We report results of the studies relating to the development of a non-invasive, label free immuno... more We report results of the studies relating to the development of a non-invasive, label free immunosensor based on nanostructured hafnium oxide (hafinia) deposited onto indium tin oxide (ITO) coated glass for oral cancer biomarker (CYFRA-21-1) detection in human saliva. The nanostructured hafnia (nHfO2) has been synthesized via one step low temperature hydrothermal process and modified with 3-aminopropyltriethoxy silane (APTES) for covalent immobilization of monoclonal antibodies (anti-CYFRA-21-1). Bovine serum albumin (BSA) was used to block non-specific sites at the anti-CYFRA-21-1/APTES/nHfO2/ITO electrode surface. The structural, morphological and spectroscopic characterization of the synthesized nanomaterials and fabricated electrodes has been carried out using X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) studies, respectively. The results of response studies conducted on BSA/anti-CYFRA-21-1/APTES/nHfO2/ITO immunoelectrode reveal that this biosensor has high sensitivity (9.28 μA mL ng−1 cm−2), wide linear detection range (2–18 ng mL−1) and fast response time (15 min). This immunosensor has been validated with enzyme linked immunosorbent assay (ELISA) in saliva samples of oral cancer patients.
CYFRA-21-1 is a novel biomarker secreted in the saliva of oral cancer patient in relatively highe... more CYFRA-21-1 is a novel biomarker secreted in the saliva of oral cancer patient in relatively higher amount (ng mL-1). We report, development of a simple, noninvasive and label free electrochemical biosensor based on nanostructured zirconia for the efficient detection of oral cancer. The silanized nanostructured zirconia has been electrophoretically deposited over ITO electrode that acts as an efficient support for covalent immobilization of monoclonal antibodies (anti-CYFRA-21-1). The fabricated platform has been characterized through AFM, XPS and FT-IR techniques. The results of the electrochemical response studies reveal that the BSA/anti-CYFRA-21-1/APTES/ZrO2/ITO immunoelectrode can be used to estimate the CYFRA-21-1 with a high sensitivity of 2.2 mA mL ng-1, linear detection range of 2-16 ng mL-1 and stability of 8 weeks. The observed results have been validated using ELISA and have implications towards the fabrication of point of care device for oral cancer monitoring.
The human CD99 protein is a 32-kDa type I transmembrane glycoprotein, while CD98 is a disulfide-l... more The human CD99 protein is a 32-kDa type I transmembrane glycoprotein, while CD98 is a disulfide-linked 125-kDa heterodimeric type II transmembrane glycoprotein. It has been previously shown that CD99 and CD98 oppositely regulate β1 integrin signaling, though the mechanisms by which this regulation occurs are not known. Our results revealed that antibody-mediated crosslinking of CD98 induced FAK phosphorylation at Y397 and facilitated the formation of the protein kinase Cα (PKCα)-syntenin-focal adhesion kinase (FAK), focal adhesions (FAs), and IPP-Akt1-syntenin complex, which mediates β1 integrin signaling. In contrast, crosslinking of CD99 disrupted the formation of the PKCα-syntenin-FAK complex as well as FA via FAK dephosphorylation. The CD99-induced dephosphorylation of FAK was apparently mediated by the recruitment of Src homology region 2 domain-containing phosphatase-2 (SHP2) to the plasma membrane and subsequent activation of its phosphatase activity. Further consequences of ...
Glanzmann&amp... more Glanzmann's thrombasthenia (GT) is an autosomal recessive bleeding disorder which is due to a defect in platelet aggregation in response to multiple physiological agonists. It has been demonstrated that the clinical phenotype of various diseases inherited in a classic Mendelian fashion can be modulated by a series of factors, inherited as well as acquired.
