Cellular response induced by a galactose-specific adhesin of enteroaggregative Escherichia coli in INT-407 cells.

In the present study, the role of a fimbrial galactose-specific adhesin of the T7 strain of enteroaggregative Escherichia coli (EAEC-T7) in the signal transduction pathways in human small intestinal epithelial cells (INT-407) was explored. The adhesin was purified by anion exchange chromatography using a Mono Q HR5/5 column in the AKTA purifier system. The characteristic stacked brick pattern of aggregative adherence of EAEC-T7 to INT-407 cells was found to be inhibited in the presence of immunoglobulin G against the purified adhesin as well as d-galactose. The adhesin induced a significant increase in the intracellular calcium concentration [Ca(2+)](i) in INT-407 cells, which was reduced in the presence of dantrolene (inhibitor of intracellular calcium stores), verapamil, calciseptin (calcium channel blockers) as well as neomycin [inhibitor of phospholipase C (PLC)]. Further, an increased level of PLCgamma1 and inositol 1,4,5-tri phosphate as well as enhanced activity of protein kinase C (PKC) in the adhesin-stimulated cells were found to be downregulated in the presence of neomycin and U73122 (inhibitors of PLC) and H-7 (inhibitor of PKC), respectively. The adhesin could also induce interleukin-8 secretion from INT-407 cells, which was inhibited in the presence of dantrolene as well as staurosporin (inhibitor of PKC). Collectively, our results have suggested that the galactose-specific adhesin-induced signal transduction pathway might play a crucial role in the EAEC-induced pathogenesis.

Apoptosis induction by Maackia amurensis agglutinin in childhood acute lymphoblastic leukemic cells.

Malignant transformation is known to be associated with changes in cell surface carbohydrate-architecture, which can be detected by lectins. In the present study, Maackia amurensis agglutinin (MAA), specific for NeuNAcalpha(2-->3)Gal/GalNAc showed strong binding with lymphoblasts of children having acute lymphoblastic leukemia (ALL) as compared to cells from children with non-hematological disorders ("Controls"). MAA recognized a 66 kDa sialoglycoprotein present in membrane fraction of ALL cells. Moreover, MAA induced apoptosis in ALL cells was found to be reduced significantly in presence of GM2/IgG(MAA). Thus, MAA has a potential to be used as diagnostic and therapeutic agent in case of childhood-ALL.

Serum soluble Fas levels and prediction of response to platinum-based chemotherapy in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is treated mainly by platinum-based combination chemotherapy. Chemotherapy induces apoptosis in which the Fas/Fas ligand pathway is important. Serum soluble Fas (sFas) is a biomarker of this pathway and functionally inhibits Fas-/FasL-mediated apoptosis. In this study, we have investigated the role of sFas in prediction of response to chemotherapy in EOC. Thirty-five patients were recruited and their serum sFas levels were estimated by ELISA at 4 time points-preoperative (sFas1), postoperative (sFas2), midchemotherapy (sFas3) and at the end of chemotherapy (sFas4). The response to chemotherapy was documented clinically, radiologically and by CA-125 levels, based on which, 2 groups were identified: primary chemosensitive (n = 24) and primary chemoresistant (n = 11). Based on the disease status at last follow-up, 2 groups were identified: No Evidence of Disease (n = 15) and Evidence of Disease (n = 20). The primary chemoresistant tumors showed significantly higher median sFas2 levels (p = 0.033) with the sFas2/sFas1 ratio > or =1 (p = 0.001). A multivariate Cox proportional hazards regression model identified sFas2/sFas1 ratio as a significant factor for the prediction of response to platinum-based chemotherapy (p = 0.011). Receiver operating characteristic (ROC) analysis showed that at a ratio of 1.2, sFas2/sFas1 achieved a sensitivity of 82% and specificity of 100% for prediction of chemotherapeutic response. sFas2/sFas1 and sFas3/sFas1 ratio was also higher in patients with evidence of disease (p = 0.018 and p = 0.028, respectively). Progression-free survival rates in patients with sFas2/sFas1 ratio <1 exceeded those with ratio > or =1 (p = 0.004). In conclusion, serum sFas is a useful biomarker for predicting response to platinum-based chemotherapy in EOC.

