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Biomedical subjects

S Pillai

Publications and source records attributed to S Pillai.

At least 37 records · Page 2Linked to original sources
PubMed2000

Value of troponin-T rapid assay, cardiac enzymes, electrocardiogram and history of chest pain in the initial diagnosis of myocardial infarction in the emergency department.

We conducted a prospective study of 152 adult patients presenting to an emergency department with chest pain or symptoms suggestive of acute myocardial infarction (AMI) to evaluate the first electrocardiogram (ECG), creatine kinase (CK)-MB and Troponin-T Rapid Assay (TnT) alone or in combination with chest pain in the initial diagnosis of AMI. A provisional diagnosis was made after the history, physical examination and the first ECG reading. Blood specimens were taken for TnT, CK and CK-MB mass. A final discharge diagnosis of AMI was made according to World Health Organization criteria. Seventy-six (50%) of patients had a final diagnosis of AMI. The sensitivities of the first ECG, first CK-MB mass and first TnT were 76.3% (95% confidence interval (CI), 66.8-85.9), 38.2% (95% CI, 27.2-49.1) and 31.6% (95% CI, 21.2-42.0) respectively. The area under the curve for a combination of ECG, CK-MB mass, TnT and chest pain was the highest at 0.937 when compared with chest pain with varying combinations of tests. A combination of the first ECG, CK-MB mass and TnT had a negative predictive value (NPV) of 87.9% (95% CI, 80.0-95.8). The first ECG was the most sensitive test while the combination of chest pain, ECG, cardiac enzymes and TnT gave the best results in the initial diagnosis of AMI. If the first ECG, CK-MB mass and TnT are all negative, the probability of having an AMI is 12%.

Adult↗
PubMed2000

Toxoids of streptococcal pyrogenic exotoxin A are protective in rabbit models of streptococcal toxic shock syndrome.

Streptococcal pyrogenic exotoxins (SPEs) are superantigens that have been implicated in causing streptococcal toxic shock syndrome (STSS). Most notably, SPE serotype A is made by nearly all M-protein serotype 1 and 3 streptococci, the M types most associated with the illness (these strains contain one or more other SPEs, and those proteins are likely also to contribute to disease). We have prepared double-, triple-, and hexa-amino-acid mutants of SPE A by PCR and other mutagenesis procedures. The sites chosen for mutation were solvent-exposed residues thought to be important for T-cell receptor (TCR) or major histocompatibility complex (MHC) class II interaction. These mutants were nonsuperantigenic for human peripheral blood mononuclear cells and rabbit and mouse splenocytes and were nonlethal in two rabbit models of STSS. In addition, these mutants stimulated protective antibody responses. Interestingly, mutants that altered toxin binding to MHC class II were more immunogenic than mutants altering TCR binding. Collectively, these studies indicate that multiple-site mutants of SPE A are toxoids that may have use in protecting against the toxin's effects in STSS.

Animals↗
PubMed1999

Accelerated emigration of B lymphocytes in the Xid mouse.

The B cell receptor is required for the emigration of newly generated B lymphocytes and for their maintenance in the periphery. A specific maintenance defect was noted in fraction I (IgDhighIgMlow) B cells in Xid mice (which harbor a mutation in Btk). Although Bcl-2 levels in fractions I and II (IgDhighIgMhigh) are equivalent in normal and Xid B cells, a novel peak of Bcl-2low fraction III (IgDlowIgMhigh) B cells was noted in the Xid mouse. Since this B cell population resembled bone marrow immature B cells, we examined the emigration of newly formed B cells in normal and Xid mice. These studies revealed the accelerated emigration of newly formed Xid B cells. We conclude that distinct Btk-independent and Btk-dependent signals mediate emigration and maintenance events during peripheral B cell maturation.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1999

Ceramide potentiates, but sphingomyelin inhibits, vitamin D-induced keratinocyte differentiation: comparison between keratinocytes and HL-60 cells.