ABSTRACT Background. Elevated homocysteine levels have been seen to be associated with the presen... more ABSTRACT Background. Elevated homocysteine levels have been seen to be associated with the presence of thrombotic events. These may include arterial thrombotic events like stroke and myocardial infarctions or venous events like deep vein thrombosis, more so arterial as its association with thrombosis is well established. Aims. Lowering of homocysteine through administration of appropriate vitamin supplements has been documented in Cardiovascular disorders in India. These are primarily arterial conditions. The role of homocysteine in venous thrombotic conditions is less well documented and very little data is available in Deep Vein Thrombosis in Indian patients in this regard. Our present study takes a retrospective look at Deep Vein Thrombosis patients attending the Hematology Department of our hospital, which apart from looking into issues of Vitamin supplementation and Homocysteine, includes data on polymorphisms as well. Methods. 120 consecutive patients with deep venous thrombosis, confirmed by Doppler ultrasonography, were the subjects of study. All patients and equal number of healthy age and sex-matched controls were investigated for Homocysteine levels and also screened for MTHFR C677T, A1298C, MTR A2756G and MTRR G66A polymorphisms. Patients who were seen to have high levels of homocysteine were prescribed vitamin supplementation (Folic acid 5mg O.D, Vitamin B12 5µg O.D, Vitamin B6 2mg O.D) and were asked to report for a follow-up homocysteine check up post 3 months. 61 patients were found to have high levels of homocysteine of which only 23 reported for a follow-up homocysteine checkup. Differences between allelic and genotypic frequencies of individual polymorphisms between cases and controls and Hardy-Weinberg equilibrium (HWE) were tested using chi square tests. Statistical tests were done using SPSS version 12. Results. Most cases (97) were of lower limb thrombosis, 11 cases were of upper limb thrombosis. Thrombosis at unusual sites was present in 12 cases. These included the cerebral (5), abdominal (3), subclavian(2), renal(1) and retinal vein(1). High levels of baseline homocysteine were seen to be significantly associated with Deep Vein Thrombosis (p<0.001). All of the patients responded to the Folic acid dosage and showed reduction in the levels of plasma homocysteine(of the order of 13.1 umol/L i.e. 60.6% of the mean baseline plasma homocysteine of responders). However in some of these (7 out of 23) only a partial response was seen (plasma homocysteine levels reduced but not normalized i.e. between 5-15 umol/L). In these partial responders none of the patients were seen to carry the mutant homozygous forms of the MTHFRC677T, MTRA2756G or MTRRG66A polymorphisms. Only one patient amongst them carried the CC (mutant) genotype of the A1298C polymorphism. None of the complete responders carried the mutant homozygous forms of any of the four polymorphisms studied. The partial response to Folic acid dosage may be explained by the short duration of follow-up time(3-6 months) involved. Only MTHFR C677T & A1298C polymorphisms were seen to be associated with homocysteine levels (p<0.01). However none of the investigated four polymorphisms showed any association with Deep Vein Thrombosis. Summary and Conclusions. Nutritional deficiency and not genetic variation maybe the primary contributor to the prevalent hyperhomocysteinemic condition in Deep Vein Thrombosis patients of Asian Indian origin.
Multiple micronutrient supplementation of Nepalese women during pregnancy is associated with a si... more Multiple micronutrient supplementation of Nepalese women during pregnancy is associated with a significant increase in birth weight. We tested the hypothesis that improved birth weight in infants of mothers supplemented with micronutrients is associated with a decrease in inflammatory responses and an increase in the production of T helper 1 cells and T helper 2 cells. The study was embedded in a randomized controlled trial of 15 micronutrients, compared with iron-folate supplementation (control), given during pregnancy with the aim of increasing birth weight. Blood samples were collected at 32 wk of gestation, 12-20 wk after supplementation began, for the measurement of inflammatory markers. Breast-milk samples were collected 1 mo after delivery for the measurement of the ratio of milk sodium to potassium (milk Na:K). In an opportunistically selected subgroup of 70 women, mitogen-stimulated cytokine production was measured ex vivo in whole blood. Blood eosinophils; plasma concentra...
