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R Rai

Publications and source records attributed to R Rai.

At least 19 recordsLinked to original sources
PubMed2025

HLA-E and NKG2A Mediate Resistance to BCG Immunotherapy in Non-Muscle-Invasive Bladder Cancer.

Bacillus Calmette-Guérin (BCG) is the first-line therapy for high-grade non-muscle-invasive bladder cancer (NMIBC), yet many patients experience recurrence due to immune evasion. We identify HLA-E and NKG2A as mediators of adaptive resistance involving chronic activation of NK and T cells in BCG-unresponsive tumors. Prolonged IFN-γ exposure enhances HLA-E and PD-L1 expression on recurrent tumors, accompanied by the accumulation of NKG2A+ NK and CD8 T cells. HLA-Ehigh tumor cells preferentially cluster near CXCL12-rich stromal regions with dense effector cell presence, underscoring a spatially segregated tumor architecture. Although cytotoxic lymphocytes retain effector potential, their activity is restrained by HLA-E/NKG2A and PD-L1/PD-1 pathways located in their immediate neighborhood within the bladder tumor microenvironment. These data reveal a spatially organized immune escape program that limits anti-tumor immunity. Our findings support dually targeting NKG2A and PD-L1 checkpoint blockade as a rational, bladder-sparing strategy for patients with BCG-unresponsive NMIBC.

BCG-unresponsive↗
PubMed2002

Pinopode expression during human implantation.

Implantation failure is considered the major impediment to successful in vitro fertilization pregnancy rate. Despite recent advances in reproductive medicine, the role played by uterine receptivity in contributing to human infertility remains unclear. Several clinical studies suggest the importance of blastocyst implantation in a narrow window of uterine receptivity, between 6 and 8 days after ovulation. This brief and precise period, called "implantation or nidation window", lasts less than 48 h and coincides with the formation of large and smooth projections, called "pinopodes", on the apical membranes of the endometrial epithelial cells. Pinopode expression appears to advance or retard depending on the hormonal treatments and an individual's particular response. This displacement can be followed through endometrial biopsy and scanning electron microscopy (SEM). Therefore, pinopode expression could be considered a useful biological marker to assess endometrial receptivity and to locate the implantation window.

Embryo Implantation↗
PubMed2002

Factor V Leiden and recurrent miscarriage-prospective outcome of untreated pregnancies.

BACKGROUND: Some cases of recurrent miscarriage and later pregnancy complications have a thrombotic basis. Factor V Leiden is a common thrombophilic mutation. METHODS: The prospective outcome of untreated pregnancies amongst 25 women heterozygous for the Factor V Leiden allele who had a history of either recurrent early miscarriages only (three or more miscarriages at <12 weeks gestation; n = 19) or of late miscarriage (>12 weeks gestation; n = 9) was studied. Control groups of women with a similar pregnancy history but who had a normal Factor V genotype were also studied. RESULTS: The live birth rate was significantly lower amongst women with a history of recurrent early miscarriage who carried the Factor V Leiden allele (6/16; 37.5%) compared with that amongst those with a normal Factor V genotype (106/153; 69.3%; odds ratio 3.75, 95% confidence intervals 1.3-10.9). The live birth rate was 11.1% (1/9) amongst those with a history of late miscarriage carrying the Factor V Leiden allele and 48.9% (22/45) amongst those with a normal Factor V genotype. CONCLUSIONS: Attention should be directed at screening women with recurrent miscarriage associated with placental thrombosis for Factor V Leiden and a policy of targeted thromboprophylaxis during future pregnancies should be assessed in the form of a randomized controlled trial.

Abortion, Habitual↗
PubMed2002

Defective endovascular trophoblast invasion in primary antiphospholipid antibody syndrome-associated early pregnancy failure.