Differentiation of epidermal keratinocytes and leukemia HL-60 cells induced by 1,25-dihydroxyvitamin D [1,25(OH)2D] has been reported to be mediated, at least in part, by increases in cellular ceramide levels. Ceramides produced by 1,25(OH)2D-induced sphingomyelin (SM) hydrolysis also contribute to the permeability barrier lipids in keratinocytes. Exogenously supplied SM is taken up by mammalian cells, including keratinocytes, and is incorporated into cellular pools. However, the effects of exogenously added SM on keratinocyte differentiation have not been studied. Therefore, in this study, we compared exogenously added SM with a cell-permeable ceramide for their ability to stimulate keratinocyte differentiation induced by 1,25(OH)2D. Both short-chain ceramide (C2-cer) and SM stimulated the differentiation and inhibited the proliferation of HL-60 cells. As expected, this effect was potentiated by 1,25(OH)2D. However, SM inhibited the differentiation and stimulated the proliferation of keratinocytes. While C2-cer potentiated the effects of 1,25(OH)2D, SM reversed the effects of 1,25(OH)2D on keratinocytes. The ratio of SM to ceramide was significantly different between keratinocytes and HL-60 cells. While the SM level of HL-60 cells were twice that of keratinocytes, keratinocytes contained ten times more ceramides than HL-60 cells, resulting in a ceramide/SM ratio 17 times higher in keratinocytes. Thus, we identified similarities and significant differences in the sphingolipid-mediated cell signaling pathway between keratinocytes and HL-60 cells. While SM stimulated HL-60 cell differentiation, presumably by incorporation into SMase-accessible membrane pools, it inhibited keratinocyte differentiation. In keratinocytes, SM was possibly incorporated into a different cellular pool (barrier lipid pool) or altered membrane phospholipid metabolism and membrane fluidity.

Calcitriol↗
PubMed1999

Membrane anchoring of calnexin facilitates its interaction with its targets.

Calnexin, a chaperone that resides in the endoplasmic reticulum, participates in the quality control function of this compartment. Many glycoproteins in the process of folding associate transiently with this chaperone via interactions involving the recognition of their mono-glucosylated glycans. Some misfolded proteins which are retained in the endoplasmic reticulum exhibit prolonged association with calnexin. We have examined whether the transmembrane and cytoplasmic domains of calnexin influence the association of this chaperone with its targets. Interactions of wild type and truncated calnexin with a glycoprotein that is retained in the endoplasmic reticulum (the lymphocyte tyrosine kinase, Ltk), with membrane IgM heavy chains, and with the MHC class I heavy chain protein were investigated. A soluble calnexin molecule lacking the transmembrane domain and cytoplasmic tail does not associate with any of these proteins. When a heterologous transmembrane domain is fused to the lumenal portion of calnexin, this membrane-bound protein can bind Ltk, IgM heavy chains, and MHC class I heavy chain proteins. These results suggest that calnexin must be membrane-anchored in order to recognize its substrates.

Animals↗
PubMed1999

Direct hyperplasia does not enhance the kinetics of liver repopulation in a new model of hepatocyte transplantation in the rat.

BACKGROUND/AIMS: We have recently developed a new model of extensive liver repopulation by transplanted hepatocytes following exposure to pyrrolizidine alkaloids. In the present study, the effect of 2/3 partial hepatectomy (PH) and that of a potent direct liver mitogen, lead nitrate, were compared in their ability to modulate the kinetics of liver repopulation. METHODS: Fischer 344 rats deficient in enzymatic activity for dipeptidyl-peptidase IV (DPPIV-) were used as cell transplantation recipients. They were given 2 doses of the pyrrolizidine alkaloid retrorsine (30 mg/kg, i.p.), 2 weeks apart, followed 2 weeks later by transplantation of 2 x 10(6) hepatocytes (via the portal vein), freshly isolated from a normal congeneic DPPIV+ donor. PH was carried out or a single injection of lead nitrate (100 micromol/kg, i.v.) was administered 2 weeks post-transplantation. Liver samples obtained at different time points post-treatment were processed histochemically for DPPIV activity. RESULTS: The percent of liver sections occupied by DPPIV+ hepatocytes was <1% at the time of PH or lead nitrate administration. In animals which underwent PH, it increased to 33.4+/-5.7% at 2 weeks and to 55.6+/-8.5% at 1 month. However, in animals receiving lead nitrate, these percentages were only 3.3+/-1.3% at 2 weeks and 16.2+/-3.9% at 1 month. Repeated injections of lead nitrate had no additional effect. Further experiments indicated that an acute mitogenic response to lead nitrate was present in transplanted cells, while resident hepatocytes were inhibited by retrorsine. CONCLUSIONS: These results indicate that direct mitogenic signals (such as those induced by lead nitrate), and compensatory signals (such as those elicited by PH), are not equally effective on kinetics of liver repopulation in this system. The possible reasons for these differential effects are discussed.