CD99 is known to be involved in the regulation of cell-cell adhesion. However, it remains unclear... more CD99 is known to be involved in the regulation of cell-cell adhesion. However, it remains unclear whether CD99 controls cell-extracellular matrix adhesion. In this study, the effects of CD99 activation on cell-extracellular matrix adhesion were investigated. It was found that engagement of CD99 with the stimulating antibody YG32 downregulated the adhesion of MCF-7 cells to fibronectin, laminin and collagen IV in a dose-dependent manner. The CD99 effect on cell-ECM adhesion was inhibited by overexpression of the dominant negative form of CD99 or CD99 siRNA transfection. Treatment of cells with Mn(2+) or by β(1) integrin-stimulating antibody restored the inhibitory effect of CD99 on cell-ECM adhesion. Cross-linking CD99 inactivated β(1) integrin through conformational change. CD99 activation caused dephosphorylation at Tyr-397 in FAK, which was restored by the β(1) stimulating antibody. Taken together, these results provide the first evidence that CD99 inhibits cell-extracellular matr...
British Journal of Haematology - BRIT J HAEMATOL, 2008
Thrombin activatable fibrinolysis inhibitor [carboxypeptidase B2 (plasma), CPB2] is a basic carbo... more Thrombin activatable fibrinolysis inhibitor [carboxypeptidase B2 (plasma), CPB2] is a basic carboxypeptidase, which inhibits fibrinolysis by cleaving the C-terminal lysine residues on plasmin-modified partially degraded fibrin. Plasma CPB2 concentrations have been reported to be under the control of numerous single nucleotide polymorphisms located in the regulatory and coding regions of the gene encoding CPB2 (CPB2). High functional CPB2 levels have been found to be associated with an increased risk for ischemic stroke. The present study investigated CPB2 antigen levels and associated CPB2 polymorphisms in an acute onset non-cardioembolic stroke population compared with an age- and sex-matched healthy control population. This is, to the best of our knowledge, the first such study done in an Asian Indian population. CPB2 antigen levels were significantly associated with the disease phenotype (P < 0.001) and with CPB2 polymorphisms (P < 0.001). The haplotypes generated on analysis of the genotypic data accounted for 21% of the natural variation in the CPB2 antigenic levels. However none of the haplotype combinations generated showed any association with disease phenotype and therefore could not explain for the difference in CPB2 antigen levels between cases and controls.
Aspirin and Clopidogrel are used in prophylaxis of patients undergoing percutaneous coronary inte... more Aspirin and Clopidogrel are used in prophylaxis of patients undergoing percutaneous coronary intervention and long-term prevention of cardiovascular and cerebrovascular events. Clopidogrel resistance has been attributed to P2Y1 and P2Y12 adenosine diphosphate (ADP) receptor polymorphisms. This study enrolled 100 patients of coronary artery disease (CAD) who were on the maintenance dose of clopidogrel (75 mg OD) with or without aspirin. In addition, 10 received loading dose (300 mg) prior to percutaneous coronary intervention. Relevant clinical and drug history were elicited. ADP-induced platelet aggregation study and PCR-RFLP for P2Y1 (1622A > G) and P2Y12 (i-T744C) polymorphisms were performed. Two groups of controls were used for defining cut-off for platelet aggregation response. Follow-up data, wherever available was recorded. The most common pattern of aggregation response was disaggregation, either complete (46.4%) or partial (53.6%). A frequency of 13% clopidogrel non-responders and 19% semi-responders was found. All the cases were H1/H1 haplotype for P2Y12 gene polymorphism and 28 (29.2%) patients carried P2Y1 1622A > G (21(21.9%) AG and 7(7.3%) GG) gene polymorphism, the frequency being greater in clopidogrel responders compared to semi/non-responders but difference was not statistically significant. There was no statistically significant difference between responders and semi/non-responders in terms of the history of risk factor for CAD, concurrent atorvastatin use or past history of an acute vascular event. On follow up, the two patients who developed myocardial infarction/acute coronary syndromes (MI/ACS) were clopidogrel semi- and non-responder, respectively. Variability in clopidogrel response with 13% non-responders and 19% semi-responders was seen in this study with adverse outcome (MI/ACS) on follow up seen in two patients. Hence, poor response to clopidogrel may be related to increased likelihood of adverse long-term coronary event that may benefit from additional or alternative anti-platelet therapy. Clopidogrel resistance was not associated with ADP receptor P2Y1 and P2Y12 gene polymorphisms. Hence, it is postulated that clopidogrel resistance in CAD patients is multifactorial and not caused by single-gene polymorphisms.