A potential epidemic factor from the bacteria, Vibrio cholerae WO7.

Certain species of Vibrio cholerae have evolved mechanisms to become pathogenic to humans, with the potential to cause a severe life-threatening diarrheal disease, cholera. Cholera can emerge as explosive outbreaks in the human population. V. cholerae illness is produced primarily through the expression of a potent toxin (cholera toxin) within the human intestine. The present study has been carried out on a novel toxin purified from V. cholerae W07, an epidemic cholera strain devoid of cholera toxin gene (ctx). A modified method of purification improved purification fold as well as yield of this toxin. Heating was found to be the essential and sufficient condition for dissociation of the two subunits (58 kDa and 40 kDa) of this toxin (pI 5.2). The 40-kDa subunit of the purified toxin was identified as the carbohydrate binding subunit. This toxin was found to induce apoptosis in HEp-2 cells. Thus, the WO7 toxin seems to have potential importance in the pathogenesis of disease associated with Vibrio cholerae WO7.

Telomerase activity in sputum and telomerase and its components in biopsies of advanced lung cancer.

PURPOSE: In this study, we explored the diagnostic utility of sputum telomerase activity as a non-invasive biomarker of lung cancer. In biopsies of lung cancer, the relationship of telomerase activity to telomerase reverse transcriptase (hTERT) and telomerase RNA component (hTERC) and to c-Myc expression was also evaluated. METHODS: Paired biopsy and sputum samples were evaluated for telomerase activity by the telomerase repeat amplification protocol (TRAP) assay in 34 cases of lung cancer and in 30 control subjects without any evidence of lung cancer. hTERT and hTERC transcript expression was evaluated in 42 cases of lung cancer and compared to telomerase activity and c-Myc transcript expression. RESULTS: Telomerase activity was present in 85.2% of biopsies and in 67.6% of paired sputum with a good concordance. Three out of the 30 negative controls showed a weak telomerase activity, all of whom had sarcoidosis. Thus, sputum telomerase activity had sensitivity, specificity, Negative Predictive Value and Positive Predictive Value of 67.6%, 90%, 71% and 88.46%, respectively. The hTERT levels correlated to the telomerase activity but not to the c-Myc oncogene expression. CONCLUSIONS: In lung cancer, sputum telomerase activity is a candidate non-invasive biomarker of malignancy.

Galactose-specific fimbrial adhesin of enteroaggregative Escherichia coli: a possible aggregative factor.

A galactose-specific adhesin was isolated from the fimbriae of an enteroaggregative Escherichia coli (EAEC) strain. The adhesin was found to be a high molecular weight aggregate of the 18-kDa monomer. The dimeric (36 kDa) and tetrameric (76 kDa) forms appeared in sodium dodecyl sulphate polyacrylamide gel electrophoresis when a higher concentration of the adhesin was used. The IgGAD (IgG against adhesin) obtained from the immune sera raised in rabbits against purified adhesin could detect all three forms of the adhesin even from the crude fimbrial preparation. The IgGAD failed to recognize the adhesin in the presence of galactose, thereby suggesting the antibody-binding site and the sugar-binding site on the adhesin might be same or overlapping. Furthermore, the IgGAD could localize the adhesin exclusively on the fimbriae as observed in immunogold electron microscopy. The aggregative adherence of the bacteria to HEp-2 cells was reduced to 70% in the presence of the IgGAD. A glycoprotein (34 kDa) present in the membrane fraction of HEp-2 cells interacted with the purified adhesin in a galactose-specific manner. The IgGAD could recognize the adhesin from the crude fimbrial preparation of 9 out of 10 clinical isolates of EAEC strains but failed to identify any protein from the crude fimbrial preparation of Salmonella typhimurium (fim +ve as well as fim -ve strain), Vibrio cholerae (WO7) or Escherichia coli DH5alpha.