BACKGROUND: Primary antiphospholipid antibody syndrome (PAPS) is an established cause of recurrent pregnancy loss, traditionally presumed to be due to 'intraplacental thromboses'. This study examines products of conception (POC) from early pregnancy failures to investigate the mechanism of pregnancy loss. METHODS: POC from patients attending a recurrent miscarriage clinic and from terminations of pregnancy for non-medical reasons were examined histologically with particular regard to the presence or absence of vascular or intervillous thromboses and decidual endovascular trophoblast invasion. RESULTS: There were 31 PAPS-positive, 50 PAPS-negative, 34 aneuploid and 20 control cases at 6-14 weeks gestation. Villous morphology and frequency of intervillous thrombosis were not different among groups. Normal intradecidual endovascular trophoblast invasion was identified significantly less frequently in PAPS+ cases (24%), compared with controls (75%), aneuploid (53%), or PAPS- cases (61%; Z = -3.0, P < 0.01). In all cases there was apparently normal interstitial extravillous trophoblast invasion. CONCLUSIONS: Defective decidual endovascular trophoblast invasion, rather than excessive intervillous thrombosis, is the most frequent histological abnormality in PAPS+ associated early pregnancy loss. Furthermore, endovascular trophoblast invasion is not significantly reduced in the majority of fetal aneuploidy-associated pregnancy failures.

Abortion, Habitual↗
PubMed2001

Development of serine protease inhibitors displaying a multicentered short (<2.3 A) hydrogen bond binding mode: inhibitors of urokinase-type plasminogen activator and factor Xa.

Novel scaffolds that bind to serine proteases through a unique network of short hydrogen bonds to the catalytic Ser195 have been developed. The resulting potent serine protease inhibitors were designed from lead molecule 2-(2-hydroxyphenyl)1H-benzoimidazole-5-carboxamidine, 6b, which is known to display several modes of binding. For instance, 6b can recruit zinc and bind in a manner similar to that reported by bis(5-amidino-2-benzimidazolyl)methane (BABIM) (Nature 1998, 391, 608-612).(1) Alternatively, 6b can bind in the absence of zinc through a multicentered network of short (<2.3 A) hydrogen bonds. The lead structure was optimized in the zinc-independent binding mode toward a panel of six human serine proteases to yield optimized inhibitors such as 2-(3-bromo-2-hydroxy-5-methylphenyl)-1H-indole-5-carboxamidine, 22a, and 2-(2-hydroxybiphenyl-3-yl)-1H-indole-5-carboxamidine, 22f. Structure-activity relationships determined that, apart from the amidine function, an indole or benzimidazole and an ortho substituted phenol group were also essential components for optimal potency. The affinities (K(i)) of 22a and 22f, for example, bearing these groups ranged from 8 to 600 nM toward a panel of six human serine proteases. High-resolution crystal structures revealed that the binding mode of these molecules in several of the enzymes was identical to that of 6b and involved short (<2.3 A) hydrogen bonds among the inhibitor hydroxyl oxygen, Ser195, and a water molecule trapped in the oxyanion hole. In summation, novel and potent trypsin-like serine protease inhibitors possessing a unique mode of binding have been discovered.

Amidines↗
PubMed2001

Gln3p nuclear localization and interaction with Ure2p in Saccharomyces cerevisiae.

Gln3p is one of two well characterized GATA family transcriptional activation factors whose function is regulated by the nitrogen supply of the cell. When nitrogen is limiting, Gln3p and Gat1p are concentrated in the nucleus where they bind GATA sequences upstream of nitrogen catabolite repression (NCR)-sensitive genes and activate their transcription. Conversely, in excess nitrogen, these GATA sequences are unoccupied by Gln3p and Gat1p because these transcription activators are excluded from the nucleus. Ure2p binds to Gln3p and Gat1p and is required for NCR-sensitive transcription to be repressed and for nuclear exclusion of these transcription factors. Here we show the following. (i) Gln3p residues 344-365 are required for nuclear localization. (ii) Replacing Ser-344, Ser-347, and Ser-355 with alanines has minimal effects on GFP-Gln3p localization. However, replacing Gln3p Ser-344, Ser-347, and Ser-355 with aspartates results in significant loss of its ability to be concentrated in the nucleus. (iii) N and C termini of the Gln3p region required for it to complex with Ure2p and be excluded from the nucleus are between residues 1-103 and 301-365, respectively. (iv) N and C termini of the Ure2p region required for it to interact with Gln3p are situated between residues 101-151 and 330-346, respectively. (v) Loss of Ure2p residues participating in either dimer or prion formation diminishes its ability to carry out NCR-sensitive regulation of Gln3p activity.

Amino Acid Sequence↗
PubMed2001

A novel serine protease inhibition motif involving a multi-centered short hydrogen bonding network at the active site.