Thirty-three patients of Glanzmann&am... more Thirty-three patients of Glanzmann's thrombasthenia (GT) and their families were assessed for the expression of alphaIIbbeta3 on platelet surface, by flow cytometry, to determine the common subtypes in North Indians as well as to assess the carrier status in family members of GT patients. GT was diagnosed in patients with bleeding manifestations accompanied by absent/reduced platelet aggregation, secondary to adenosine-di-phosphate, adrenaline, arachidonic acid and collagen. Based on alphaIIbbeta3 levels, 21 patients (64%) were classified as type I (as alphaIIbbeta3 was absent), 4 patients (12%) as type II and 8 patients (24%) as type III. Eight out of 20 fathers, 10 out of 20 mothers and 20 out of 31 siblings were found to have reduced alphaIIbbeta3 levels. Reduced alphaIIbbeta3 expression was seen in 63% of parents and 65% of siblings. It is possible that low alphaIIbbeta3 levels in family members may reflect their carrier status. It is postulated that flow cytometry estimation of alphaIIbbeta3 in parents/siblings may detect carrier status in GT. It is also revealed that type I GT is the commonest subtype in North Indians.
Indian Journal of Community Medicine, the official organ of the Indian Association of Preventive ... more Indian Journal of Community Medicine, the official organ of the Indian Association of Preventive and Social Medicine (IAPSM), India.
A number of prothrombotic and fibrinolytic disorders may lead to venous thrombosis. A 4G/5G polym... more A number of prothrombotic and fibrinolytic disorders may lead to venous thrombosis. A 4G/5G polymorphism located in the promoter region of plasminogen activator inhibitor-1 (PAI-1) gene has been found to be commonly associated with the levels of PAI-1 and might be a risk factor for deep vein thrombosis (DVT). The aim of this study was to look for the potential association of this polymorphism with DVT in the Asian Indian population. A total of 110 consecutive patients (M:F = 62:48) with idiopathic DVT and equal number of age- and sex-matched healthy controls were the study participants. All study participants were typed for the PAI-1 4G/ 5G polymorphism, factor V Leiden, factor V Hong Kong/Cambridge mutations, and HR2 haplotype. The variant allele for the PAI-1 4G/5G polymorphism showed both genotypic (P = .0013, chi(2) = 10.303; odds ratio [OR] = 3.75) as well as allelic association (P = .0004, chi(2) = 12.273; OR = 1.99) with DVT. Factor V Leiden and factor V HR2 haplotype were observed in 10 (9.0%) and 13 (11.8%) patients, respectively. None of the study participants showed the factor V Hong Kong Cambridge mutations. Our study shows the association of 4G allele with DVT in Asian Indian population. The higher prevalence of 4G polymorphism in patients with DVT (compared with controls) seen in our study is in concordance with previous reports from the Caucasian population.
Hemophilia A is a common hereditary bleeding disorder caused mainly by mutations in the Factor VI... more Hemophilia A is a common hereditary bleeding disorder caused mainly by mutations in the Factor VIII (FVIII) gene, which results in defective or absent FVIII protein. Most of the causative mutations arise from the germ cells, which leads to either heterozygous or hemizygous state for the mutation in the next generation. Germline or somatic mosaic may result due to a de novo mutation during early embryogenesis. We analyzed 14 families of Indian origin with Hemophilia A [sporadic and severe] for the presence of mosaic individuals by employing Allele Specific PCR, mutation enrichment experiment and sequencing. Nine families had point mutations, 3 families had small deletions or insertions, 2 families had splice site mutations. The origin of the de novo mutation was assigned to the patients' mother in 8 families. For 4 families it was assigned to the maternal grandmother and to the maternal grandfather in 2 families. In a single family somatic mosaic was detected. The presence of somatic mosaic in families with sporadic Hemophilia A in India may confound risk estimation during genetic counseling.