Animals↗
PubMed1999

Fibrinogen cleavage by the Streptococcus pyogenes extracellular cysteine protease and generation of antibodies that inhibit enzyme proteolytic activity.

The extracellular cysteine protease from Streptococcus pyogenes is a virulence factor that plays a significant role in host-pathogen interaction. Streptococcal protease is expressed as an inactive 40-kDa precursor that is autocatalytically converted into a 28-kDa mature (active) enzyme. Replacement of the single cysteine residue involved in formation of the enzyme active site with serine (C192S mutation) abolished detectable proteolytic activity and eliminated autocatalytic processing of zymogen to the mature form. In the present study, we investigated activity of the wild-type (wt) streptococcal protease toward human fibrinogen and bovine casein. The former is involved in blood coagulation, wound healing, and other aspects of hemostasis. Treatment with streptococcal protease resulted in degradation of the COOH-terminal region of fibrinogen alpha chain, indicating that fibrinogen may serve as an important substrate for this enzyme during the course of human infection. Polyclonal antibodies generated against recombinant 40- and 28-kDa (r40- and r28-kDa) forms of the C192S streptococcal protease mutant exhibited high enzyme-linked immunosorbent assay titers but demonstrated different inhibition activities toward proteolytic action of the wt enzyme. Activity of the wt protease was readily inhibited when the reaction was carried out in the presence of antibodies generated against r28-kDa C192S mutant. Antibodies produced against r40-kDa C192S mutant had no significant effect on proteolysis. These data suggest that the presence of the NH(2)-terminal prosegment prevents generation of functionally active antibodies and indicate that inhibition activity of antibodies most likely depends on their ability to bind the active-site region epitope(s) of the protein.

Animals↗
PubMed1999

Regulation of nuclear localization and transcriptional activity of TFII-I by Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk) is required for normal B-cell development, as defects in Btk lead to X-linked immunodeficiency (xid) in mice and X-linked agammaglobulinemia (XLA) in humans. Here we demonstrate a functional interaction between the multifunctional transcription factor TFII-I and Btk. Ectopic expression of wild-type Btk enhances TFII-I-mediated transcriptional activation and its tyrosine phosphorylation in transient-transfection assays. Mutation of Btk in either the PH domain (R28C, as in the murine xid mutation) or the kinase domain (K430E) compromises its ability to enhance both the tyrosine phosphorylation and the transcriptional activity of TFII-I. TFII-I associates constitutively in vivo with wild-type Btk and kinase-inactive Btk but not xid Btk. However, membrane immunoglobulin M cross-linking in B cells leads to dissociation of TFII-I from Btk. We further show that while TFII-I is found in both the nucleus and cytoplasm of wild-type and xid primary resting B cells, nuclear TFII-I is greater in xid B cells. Most strikingly, receptor cross-linking of wild-type (but not xid) B cells results in increased nuclear import of TFII-I. Taken together, these data suggest that although the PH domain of Btk is primarily responsible for its physical interaction with TFII-I, an intact kinase domain of Btk is required to enhance transcriptional activity of TFII-I in the nucleus. Thus, mutations impairing the physical and/or functional association between TFII-I and Btk may result in diminished TFII-I-dependent transcription and contribute to defective B-cell development and/or function.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

The calcium sensing receptor and its alternatively spliced form in keratinocyte differentiation.