The role of a heat shock protein from V. cholerae 0139 in the gut immune response.

An immunodominant heat shock protein (Hsp 24) was purified from Vibrio cholerae O139 at 42 degrees C and used as an immunomodulator for studying the gut immune response. T cell clone and T cell line specific for the Hsp 24 were generated from the lymphocytes of lamina propria and intra-epithelial lymphocytes of mice orally infected with V. cholerae O139, respectively. The T cell clone was TCR alphabeta(+), CD4(+) and appeared to play an important role in the functioning of gut B-lymphocytes. The T cell line had heterogenous population of CD8+ and CD4+ cells, most of which were found to be TCR alphabeta(+) and a minor population was TCR gammadelta(+). The lymphokine profile of T cell line showed IFN-gamma to be the most abundant lymphokine followed by IL-2 and IL-4. The possible involvement of alternative pathway of activation for T cell clone was also addressed in this study. The splenocytes showed an up-regulation of their CD2 receptor expression on stimulation with the Hsp-24. The pattern of lymphokines released by splenocytes stimulated with the Hsp-24 showed no particular cell type to be responsible for mounting immune response. Thus, there is involvement of both, mucosal and peripheral arm of the immune system.

Characterization of adhesin variants in Indian isolates of enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) are causative agents of diarrhea, being characterized by aggregative adherence to cultured epithelial cells. In this study, phenotypic properties of EAEC were analyzed with respect to AA, hemagglutination, clump and biofilm formation, all of which are mediated by aggregative adherence fimbriae (AAF). The strains were also screened for AAF types, AAF adhesin variants and Dr adhesin by PCR. Of the three known AAF types, AAF/I and AAF/II adhesin variants were identified. An association between the AAF/adhesin genotypes and the subtypes/scores of phenotypic properties was sought and it was observed that strains harboring same adhesins displayed different subtypes/scores and vice versa.

Loss of fragile histidine triad gene expression in advanced lung cancer is consequent to allelic loss at 3p14 locus and promoter methylation.

The fragile histidine triad (FHIT) gene located at the 3p14.2 locus plays an important role in the pathogenesis of lung cancer. The objective of this study was to analyze loss of heterozygosity and FHIT gene methylation status and correlate them to fhit expression. Bronchoscopically obtained lung biopsies from 30 cases of histologically proven carcinoma of the lung in stage III were assessed for the alterations in the FHIT gene. Fhit protein expression was determined by immunohistochemistry, and transcript levels were determined by reverse transcription-PCR. Microsattelite alterations and methylation status of the Fhit gene promoter was determined by PCR. Loss of heterozygosity at the 3p14 locus was observed in all the 30 cases at least by one of the three microsatellite polymorphic markers. The FHIT gene promoter showed complete methylation in 37% cases and partial methylation in 47% cases, and 16% cases showed no promoter methylation. FHIT full-length coding region (exons 5-9) transcripts were present in eight cases (26.6%), and aberrant transcripts were additionally seen in four cases. Loss of FHIT mRNA expression correlated to FHIT promoter methylation but not to loss of heterozygosity at the 3p14 locus. There was a strong correlation between the expression of FHIT at the transcript and protein level. The apoptotic index estimated by the terminal deoxynucleotidyl transferase-mediated nick end labeling assay was significantly correlated to the fhit protein expression. The results of this study indicate that in locally advanced carcinoma of the lung, there is frequent loss of FHIT expression, and methylation of the FHIT gene promoter is an important mechanism of its inactivation.

Evaluation of renal functions in asphyxiated newborns.