We report results of the studies relating to the development of a non-invasive, label free immuno... more We report results of the studies relating to the development of a non-invasive, label free immunosensor based on nanostructured hafnium oxide (hafinia) deposited onto indium tin oxide (ITO) coated glass for oral cancer biomarker (CYFRA-21-1) detection in human saliva. The nanostructured hafnia (nHfO2) has been synthesized via one step low temperature hydrothermal process and modified with 3-aminopropyltriethoxy silane (APTES) for covalent immobilization of monoclonal antibodies (anti-CYFRA-21-1). Bovine serum albumin (BSA) was used to block non-specific sites at the anti-CYFRA-21-1/APTES/nHfO2/ITO electrode surface. The structural, morphological and spectroscopic characterization of the synthesized nanomaterials and fabricated electrodes has been carried out using X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) studies, respectively. The results of response studies conducted on BSA/anti-CYFRA-21-1/APTES/nHfO2/ITO immunoelectrode reveal that this biosensor has high sensitivity (9.28 μA mL ng−1 cm−2), wide linear detection range (2–18 ng mL−1) and fast response time (15 min). This immunosensor has been validated with enzyme linked immunosorbent assay (ELISA) in saliva samples of oral cancer patients.
CYFRA-21-1 is a novel biomarker secreted in the saliva of oral cancer patient in relatively highe... more CYFRA-21-1 is a novel biomarker secreted in the saliva of oral cancer patient in relatively higher amount (ng mL-1). We report, development of a simple, noninvasive and label free electrochemical biosensor based on nanostructured zirconia for the efficient detection of oral cancer. The silanized nanostructured zirconia has been electrophoretically deposited over ITO electrode that acts as an efficient support for covalent immobilization of monoclonal antibodies (anti-CYFRA-21-1). The fabricated platform has been characterized through AFM, XPS and FT-IR techniques. The results of the electrochemical response studies reveal that the BSA/anti-CYFRA-21-1/APTES/ZrO2/ITO immunoelectrode can be used to estimate the CYFRA-21-1 with a high sensitivity of 2.2 mA mL ng-1, linear detection range of 2-16 ng mL-1 and stability of 8 weeks. The observed results have been validated using ELISA and have implications towards the fabrication of point of care device for oral cancer monitoring.
The human CD99 protein is a 32-kDa type I transmembrane glycoprotein, while CD98 is a disulfide-l... more The human CD99 protein is a 32-kDa type I transmembrane glycoprotein, while CD98 is a disulfide-linked 125-kDa heterodimeric type II transmembrane glycoprotein. It has been previously shown that CD99 and CD98 oppositely regulate β1 integrin signaling, though the mechanisms by which this regulation occurs are not known. Our results revealed that antibody-mediated crosslinking of CD98 induced FAK phosphorylation at Y397 and facilitated the formation of the protein kinase Cα (PKCα)-syntenin-focal adhesion kinase (FAK), focal adhesions (FAs), and IPP-Akt1-syntenin complex, which mediates β1 integrin signaling. In contrast, crosslinking of CD99 disrupted the formation of the PKCα-syntenin-FAK complex as well as FA via FAK dephosphorylation. The CD99-induced dephosphorylation of FAK was apparently mediated by the recruitment of Src homology region 2 domain-containing phosphatase-2 (SHP2) to the plasma membrane and subsequent activation of its phosphatase activity. Further consequences of ...
Glanzmann&amp... more Glanzmann's thrombasthenia (GT) is an autosomal recessive bleeding disorder which is due to a defect in platelet aggregation in response to multiple physiological agonists. It has been demonstrated that the clinical phenotype of various diseases inherited in a classic Mendelian fashion can be modulated by a series of factors, inherited as well as acquired.