We have recently reported the presence of the calcium sensing receptor (CaR) in keratinocytes and suggested that it signaled calcium-induced differentiation of these cells. cDNA clones encoding the full-length CaR were isolated from human keratinocytes. In addition, an alternatively spliced form that lacks exon 5, encoding a portion of the extracellular domain, also was found. The in frame deletion of 231 nucleotides of exon 5 resulted in the loss of function of the CaR as measured by calcium-stimulated production of inositol phosphates when transfected into HEK293 cells or keratinocytes. This variant produced a smaller CaR protein with an altered glycosylation pattern compared with the full-length CaR. Coexpression of the spliced variant with the full-length CaR reduced the function of the full-length CaR. The full-length CaR was expressed in undifferentiated keratinocytes consistent with their greater response to elevated extracellular calcium in terms of increased intracellular free calcium and production of inositol phosphates. The full-length CaR decreased as the keratinocytes differentiated with an increase in the ratio of the spliced variant to the full-length form. The relative proportions of these two forms of CaR may regulate the calcium responsiveness of keratinocytes during their differentiation.

Alternative Splicing↗
PubMed1998

Inhibition of caspase activity prevents anti-IgM induced apoptosis but not ceramide generation in WEHI 231 B cells.

In apoptosis induced by Reaper in Drosophila, as well as in a number of other systems, it has been suggested that the increased synthesis of ceramide might be a consequence of the activation of the caspase/ICE (Interleukin-1beta converting enzyme) protease pathway involved in cell death, implying that ceramide generation might often be the result rather than the cause of apoptosis. WEHI 231 B cells have previously been shown to undergo apoptosis following exposure to exogenous ceramide and to produce increased amounts of ceramide in response to anti-IgM crosslinking. We show here that in WEHI 231 cells a peptide inhibitor of caspase activity blocks cell death in response to both anti-IgM and exogenous ceramide. However, the induction of ceramide synthesis by WEHI 231 cells in response to anti-IgM crosslinking is not blocked by this peptide. These results indicate that antigen receptor induced ceramide generation in WEHI 231 cells does not require caspase activation, and support the view that ceramide generation in immature B cells may be the cause rather than the consequence of activation of the caspase dependent death pathway.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Aiolos regulates B cell activation and maturation to effector state.

Aiolos encodes a zinc finger DNA-binding protein that is highly expressed in mature B cells and is homologous to Ikaros. In the periphery of mice homozygous for an Aiolos-null mutation, B cells exhibit an activated cell surface phenotype and undergo augmented antigen receptor (BCR)-mediated in vitro proliferative responses, even at limiting amounts of stimulant. In vivo, T cell-dependent B cell responses, including the formation of germinal centers and elevated serum IgG and IgE, are detected in Aiolos-deficient mice in the absence of immunization. Auto-antibodies and development of B cell lymphomas are frequently seen among aging Aiolos mutants. In sharp contrast to conventional B cells, B cells of the peritoneum, of the marginal zone, and the recirculating bone marrow population are greatly reduced.

Animals↗
PubMed1998

Sperm acrosome status and sperm antibodies in infertility.

PURPOSE: We studied whether the spermatozoa from sperm autoimmune infertile men undergo premature acrosomal loss and whether this relates to the presence of sperm antibodies in wives. MATERIALS AND METHODS: We evaluated acrosome status of live washed native and overnight capacitated spermatozoa from 17 sperm nonautoimmune fertile and 23 sperm autoimmune infertile men using an immunofluorescent peanut lectin binding assay. We used cytotoxic and immunobead binding assays to prescreen the serum and seminal plasma of these men, and serum and cervical mucus of the wives for immunological infertility. We performed immunofluorescent sperm antibody assays on all study samples to ascertain sperm antibody isotype levels in each sample. Levels of acrosomal loss in husband native and capacitated spermatozoa were correlated with levels of IgG, IgA and IgM sperm antibodies in the study samples. RESULTS: Sperm autoimmune infertile men had a significantly larger percentage of sperm (p <0.0001) that had lost the acrosome and a lower percentage of sperm with intact acrosome (p <0.0001) in native and capacitated preparations in contrast to those of fertile controls. Levels of cytotoxic and IgA antibodies, especially in seminal plasma and cervical mucus, correlated significantly with percentages of sperm with a total loss of acrosome in native and capacitated sperm preparations (p < or = 0.01). CONCLUSION: Infertile men with sperm antibodies in serum and seminal plasma undergo premature acrosome loss. This loss may expose the reproductive tract immune system, especially that involving IgA, in autoimmune infertile men and the wives to high immunogenic levels of sperm acrosome membrane antigens, thereby rendering them immunologically infertile.