Renal injury due to perinatal asphyxia has not been systematically evaluated. The available studies have used variable definitions, incomplete investigations and none had a control group. The aim of this study was to evaluate systematically the renal functions in severely asphyxiated newborns and to find if abnormal renal function tests can predict adverse outcome (death or neurologic abnormality at discharge). In a prospective case-control design, 25 inborn babies>or=34 weeks gestation having asphyxia (5 min Apgaror=5 min) were enrolled as 'cases'. Simultaneously 25 gestation and weight matched babies with no asphyxia were enrolled as 'controls'. Renal function tests, calculated renal indices using timed urine collections and excretion of beta2-microglobulin and N-acetyl-beta-D-glucosaminidase (NAG) were monitored in both the groups for first 4 days of life. Fourteen (56 per cent) asphyxiated babies had acute renal failure (ARF) as compared to 1 (4 per cent) control (p=0.002). Blood urea and serum creatinine values were significantly higher in asphyxiated babies on day 4 but not on day 2. Renal failure index and FeNa were higher in asphyxiated babies on both day 2 and day 4, but creatinine clearance was not different. Urinary excretion of both beta2-microglobulin and NAG was higher in the asphyxiated babies on day 2 as well as day 4. Five minute Apgar1.5 mg/dl alone had the best specificity and positive predictive value. The renal parameters were however poorer predictors of adverse outcome in comparison to clinical markers like 5 min Apgar

Alterations of tumor suppressor gene p16INK4a in pancreatic ductal carcinoma.

BACKGROUND: Cell cycle inhibitor and tumor suppressor gene p16/MTS-1 has been reported to be altered in a variety of human tumors. The purpose of the study was to evaluate primary pancreatic ductal adenocarcinomas for potentially inactivating p16 alterations. METHODS: We investigated the status of p16 gene by polymerase chain reaction (PCR), nonradioisotopic single strand conformation polymorphism (SSCP), DNA sequencing and hypermethylation analysis in 25 primary resected ductal adenocarcinomas. In addition, we investigated p16 protein expression in these cases by immunohistochemistry (IHC) using a monoclonal antibody clone (MS-887-PO). RESULTS: Out of the 25 samples analyzed and compared to normal pancreatic control tissues, the overall frequency of p16 alterations was 80% (20/25). Aberrant promoter methylation was the most common mechanism of gene inactivation present in 52% (13/25) cases, followed by coding sequence mutations in 16% (4/25) cases and presumably homozygous deletion in 12% (3/25) cases. These genetic alterations correlated well with p16 protein expression as complete loss of p16 protein was found in 18 of 25 tumors (72%). CONCLUSION: These findings confirm that loss of p16 function could be involved in pancreatic cancer and may explain at least in part the aggressive behaviour of this tumor type.

Nitric oxide metabolites in induced sputum: a noninvasive marker of airway inflammation in asthma.

OBJECTIVE: This study was designed to examine for nitric oxide (NO) metabolites in induced sputum as a marker of airway inflammation in asthmatic children. DESIGN. Prospective interventional SETTING: Pediatric Allergy and Asthma Clinic of a tertiary care referral hospital in Northern India. SUBJECTS: Twenty-one children with asthma who were not receiving corticosteroids for the preceding 3 months and 10 healthy controls were enrolled. METHODS: Hypertonic saline-induced sputum was obtained at study entry in controls, and at study entry and after 6 weeks of inhaled corticosteroid (ICS) therapy in asthmatic children. Fresh expectorated sputum was treated with dithiothreitol and cytospinned for cell count. NO metabolites were measured in the supernatant by the modified Griess reaction. RESULTS: Asthmatic children, compared with controls, had significantly higher concentration of NO metabolites (22.4 +/- 209.69 vs 39.2 +/- 15.9 (moL/L, P <0.01) and a higher percentage of eosinophils (15.3 +/- 12.0 vs 0.8 +/- 1.1%, P <0.01) in induced sputum. Both NO metabolites and eosinophil percentage declined following treatment with ICS for 6 weeks (P <0.01). CONCLUSION: The study confirms that the level of NO metabolites is increased in the tracheobronchial secretions of asthmatic children and decreases following ICS therapy. Measurement of NO metabolites in induced sputum may be useful for monitoring airway inflammation in children with asthma.