ABSTRACT Background. Elevated homocysteine levels have been seen to be associated with the presen... more ABSTRACT Background. Elevated homocysteine levels have been seen to be associated with the presence of thrombotic events. These may include arterial thrombotic events like stroke and myocardial infarctions or venous events like deep vein thrombosis, more so arterial as its association with thrombosis is well established. Aims. Lowering of homocysteine through administration of appropriate vitamin supplements has been documented in Cardiovascular disorders in India. These are primarily arterial conditions. The role of homocysteine in venous thrombotic conditions is less well documented and very little data is available in Deep Vein Thrombosis in Indian patients in this regard. Our present study takes a retrospective look at Deep Vein Thrombosis patients attending the Hematology Department of our hospital, which apart from looking into issues of Vitamin supplementation and Homocysteine, includes data on polymorphisms as well. Methods. 120 consecutive patients with deep venous thrombosis, confirmed by Doppler ultrasonography, were the subjects of study. All patients and equal number of healthy age and sex-matched controls were investigated for Homocysteine levels and also screened for MTHFR C677T, A1298C, MTR A2756G and MTRR G66A polymorphisms. Patients who were seen to have high levels of homocysteine were prescribed vitamin supplementation (Folic acid 5mg O.D, Vitamin B12 5µg O.D, Vitamin B6 2mg O.D) and were asked to report for a follow-up homocysteine check up post 3 months. 61 patients were found to have high levels of homocysteine of which only 23 reported for a follow-up homocysteine checkup. Differences between allelic and genotypic frequencies of individual polymorphisms between cases and controls and Hardy-Weinberg equilibrium (HWE) were tested using chi square tests. Statistical tests were done using SPSS version 12. Results. Most cases (97) were of lower limb thrombosis, 11 cases were of upper limb thrombosis. Thrombosis at unusual sites was present in 12 cases. These included the cerebral (5), abdominal (3), subclavian(2), renal(1) and retinal vein(1). High levels of baseline homocysteine were seen to be significantly associated with Deep Vein Thrombosis (p<0.001). All of the patients responded to the Folic acid dosage and showed reduction in the levels of plasma homocysteine(of the order of 13.1 umol/L i.e. 60.6% of the mean baseline plasma homocysteine of responders). However in some of these (7 out of 23) only a partial response was seen (plasma homocysteine levels reduced but not normalized i.e. between 5-15 umol/L). In these partial responders none of the patients were seen to carry the mutant homozygous forms of the MTHFRC677T, MTRA2756G or MTRRG66A polymorphisms. Only one patient amongst them carried the CC (mutant) genotype of the A1298C polymorphism. None of the complete responders carried the mutant homozygous forms of any of the four polymorphisms studied. The partial response to Folic acid dosage may be explained by the short duration of follow-up time(3-6 months) involved. Only MTHFR C677T & A1298C polymorphisms were seen to be associated with homocysteine levels (p<0.01). However none of the investigated four polymorphisms showed any association with Deep Vein Thrombosis. Summary and Conclusions. Nutritional deficiency and not genetic variation maybe the primary contributor to the prevalent hyperhomocysteinemic condition in Deep Vein Thrombosis patients of Asian Indian origin.
Multiple micronutrient supplementation of Nepalese women during pregnancy is associated with a si... more Multiple micronutrient supplementation of Nepalese women during pregnancy is associated with a significant increase in birth weight. We tested the hypothesis that improved birth weight in infants of mothers supplemented with micronutrients is associated with a decrease in inflammatory responses and an increase in the production of T helper 1 cells and T helper 2 cells. The study was embedded in a randomized controlled trial of 15 micronutrients, compared with iron-folate supplementation (control), given during pregnancy with the aim of increasing birth weight. Blood samples were collected at 32 wk of gestation, 12-20 wk after supplementation began, for the measurement of inflammatory markers. Breast-milk samples were collected 1 mo after delivery for the measurement of the ratio of milk sodium to potassium (milk Na:K). In an opportunistically selected subgroup of 70 women, mitogen-stimulated cytokine production was measured ex vivo in whole blood. Blood eosinophils; plasma concentra...
