased as the outflow facility improved after surgery, while the intraocular pressure remained stable over time. CONCLUSION: This conjunction of UBM, angiography and outflow measurement allowed us to study and compare three types of implants used as space maintainer after deep sclerectomy, and analyse their effect on the aqueous filtration and outflow facility.
BACKGROUND: A new technique for permanent sectioning of the human spinal cord has provided superior images over those produced with traditional methods. Application of this technique for sections of the human larynx may yield cost-effective, efficient, and accurate laryngeal anatomic dissections. STUDY DESIGN AND METHODS: This study was designed to evaluate this technique for dissections of the human larynx. Laryngeal sections from cadavers were submerged in a celloidin solution, a derivative of wallpaper plaster, and frozen to -15 degrees C. After preparation, axial and coronal cuts of 100 microm were made with a Macrocut Tome sectioning system. RESULTS: Sections were completed in approximately 30 hours. Digitized photographs of the laryngeal sections provide detailed images of precise anatomic relationships. CONCLUSION: Celloidin-based sectioning of the human larynx yields precise anatomic information beyond standard radiographic imagining and previous permanent laryngeal sectioning techniques in a cost-efficient and timely manner. Black and white fine-section photographs are provided.
BACKGROUND: Failure of cell-mediated immunity is thought to increase the morbidity and mortality rates after trauma and major surgical procedures and to be the result, in part, of a redirection of CD4(+) T cells toward T(H2) differentiation. We tested the hypothesis that morphine treatment after injury promotes T(H2) differentiation of precursor T cells through the mu-opioid receptor. METHODS: Human peripheral blood mononuclear cells (PBMCs) or splenocytes from either wild type or mu-opioid receptor knock-out mice were treated in vitro with either vehicle or morphine and then stimulated with anti-CD3/anti-CD28. The supernatant was assayed for T(H1) (interleukin-2 [IL-2], interferon gamma [IFN gamma]) and T(H2) (IL-4, IL-5) cytokines (enzyme-linked immunosorbent assay). Morphine regulation of IL-4 transcription was investigated in PBMCs (IL-4 messenger RNA, nuclear factor of activated T-cells) and Jurkat T cells transfected with a murine IL-4 promoter-luciferase construct. Morphine-induced nuclear factor of activated T-cell (NFAT) binding was assayed with the electromobility shift assay in Jurkat T cells. RESULTS: Morphine treatment of PBMCs decreases IL-2 and IFN gamma and increases IL-4 and IL-5 as a function of morphine concentration. Morphine treatment in wild type splenocytes inhibited IFN gamma and stimulated IL-4 protein synthesis. Changes in cytokine synthesis were abolished in mu-opioid receptor knockout mice. Morphine treatment increases IL-4 messenger RNA accumulation in PBMCs and increases IL-4 promoter activity in Jurkat T cells. Morphine increases NFAT nuclear protein binding to an NFAT DNA response element. CONCLUSIONS: We conclude that morphine treatment promotes T(H2) differentiation through a mu-opioid receptor mechanism and that morphine treatment increases IL-4 transcription, in part, through an NFAT mechanism.
PURPOSE: In this study the acute effect of the topically-delivered non-selective beta-blockers timolol and carteolol, and the selective beta-blocker betaxolol, were evaluated with respect to ocular blood flow, intraocular pressure (IOP) and vessel resistance in rabbits' eyes. METHODS: In a double masked randomized design, one eye of each subject (n = 9) received two drops of 0.5 % timolol or 2 % cartelol or 0.5 % betaxolol ophthalmic solution and a separate group of nine rabbits received two drops of placebo consisting of physiological saline in both eyes to serve as control. Using hydrogen clearance method, ciliary body blood flow (CiBF), choroidal blood flow (CBF), and retinal blood flow (RBF) were measured. IOP and systemic mean arterial pressure (MAP) of each subject were measured under same condition before and after the administration of respective drugs to calculate the ocular perfusion pressure (OPP) and vessel resistance. RESULTS: In timolol- and carteolol-treated eyes significant reduction was observed in IOP (p < 0.01), CiBF (p < 0.01), CBF (p < 0.01) and RBF (p < 0.01) compared to control eyes. However, in betaxolol-treated eyes a marginal reduction in IOP was observed accompanied by significant increase in CiBF (p < 0.01) and RBF (p < 0.05). The non-selective beta-blocker-treated eyes tended to have increased vessel resistance, whereas, selective beta-blocker-treated eyes tended to have decreased vessel resistance. CONCLUSIONS: Our current results comparing non-selective and selective beta-blockers suggest that the selective beta-blocker betaxolol may be more appropriate for maintenance of retinal blood flow in situations with low perfusion. Currently the mechanism for regulation of IOP is unclear; however, the findings from this study indicate that decreased CiBF may contribute to reduction in IOP.