We describe a new serine protease inhibition motif in which binding is mediated by a cluster of very short hydrogen bonds (<2.3 A) at the active site. This protease-inhibitor binding paradigm is observed at high resolution in a large set of crystal structures of trypsin, thrombin, and urokinase-type plasminogen activator (uPA) bound with a series of small molecule inhibitors (2-(2-phenol)indoles and 2-(2-phenol)benzimidazoles). In each complex there are eight enzyme-inhibitor or enzyme-water-inhibitor hydrogen bonds at the active site, three of which are very short. These short hydrogen bonds connect a triangle of oxygen atoms comprising O(gamma)(Ser195), a water molecule co-bound in the oxyanion hole (H(2)O(oxy)), and the phenolate oxygen atom of the inhibitor (O6'). Two of the other hydrogen bonds between the inhibitor and active site of the trypsin and uPA complexes become short in the thrombin counterparts, extending the three-centered short hydrogen-bonding array into a tetrahedral array of atoms (three oxygen and one nitrogen) involved in short hydrogen bonds. In the uPA complexes, the extensive hydrogen-bonding interactions at the active site prevent the inhibitor S1 amidine from forming direct hydrogen bonds with Asp189 because the S1 site is deeper in uPA than in trypsin or thrombin. Ionization equilibria at the active site associated with inhibitor binding are probed through determination and comparison of structures over a wide range of pH (3.5 to 11.4) of thrombin complexes and of trypsin complexes in three different crystal forms. The high-pH trypsin-inhibitor structures suggest that His57 is protonated at pH values as high as 9.5. The pH-dependent inhibition of trypsin, thrombin, uPA and factor Xa by 2-(2-phenol)benzimidazole analogs in which the pK(a) of the phenol group is modulated is shown to be consistent with a binding process involving ionization of both the inhibitor and the enzyme. These data further suggest that the pK(a) of His57 of each protease in the unbound state in solution is about the same, approximately 6.8. By comparing inhibition constants (K(i) values), inhibitor solubilities, inhibitor conformational energies and corresponding structures of short and normal hydrogen bond-mediated complexes, we have estimated the contribution of the short hydrogen bond networks to inhibitor affinity ( approximately 1.7 kcal/mol). The structures and K(i) values associated with the short hydrogen-bonding motif are compared with those corresponding to an alternate, Zn(2+)-mediated inhibition motif at the active site. Structural differences among apo-enzymes, enzyme-inhibitor and enzyme-inhibitor-Zn(2+) complexes are discussed in the context of affinity determinants, selectivity development, and structure-based inhibitor design.

Amino Acid Motifs↗
PubMed2001

In vitro intestinal permeability of factor Xa inhibitors: influence of chemical structure on passive transport and susceptibility to efflux.

PURPOSE: To study the in vitro intestinal permeability of a number of newly synthesised factor Xa inhibitors to better understand the poor oral absorption of these compounds. METHODS: The bidirectional transport of the fXa inhibitors was studied in the Caco-2 cell model and isolated rat ileal tissue. An attempt was made to characterize efflux mechanisms with the help of commonly used substrates and inhibitors of various transport proteins. In addition, the transport of the fXa inhibitors was studied in MDCK cells transfected with the human MDR1 gene and expressing large amounts of P-glycoprotein (Pgp). RESULTS: The in vitro absorptive permeability was low for all but one of the fXa inhibitors. For compounds with non-substituted amidine, a charge (due to ionisation at neutral pH) may have resulted in poor membrane partitioning. Neutral compounds with substituted amidines were effluxed from the epithelial cells. The significance of the secretion process was illustrated by the results obtained for a neutral analogue showing high absorptive Caco-2 cell permeability that was not obviated by efflux. Transport inhibition studies in Caco-2 and permeability studies in the MDR1-transfected MDCK cells consistently showed that Pgp is not involved in the secretion of fXa inhibitors. Besides efflux, metabolic liability limited the permeation of the neutral lipophilic analogues with a carbamate ester. CONCLUSIONS: Poor intestinal permeability may be an important factor in the incomplete oral absorption of the bisbenzimidazole-type fXa inhibitors. Poor permeability may be related to poor membrane partitioning for hydrophilic analogues, whereas susceptibility to efflux transports and gastro-intestinal enzymatic degradation may limit the permeability of some of the neutral less hydrophilic derivatives.