CD99 is known to be involved in the regulation of cell-cell adhesion. However, it remains unclear... more CD99 is known to be involved in the regulation of cell-cell adhesion. However, it remains unclear whether CD99 controls cell-extracellular matrix adhesion. In this study, the effects of CD99 activation on cell-extracellular matrix adhesion were investigated. It was found that engagement of CD99 with the stimulating antibody YG32 downregulated the adhesion of MCF-7 cells to fibronectin, laminin and collagen IV in a dose-dependent manner. The CD99 effect on cell-ECM adhesion was inhibited by overexpression of the dominant negative form of CD99 or CD99 siRNA transfection. Treatment of cells with Mn(2+) or by β(1) integrin-stimulating antibody restored the inhibitory effect of CD99 on cell-ECM adhesion. Cross-linking CD99 inactivated β(1) integrin through conformational change. CD99 activation caused dephosphorylation at Tyr-397 in FAK, which was restored by the β(1) stimulating antibody. Taken together, these results provide the first evidence that CD99 inhibits cell-extracellular matr...
British Journal of Haematology - BRIT J HAEMATOL, 2008
Thrombin activatable fibrinolysis inhibitor [carboxypeptidase B2 (plasma), CPB2] is a basic carbo... more Thrombin activatable fibrinolysis inhibitor [carboxypeptidase B2 (plasma), CPB2] is a basic carboxypeptidase, which inhibits fibrinolysis by cleaving the C-terminal lysine residues on plasmin-modified partially degraded fibrin. Plasma CPB2 concentrations have been reported to be under the control of numerous single nucleotide polymorphisms located in the regulatory and coding regions of the gene encoding CPB2 (CPB2). High functional CPB2 levels have been found to be associated with an increased risk for ischemic stroke. The present study investigated CPB2 antigen levels and associated CPB2 polymorphisms in an acute onset non-cardioembolic stroke population compared with an age- and sex-matched healthy control population. This is, to the best of our knowledge, the first such study done in an Asian Indian population. CPB2 antigen levels were significantly associated with the disease phenotype (P < 0.001) and with CPB2 polymorphisms (P < 0.001). The haplotypes generated on analysis of the genotypic data accounted for 21% of the natural variation in the CPB2 antigenic levels. However none of the haplotype combinations generated showed any association with disease phenotype and therefore could not explain for the difference in CPB2 antigen levels between cases and controls.
Aspirin and Clopidogrel are used in prophylaxis of patients undergoing percutaneous coronary inte... more Aspirin and Clopidogrel are used in prophylaxis of patients undergoing percutaneous coronary intervention and long-term prevention of cardiovascular and cerebrovascular events. Clopidogrel resistance has been attributed to P2Y1 and P2Y12 adenosine diphosphate (ADP) receptor polymorphisms. This study enrolled 100 patients of coronary artery disease (CAD) who were on the maintenance dose of clopidogrel (75 mg OD) with or without aspirin. In addition, 10 received loading dose (300 mg) prior to percutaneous coronary intervention. Relevant clinical and drug history were elicited. ADP-induced platelet aggregation study and PCR-RFLP for P2Y1 (1622A > G) and P2Y12 (i-T744C) polymorphisms were performed. Two groups of controls were used for defining cut-off for platelet aggregation response. Follow-up data, wherever available was recorded. The most common pattern of aggregation response was disaggregation, either complete (46.4%) or partial (53.6%). A frequency of 13% clopidogrel non-responders and 19% semi-responders was found. All the cases were H1/H1 haplotype for P2Y12 gene polymorphism and 28 (29.2%) patients carried P2Y1 1622A > G (21(21.9%) AG and 7(7.3%) GG) gene polymorphism, the frequency being greater in clopidogrel responders compared to semi/non-responders but difference was not statistically significant. There was no statistically significant difference between responders and semi/non-responders in terms of the history of risk factor for CAD, concurrent atorvastatin use or past history of an acute vascular event. On follow up, the two patients who developed myocardial infarction/acute coronary syndromes (MI/ACS) were clopidogrel semi- and non-responder, respectively. Variability in clopidogrel response with 13% non-responders and 19% semi-responders was seen in this study with adverse outcome (MI/ACS) on follow up seen in two patients. Hence, poor response to clopidogrel may be related to increased likelihood of adverse long-term coronary event that may benefit from additional or alternative anti-platelet therapy. Clopidogrel resistance was not associated with ADP receptor P2Y1 and P2Y12 gene polymorphisms. Hence, it is postulated that clopidogrel resistance in CAD patients is multifactorial and not caused by single-gene polymorphisms.