Acrosome↗
PubMed1998

Effects of antibodies to transferrin and alpha 2-HS glycoprotein on in vitro sperm motion: implications in infertility associated with endometriosis.

PROBLEM: Women with endometriosis have antibodies to endometrial transferrin and alpha 2-HS glycoprotein in their serum and peritoneal fluid. The objective of this study was to determine whether antibodies to transferrin and alpha 2-HS glycoprotein adversely affect sperm motility and survival. METHOD OF STUDY: Spermatozoa obtained from normal fertile donors and washed free of seminal plasma were incubated with the medium (control), 1:2 and 1:100 dilutions of antitransferrin, 1:4, 1:8 and 1:100 dilutions of anti-alpha 2-HS glycoprotein, and a 1:2 dilution of antialbumin antiserum (negative control). Sperm motion characteristics in 10 microliters aliquots were evaluated at 30 min, 1 hr, 2 hr, 4 hr, and 24 hr using computerized sperm motion analysis. A paired t-test was done to analyze the effects of the various antibodies on sperm motion characteristics. RESULTS: Antibodies to albumin failed to adversely affect sperm motility in general or the several sperm motion characteristics in particular. In contrast, antibodies to transferrin at the dilution of 1:2 adversely affected the percentage of motile and rapid spermatozoa, progressive and path velocities, straightness, linearity, track speed, and anterior-lateral head displacement (P < 0.001) at all the time intervals, whereas a 1:100 dilution of this antiserum adversely affected these parameters only at 24 hr. Elongation and beat cross-frequency were significantly affected at 4 and 24 hr by a 1:2 dilution of antitransferrin antiserum. The effects of anti-alpha 2-HS glycoprotein were more pronounced than those of antitransferrin, but they were similar. Dilutions of 1:4 and 1:8 were effective at all time intervals, whereas a 1:100 dilution was effective in reducing the track speed and the percentage of rapid cells at 24 hr (P < 0.001). CONCLUSION: Antibodies to endometrial transferrin and alpha 2-HS glycoprotein present in the peritoneal fluid, and possibly in the oviductal fluid, of patients with endometriosis may adversely affect postcoital sperm motility and sperm survival.

Antibodies↗
PubMed1998

Complement regulatory proteins on the sperm surface: relevance to sperm motility.

PROBLEM: To determine whether complement regulatory proteins are present on human spermatozoa and whether antibodies to these proteins adversely affect sperm motility. METHOD OF STUDY: Human sperm membrane proteins were solubilized and subjected to polyacrylamide gel electrophoresis followed by Western blot analysis against antibodies to complement component 1 inhibitor (C1-INH), decay-activating factor (DAF; CD55), membrane cofactor protein (MCP; CD46), and homologous restriction factor (HRF; CD59). Spermatozoa, obtained by a swim-up technique, were incubated in medium (control 1) and medium supplemented with antibodies to human albumin (control 2) and antibodies to these complement regulatory proteins. We used a computerized sperm motion analysis to determine the effect of these antibodies on sperm motion characteristics. RESULTS: Complement regulatory proteins such as C1-INH, CD55, CD46, and CD59 were found in the sperm extracts as shown by Western blot analysis. The heat-treated (56 degrees C, 30 min) IgG fraction of antibodies to these proteins significantly reduced sperm motility in general and other motion parameters. Addition of complement did not affect these results except in the antibodies to CD46 in which the reducing action was further amplified. CONCLUSIONS: Our data suggest that C1-INH, CD55, CD46, and CD59 are present on the sperm surface. These proteins may have biological functions, such as affecting sperm motility, besides the complement regulatory functions. In infertile men and women with antibodies that recognize one or more of these complement regulatory proteins, there may be problems related to poor sperm motility and survival in the reproductive tracts.