Animals↗
PubMed2001

Childhood cutaneous tuberculosis: a study over 25 years from northern India.

AIMS: We undertook this study to analyse the pattern of childhood cutaneous tuberculosis prevailing in northern India over the past 25 years and to highlight differences from and similarities to adult tuberculosis. MATERIALS AND METHODS: Clinical records of children with cutaneous tuberculosis who attended the Nehru Hospital attached to the Postgraduate Institute of Medical Education and Research, Chandigarh, India, between January 1975 to December 1999 were analysed. RESULTS: Four hundred and two patients with cutaneous tuberculosis were examined during the 25-year period of observation. These patients formed 0.1% of the total dermatology outpatients. Seventy-five (18.7%) of these 402 cases were children (</= 16 years). There were 32 (42.7%) boys and 43 (57.3%) girls with a boy : girl ratio of 0.74 : 1. The majority of the children, 41 (54.7%) were in the 10-14 years age group. There was no significant boy or girl preponderance in any group other than in scrofuloderma (SFD), where girls significantly outnumbered boys at all ages. Of the various patterns of cutaneous tuberculosis seen, 40 (53.3%) had SFD, 30 (40.0%) had lupus vulgaris (LV), 3 (4.0%) had tuberculosis verrucosa cutis (TVC), 1 (1.3%) child each had tuberculids and tubercular gumma. No child had a tubercular chancre or acute miliary cutaneous tuberculosis. The neck was the commonest site afflicted with SFD and the face was the commonest site affected with LV. No child had generalized lymphadenopathy. Eighteen (60.0%) of the 30 children with LV had regional lymphadenopathy of which 15 (83.3%) had localized disease and 3 (16.7%) had disseminated disease. Of the 16 children with systemic organ involvement, 12 (75.0%) had regional lymphadenopathy. Of the 62 children in whom the data regarding vaccination status was available, 31 (50.0%) had been vaccinated and 31 (50.0%) had not. Among the vaccinated group no child had disseminated disease. Three (9.7%) children in the nonvaccinated group had disseminated disease. Information regarding Mantoux reactivity was available in 71 (94.7%) children, 61 (86.0%) with localized disease and 10 (14.1%) with disseminated disease. Of the 61 children with localized disease, 56 (91.8%) were Mantoux positive and of the 10 children with disseminated disease, only 5 (50.0%) were Mantoux positive (> 10 mm). Histopathologic reports were available for evaluation in all 75 children. Out of 30 cases of LV, 24 (80%) showed classical tuberculous histology. In contrast, out of 40 cases with SFD, only 19 (47.5%) showed classical histology. Classical tuberculous histology was noted in all 3 (100%) cases of TVC and 1 (100%) case each with tuberculids and gumma. Tubercle bacilli could be demonstrated in 4/30 (13.3%) cases with LV and 9/40 (22.5%) cases with SFD. Systemic involvement was seen in 16 (21.3%) children, of whom 3 (18.8%) had LV and 13 (81.3%) SFD. The lungs were the most common organs involved in 8 (50.0%) children followed by bone(s) in 4 (25.0%), abdomen in 2 (12.5%), and both lung and bone in 1 (6.3%) child. In contrast to adults, girls outnumbered boys in the childhood series; SFD was a common form of presentation in contrast to LV and TVC, tuberculous gumma and tuberculids were noted less often. In both children and adults, Mantoux reactivity did not correlate with the extent of the disease; patients with disseminated disease were found to be less often vaccinated with BCG and regional lymphadenopathy was noted more often in patients with disseminated disease. CONCLUSIONS: In the whole spectrum of cutaneous tuberculosis, there is a proportion of patients with dissemination (systemic involvement) who are of great epidemiological significance as they require a change in the standard therapeutic regimens recommended for cutaneous tuberculosis.

Adolescent↗
PubMed2001

Metformin therapy in the management of polycystic ovary syndrome: endocrine, metabolic and reproductive effects.

Polycystic ovary syndrome (PCOS) is a convergence of multisystem endocrine derangements. Impairment in insulin metabolism is a prominent feature of the syndrome and appears to play a key pathogenetic role precipitating the cascade of other disorders associated with PCOS. Recent studies report that insulin-sensitizing agents, such as metformin, reduce hyperinsulinemia, reverse the endocrinopathy of PCOS and normalize endocrine, metabolic and reproductive functions, leading to the resumption of menstrual cyclicity and ovulation. In this review, we report the most recent evidence regarding metformin usage in women with PCOS.