Bcl-XL protein levels determine apoptotic index in pancreatic carcinoma.

OBJECTIVES: The present study was designed to analyze the expression of the major antiapoptotic molecules Bcl-2, Bcl-XL and the proapoptotic Bax in pancreatic ductal carcinoma and their correlation to the extent of apoptosis. METHODS: Tissue samples were obtained from patients (age, 27-78 years) having surgery for pancreatic cancer. Normal pancreatic tissue away from the main tumor mass was also analyzed. The levels of Bcl-2, Bcl-XL, and Bax mRNA expression were analyzed by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). The presence of corresponding proteins was determined by immunohistochemistry (IHC). The apoptotic index was determined by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. RESULTS: A total of 25 cases were analyzed. The apoptotic index (percentage) ranged from 0.0% to 1.8%, with a median of 0.26. Semiquantitative RT-PCR revealed variable mRNA expression, with the Bcl-2/Bax ratio ranging from 0.2 to 1.5 and the Bcl-XL/Bax ratio ranging from 0.3 to 1.8. There was no correlation of mRNA levels with the apoptotic index. Immunohistochemical analysis showed positive Bcl-2, Bax, Bcl-XL expression in 20%, 72%, and 92% of cancer samples; however, their levels were variable. Spearman rank correlation coefficient test revealed a significant inverse association for the Bcl-XL IHC score and apoptotic index (P < 0.05). In contrast, Bcl-2, Bax protein levels did not show any association with the apoptotic index. However, as compared with the normal pancreas, Bcl-2, Bcl-XL, Bax were overexpressed in most of the pancreatic cancer samples (Mann-Whitney U test, P < 0.01). CONCLUSION: In pancreatic cancer, there is an upregulation of all the apoptotic regulatory molecules and the apoptotic index is chiefly determined by Bcl-XL protein levels.

Expression of p53 protein and the apoptotic regulatory molecules Bcl-2, Bcl-XL, and Bax in locally advanced squamous cell carcinoma of the lung.

OBJECTIVE: Bcl-2 family of proteins plays a critical role in the regulation of apoptosis. This pathway may be dysregulated leading to an altered ratio of pro- and anti-apoptotic molecules, hence rendering cells resistant to chemotherapy. The objective of this study was to understand the role of Bcl-2 family members in mediation of apoptosis in squamous cell carcinoma of the lung. RESULTS: Bronchoscopically obtained lung biopsies from 30 cases of histologically proven squamous cell carcinoma of the lung in stage III were assessed for the expression of Bcl-2, Bcl-XL, and Bax at the mRNA and protein levels by semi-quantitative reverse transcription (RT-PCR) and immunohistochemistry. The apoptotic index (AI) was determined by the TUNEL assay. The AI ranged from <0.1 to 6.0% with a median of 1.3%. Bcl-2/Bax transcript ratio ranged from 1.5 to 4.5 and Bcl-XL/Bax from 1.3 to 4.0 indicating increased levels of anti-apoptotic molecules at the transcript levels. There was no correlation of the mRNA levels to the apoptotic index. (Wilcoxon-signed rank test.) Immunohistochemistry for proteins revealed that majority of the tumors were Bax predominated. p53 protein immunohistochemical expression was present in 66% cases. The apoptotic index correlated with Bax expression (P < 0.05; Wilcoxon-signed rank test and chi-square test) but not with Bcl-2, Bcl-XL or p53 levels. There was a positive association of p53 with Bax expression. CONCLUSION: The results of this study indicate that in locally advanced squamous cell carcinoma of the lung, Bax protein is up regulated and determines the level of apoptosis.