Thirty-three patients of Glanzmann&am... more Thirty-three patients of Glanzmann's thrombasthenia (GT) and their families were assessed for the expression of alphaIIbbeta3 on platelet surface, by flow cytometry, to determine the common subtypes in North Indians as well as to assess the carrier status in family members of GT patients. GT was diagnosed in patients with bleeding manifestations accompanied by absent/reduced platelet aggregation, secondary to adenosine-di-phosphate, adrenaline, arachidonic acid and collagen. Based on alphaIIbbeta3 levels, 21 patients (64%) were classified as type I (as alphaIIbbeta3 was absent), 4 patients (12%) as type II and 8 patients (24%) as type III. Eight out of 20 fathers, 10 out of 20 mothers and 20 out of 31 siblings were found to have reduced alphaIIbbeta3 levels. Reduced alphaIIbbeta3 expression was seen in 63% of parents and 65% of siblings. It is possible that low alphaIIbbeta3 levels in family members may reflect their carrier status. It is postulated that flow cytometry estimation of alphaIIbbeta3 in parents/siblings may detect carrier status in GT. It is also revealed that type I GT is the commonest subtype in North Indians.
Indian Journal of Community Medicine, the official organ of the Indian Association of Preventive ... more Indian Journal of Community Medicine, the official organ of the Indian Association of Preventive and Social Medicine (IAPSM), India.
A number of prothrombotic and fibrinolytic disorders may lead to venous thrombosis. A 4G/5G polym... more A number of prothrombotic and fibrinolytic disorders may lead to venous thrombosis. A 4G/5G polymorphism located in the promoter region of plasminogen activator inhibitor-1 (PAI-1) gene has been found to be commonly associated with the levels of PAI-1 and might be a risk factor for deep vein thrombosis (DVT). The aim of this study was to look for the potential association of this polymorphism with DVT in the Asian Indian population. A total of 110 consecutive patients (M:F = 62:48) with idiopathic DVT and equal number of age- and sex-matched healthy controls were the study participants. All study participants were typed for the PAI-1 4G/ 5G polymorphism, factor V Leiden, factor V Hong Kong/Cambridge mutations, and HR2 haplotype. The variant allele for the PAI-1 4G/5G polymorphism showed both genotypic (P = .0013, chi(2) = 10.303; odds ratio [OR] = 3.75) as well as allelic association (P = .0004, chi(2) = 12.273; OR = 1.99) with DVT. Factor V Leiden and factor V HR2 haplotype were observed in 10 (9.0%) and 13 (11.8%) patients, respectively. None of the study participants showed the factor V Hong Kong Cambridge mutations. Our study shows the association of 4G allele with DVT in Asian Indian population. The higher prevalence of 4G polymorphism in patients with DVT (compared with controls) seen in our study is in concordance with previous reports from the Caucasian population.
Hemophilia A is a common hereditary bleeding disorder caused mainly by mutations in the Factor VI... more Hemophilia A is a common hereditary bleeding disorder caused mainly by mutations in the Factor VIII (FVIII) gene, which results in defective or absent FVIII protein. Most of the causative mutations arise from the germ cells, which leads to either heterozygous or hemizygous state for the mutation in the next generation. Germline or somatic mosaic may result due to a de novo mutation during early embryogenesis. We analyzed 14 families of Indian origin with Hemophilia A [sporadic and severe] for the presence of mosaic individuals by employing Allele Specific PCR, mutation enrichment experiment and sequencing. Nine families had point mutations, 3 families had small deletions or insertions, 2 families had splice site mutations. The origin of the de novo mutation was assigned to the patients' mother in 8 families. For 4 families it was assigned to the maternal grandmother and to the maternal grandfather in 2 families. In a single family somatic mosaic was detected. The presence of somatic mosaic in families with sporadic Hemophilia A in India may confound risk estimation during genetic counseling.
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