To evaluate some of the mechanisms involved in the plasma cholesterol lowering of sitostanol (SI), male Hartley guinea pigs were fed diets containing cholesterol (0.25 g/100 g) and four doses of SI: either 0 (control), 0.75, 1.5 or 2.25 g/100 g. In addition a negative control (-C) group with dietary cholesterol (0.04 g/100 g) was included. Corn oil was used as the source of fat and the contribution of fat energy was 35 %. Plasma total cholesterol was 43, 49 and 53 % (P < 0.0001) lower after SI intake compared to the control. Plasma LDL concentrations were 47, 53 and 61 % lower with increasing doses of SI. In addition, intake of SI resulted in 26-42 % lower hepatic total cholesterol. Hepatic esterified cholesterol and triacylglycerols were 32-60 % and 55-61 % lower after SI intake. SI intake resulted in favourable plasma and hepatic cholesterol concentrations similar to those in guinea pigs fed low levels of dietary cholesterol (-C). The LDL obtained from the control group had a higher number of molecules of free and esterified cholesterol than the SI groups. SI intake resulted in 69-71 % higher cholesterol excretion compared to the control. SI treatment enhanced the total faecal neutral sterol excretion by 54-58 % compared to control and by 70-76 % compared to the (-C) group. These results suggest that SI might have its hypocholesterolaemic effect by reducing cholesterol absorption, which results in lower concentration of cholesterol in liver. This reduction in hepatic cholesterol might possibly alter hepatic cholesterol metabolism and affect lipoprotein concentration and composition.
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Fifty districts of Bangladesh and 9 districts in West Bengal, India have arsenic levels in groundwater above the World Health Organization's maximum permissible limit of 50 microg/L. The area and population of 50 districts of Bangladesh and 9 districts in West Bengal are 118,849 km2 and 104.9 million and 38,865 km2 and 42.7 million, respectively. Our current data show arsenic levels above 50 microg/ L in 2000 villages, 178 police stations of 50 affected districts in Bangladesh and 2600 villages, 74 police stations/blocks of 9 affected districts in West Bengal. We have so far analyzed 34,000 and 101,934 hand tube-well water samples from Bangladesh and West Bengal respectively by FI-HG-AAS of which 56% and 52%, respectively, contained arsenic above 10 microg/L and 37% and 25% arsenic above 50 microg/L. In our preliminary study 18,000 persons in Bangladesh and 86,000 persons in West Bengal were clinically examined in arsenic-affected districts. Of them, 3695 (20.6% including 6.11% children) in Bangladesh and 8500 (9.8% including 1.7% children) in West Bengal had arsenical dermatological features. Symptoms of chronic arsenic toxicity developed insidiously after 6 months to 2 years or more of exposure. The time of onset depends on the concentration of arsenic in the drinking water, volume of intake, and the health and nutritional status of individuals. Major dermatological signs are diffuse or spotted melanosis, leucomelanosis, and keratosis. Chronic arsenicosis is a multisystem disorder. Apart from generalized weakness, appetite and weight loss, and anemia, our patients had symptoms relating to involvement of the lungs, gastrointestinal system, liver, spleen, genitourinary system, hemopoietic system, eyes, nervous system, and cardiovascular system. We found evidence of arsenic neuropathy in 37.3% (154 of 413 cases) in one group and 86.8% (33 of 38 cases) in another. Most of these cases had mild and predominantly sensory neuropathy. Central nervous system involvement was evident with and without neuropathy. Electrodiagnostic studies proved helpful for the diagnosis of neurological involvement. Advanced neglected cases with many years of exposure presented with cancer of skin and of the lung, liver, kidney, and bladder. The diagnosis of subclinical arsenicosis was made in 83%, 93%, and 95% of hair, nail and urine samples, respectively, in Bangladesh; and 57%, 83%, and 89% of hair, nail, and urine samples, respectively in West Bengal. Approximately 90% of children below 11 years of age living in the affected areas show hair and nail arsenic above the normal level. Children appear to have a higher body burden than adults despite fewer dermatological manifestations. Limited trials of 4 arsenic chelators in the treatment of chronic arsenic toxicity in West Bengal over the last 2 decades do not provide any clinical, biochemical, or histopathological benefit except for the accompanying preliminary report of clinical benefit with dimercaptopropanesulfonate therapy. Extensive efforts are needed in both countries to combat the arsenic crisis including control of tube-wells, watershed management with effective use of the prodigious supplies of surface water, traditional water management, public awareness programs, and education concerning the apparent benefits of optimal nutrition.
Vascular basement membrane (BM) thickening is a prominent and characteristic lesion of diabetic microangiopathy. Studies suggest that increased synthesis of laminin, a BM component, is associated with the development of thickened BM in diabetic vessels. In this study, we evaluated whether an interventive strategy using laminin antisense phosphorothioate oligonucleotides (Lam AS-oligos) could specifically inhibit high-glucose-induced laminin gene overexpression in vascular endothelial cells and normalize cell proliferation. Rat endothelial cells grown in high-glucose (30 mM) medium for 7 days showed increased laminin mRNA and protein level (195% +/- 28% of control, p < 0.05; 143% +/- 26% of control, p < 0.05, respectively) and reduced cell number (79% +/- 6% of control, p < 0.05) compared with cells grown in normal (5 mM) glucose medium. When cells grown in high-glucose medium were transfected with 0.4 microM Lam AS-oligos for 48 hours in the presence of 8 microM lipofectin, the laminin mRNA and protein level decreased (121% +/- 19% and 99% +/- 15% of control, respectively), and the cell number was restored to near normal level (93% +/- 7% of control). The results indicate that the antisense strategy is effective in selectively reducing laminin overexpression and improving endothelial cell proliferation under high-glucose conditions. Thus, the As-oligos may be potentially useful for preventing the development of thickened vascular BM in diabetic microangiopathy.
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