Effect of phenobarbital on free radicals in neonates with hypoxic ischemic encephalopathy--a randomized controlled trial.

BACKGROUND: Phenobarbital is one of the oldest, cheapest and easily available cerebroprotective drugs for the hypoxic brain. However, its potential and various actions have not been fully explored. AIM: To evaluate the effects of Phenobarbital on levels of oxidants and anti-oxidants in term and near term neonates with hypoxic ischemic encephalopathy. METHODS: Design--randomized controlled trial. Setting--tertiary care referral perinatal centre. Procedure--asphyxiated neonates (gestation > or = 34 weeks) with HIE were randomized to receive Phenobarbital 20 mg/kg i.v. within first six hours of life or to control group. CSF levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx) and blood levels of vitamins A and E were estimated at 10-12 hours of age. RESULTS: CSF levels of MDA, SOD, GPx and blood levels of vitamins A and E were significantly lower in the Phenobarbital group (p<0.001). There was a trend towards lower levels of CSF MDA, SOD, GPx and blood vitamins A and E in babies with normal outcome as compared to babies with adverse outcome (death or neurologically abnormal at discharge). CONCLUSION: Phenobarbital in the dose of 20 mg/kg i.v. given within 6 hours of life in term and near-term neonates with HIE, was associated with a decrease in lipid peroxides, anti-oxidant enzymes and anti-oxidant vitamins.

Effect of W07-toxin on gut physiological response in mice.

A number of unknown secretogenic factor(s) from Vibrio cholerae have been implicated to play a role in inducing cholera-like symptoms observed in patients. The present study has been carried out on the novel W07-toxin (pI 5.2) from V. cholerae W07, an epidemic cholera strain devoid of the ctx gene. The toxin showed maximum binding to GM(1) and interacted with a 20 kDa glycoprotein present on the cell membrane of mice enterocytes in a GM(1) specific manner. The analysis of biochemical parameters in enterocytes triggered with this toxin revealed a significant increase in intracellular calcium concentration and a massive secretion of Cl(-). However, no absorption of Na(+) was observed under the same condition. This toxin also elevated the level of cyclic adenosine 3',5'-monophosphate (cAMP) as well as protein kinase A (PKA). Thus, the novel toxin, although distinct from cholera-toxin, showed some functional homology to it and may be one of the key players inducing electrolyte imbalance within intestinal cells in the cholera-like symptoms associated with V. cholerae W07.

UDPgalactose 4-epimerase from Saccharomyces cerevisiae. A bifunctional enzyme with aldose 1-epimerase activity.

UDPgalactose 4-epimerase (epimerase) catalyzes the reversible conversion between UDPgalactose and UDPglucose and is an important enzyme of the galactose metabolic pathway. The Saccharomyces cerevisiae epimerase encoded by the GAL10 gene is about twice the size of either the bacterial or human protein. Sequence analysis indicates that the yeast epimerase has an N-terminal domain (residues 1-377) that shows significant similarity with Escherichia coli and human UDPgalactose 4-epimerase, and a C-terminal domain (residues 378-699), which shows extensive identity to either the bacterial or human aldose 1-epimerase (mutarotase). The S. cerevisiae epimerase was purified to > 95% homogeneity by sequential chromatography on DEAE-Sephacel and Resource-Q columns. Purified epimerase preparations showed mutarotase activity and could convert either alpha-d-glucose or alpha-d-galactose to their beta-anomers. Induction of cells with galactose led to simultaneous enhancement of both epimerase and mutarotase activities. Size exclusion chromatography experiments confirmed that the mutarotase activity is an intrinsic property of the yeast epimerase and not due to a copurifying endogenous mutarotase. When the purified protein was treated with 5'-UMP and l-arabinose, epimerase activity was completely lost but the mutarotase activity remained unaffected. These results demonstrate that the S. cerevisiae UDPgalactose 4-epimerase is a bifunctional enzyme with aldose 1-epimerase activity. The active sites for these two enzymatic activities are located in different regions of the epimerase holoenzyme.