Antigens, CD↗
PubMed1998

Analysis of MHC class II DP, DQ and DR alleles in Crohn's disease.

BACKGROUND: Although inflammation in Crohn's disease is believed to be mediated by activated T cells, genotyping of all MHC class II alleles in white people with this disease has not been reported. AIMS: To perform a detailed molecular analysis of HLA DPB, DQB, and DRB genes in white patients with Crohn's disease and controls in order to determine if the inheritance of any class II genes confers susceptibility or resistance to this disease. METHODS: Complete molecular typing of HLA class II DPB, DQB, and DRB alleles was performed in 58 white patients with Crohn's disease and 93 healthy controls using a polymerase chain reaction-sequence specific oligonucleotide based approach. RESULTS: No significant association with any DPB or DQB alleles was noted in patients with Crohn's disease. Since our previous studies had shown a strong association of an HLA DRB3*0301/DRB1*1302 haplotype with Crohn's disease, we re-examined this association using more stringent genotyping criteria. This haplotype was present in 20.7% of patients and 5.4% of controls (p = 0.0066; relative risk = 4.59). CONCLUSIONS: The DRB3*0301/DRB1*1302 haplotype is the only significant MHC class II association noted in white people with Crohn's disease and represents the strongest association of any MHC or non-MHC locus with this disease.

Adult↗
PubMed1998

Characterization of cell-associated and soluble forms of connective tissue growth factor (CTGF) produced by fibroblast cells in vitro.

Connective tissue growth factor (CTGF) is a mitogenic and chemotactic factor for cultured fibroblasts that has been implicated in wound healing, fibrotic disorders and uterine function. Although the primary translational products of the mouse, human and pig CTGF (mCTGF, hCTGF, pCTGF) genes are predicted to be secreted and of approximate M(r) 38,000, 10 kDa biologically active forms of pCTGF have recently been described. In this report, we show that human foreskin fibroblasts (HFFs) and mouse connective tissue fibroblasts contained 2.4 kb CTGF transcripts, stained positively with an anti-CTGF[81-94] peptide antiserum, and produced a 38 kDa protein that was immunoprecipitated by an anti-CTGF[247-260] peptide antiserum. While 38 kDa CTGF was readily detected in cell lysates, it was non- or barely detectable in conditioned medium. 38 kDa CTGF remained cell-associated for at least 5 days after synthesis and was not releasable by treatment of the cells with trypsin, heparin, 1 M NaCl or low pH. Purification of CTGF from human or mouse fibroblast conditioned medium resulted in the isolation of 10-12 kDa CTGF proteins that were heparin-binding, bioactive, and reactive with anti-CTGF[247-260] on Western blots. Whereas 10 kDa CTGF stimulated DNA synthesis in 3T3 cells to the same extent as platelet-derived growth factor (PDGF)-AA, -AB, or -BB, it did not compete with 125I-PDGF-BB for binding to alpha alpha, alpha beta or beta beta PDGF receptors (PDGF-R), did not stimulate tyrosine phosphorylation of PDGF-alpha-R or -beta-R, and was not antagonized by a neutralizing PDGF-R-alpha antiserum. These data show that, in cultured fibroblasts, 38 kDa CTGF is principally cell-associated whereas low mass forms of CTGF are soluble and biologically active. They further demonstrate that, contrary to the previously proposed properties of 38 kDa CTGF, 10 kDa CTGF does not bind to PDGF-R and stimulates Balb/c 3T3 cell mitosis via a PDGF-R-independent mechanism.

3T3 Cells↗