Endocrine System↗
PubMed2001

Factor V Leiden and acquired activated protein C resistance among 1000 women with recurrent miscarriage.

Activated protein C (APC) resistance, both in its congenital form, due to the factor V Leiden mutation, and in its acquired form, are important risk factors for systemic venous thrombosis. In view of the suspected thrombotic aetiology of some cases of recurrent miscarriage, the prevalence of APC resistance was determined among 1111 consecutive Caucasian women with a history of either recurrent early miscarriage (three or more consecutive pregnancy losses at <12 weeks gestation; n = 904) or a history of at least one late miscarriage (>12 weeks gestation; n = 207). A control group of 150 parous Caucasian women with no previous history of adverse pregnancy outcome was also studied. Acquired APC resistance was significantly more common among both women with recurrent early miscarriage (8.8%: 80/904; P = 0.02) and those with late miscarriage (8.7%: 18/207; P = 0.04) compared with controls (3.3%: 5/150). In contrast, the frequency of the factor V Leiden allele was similar among (i) women with recurrent early miscarriage (3.3%:60/1808; 58 heterozygotes and one homozygote), (ii) those with late miscarriage (3.9%:16/414; 14 heterozygotes and one homozygote) and (iii) the control group (4.0%:12/300; 12 heterozygotes). Acquired but not congenital APC resistance (due to the factor V Leiden mutation) is associated with both early and late miscarriage.

Abortion, Habitual↗
PubMed2001

Green fluorescent protein-Dal80p illuminates up to 16 distinct foci that colocalize with and exhibit the same behavior as chromosomal DNA proceeding through the cell cycle of Saccharomyces cerevisiae.

Four GATA family DNA binding proteins mediate nitrogen catabolite repression-sensitive transcription in Saccharomyces cerevisiae. Gln3p and Gat1p are transcriptional activators, while Dal80p and Deh1p repress Gln3p- and Gat1p-mediated transcription by competing with these activators for binding to DNA. Strong Dal80p binding to DNA is thought to result from C-terminal leucine zipper-mediated dimerization. Many Dal80p binding site-homologous sequences are relatively evenly distributed across the S. cerevisiae genome, raising the possibility that Dal80p might be able to "stain" DNA. We demonstrate that cells containing enhanced green fluorescent protein-Dal80p (EGFP-Dal80p) exhibit up to 16 fluorescent foci that colocalize with DAPI (4',6'-diamidino-2-phenylindole)-positive material and follow DNA movement through the cell cycle, suggesting that EGFP-Dal80p may indeed be useful for monitoring yeast chromosomes in live cells and in real time.

Cell Cycle↗
PubMed2001

Dial 1097 (toll free).

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Acquired Immunodeficiency Syndrome↗
PubMed2001

Perspectives on factor Xa inhibition.

Blood coagulation involves a complex cascade of enzymatic reactions, ultimately generating fibrin, the basis of all blood clots. This cascade is comprised of two arms, the intrinsic and extrinsic pathways which converge at factor Xa to form the common pathway. Factor Xa activates prothrombin to thrombin, which in turn catalyzes the conversion of fibrinogen to fibrin. Recently, both natural and synthetic factor Xa inhibitors have shown promising pharmacological effects in animal models of thrombosis. Accordingly, factor Xa has emerged as a compelling target for pharmacological intervention and much recent effort has focused on selective and potent inhibition of this key enzyme. Factor Xa and other enzymes in the coagulation cascade belong to the trypsin-like serine protease family, the various members of which are involved in numerous physiological functions in the body. Hence, to avoid toxicity and adverse side effects, it is important to selectively inhibit the target enzyme. Achieving the needed selectivity has proved challenging due to the high degree of structural homology around the active site of this class of enzymes. This article provides a brief review of the strategies currently being employed to develop oral anticoagulants and, more specifically, the structural features of protein-ligand binding that have been utilized to achieve potency and selectivity toward factor Xa. Additionally, selected lead molecules will be discussed to highlight binding motifs used to attain both potency and selectivity in drug candidates.

Animals↗