Role of the W07-toxin on Vibrio cholerae-induced diarrhoea.

Vibrio cholerae W07 strain isolated from a cholera epidemic in South India, lacked the ctx gene but could still secrete a novel toxin, the W07-toxin that could cause fluid accumulation in ligated rabbit ileal loop. The important intracellular messengers implicated in this study were Ca(2+), cyclic AMP, inositol triphosphate and protein kinase C (PKC). A number of inhibitors/channel blockers have further shown the major role of [Ca(2+)](i) in modulation of the toxin-induced cellular response. An increase in the level of reactive oxygen species (ROS) in the W07-toxin-stimulated enterocytes correlated with the decrease in the levels of antioxidant enzymes, catalase and superoxide dismutase (SOD). The reactive nitrogen intermediates (RNI) detected by measuring the levels of nitrite and citrulline, were found to be high in the enterocytes triggered with the W07-toxin, thereby indicating their role in toxin-mediated change in mucosal permeability. The precise role of the toxin has also been authenticated by conducting the experiments with W07-toxin preincubated in the presence of IgG(WT) (IgG isolated from antitoxin sera) or GM(1). Thus, a significant increase in the levels of second messengers and a decrease in antioxidant defenses appear to be important in mediating the fluid secretion caused by this novel toxin from V. cholerae W07.

A possible role of HSP70 in mediating cardioprotection in patients undergoing CABG.

Heat shock protein 70 (HSP70) has been reported to be involved in myocardial self-preservation system. This study shows direct evidence of the effect of HSP70 on lymphocytes during ischemia and reperfusion in CABG (coronary artery bypass graft) surgery. Lymphocytes were separated from the blood obtained from 10 patients undergoing CABG at different time intervals. (i) Baseline samples (drawn before onset of bypass), (ii) ischemic samples (30 min after cross-clamp) and (iii) reperfusion samples (10 min after the cross clamp removal) were incubated with recombinant HSP70 and the cells were harvested after 36 h. The effect of HSP70 was monitored by measuring second messengers such as intracellular calcium, protein kinase C (PKC) and inositol triphosphate (IP3). In addition CD69 expression was also measured. The results showed a significant decrease in intracellular calcium and CD69 expression in ischemia and further in reperfusion samples as compared to their respective untriggered controls. PKC and IP3 levels however remained unaffected. The protective effect of HSP70 during ischemia and reperfusion could thus be attributed to decreasing intracellular calcium and CD69 expression. This study could therefore provide a mechanism of cardioprotection afforded by HSP70.

Release of pro-inflammatory mediators during myocardial ischemia/reperfusion in coronary artery bypass graft surgery.

Inflammation has been reported to play an important role in cardiac surgery under cardiopulmonary bypass due to systemic endotoxemia. In order to develop strategies against this injury in future we studied the combined effect of a number of inflammatory mediators in myocardial ischemia/reperfusion. Coronary sinus blood samples of ten patients undergoing coronary artery bypass graft surgery (CABG) were obtained at three time intervals (1) before onset of bypass (2) 30 min after cross clamp, and (3) 10 min after removal of cross clamp. The samples were subjected to evaluate levels of nitric oxide byproducts (nitrite and nitrate and citrulline), inflammatory cytokines (interleukin-2, interferon-gamma and interleukin-6), adhesion molecules, (CD62L and CD54), ratio of cell surface markers (CD4/CD8 and TCRalphabeta/gammadelta) cell activation markers (CD69 and HLA DR) and second messengers (protein kinase C, inositol 1,4,5 triphosphate and intracellular calcium levels). Ischemia and further reperfusion resulted in significant rise in nitrite and nitrate levels (p < 0.001), interleukin-6 (p < 0.01), CD62L (p < 0.001), CD69 (p < 0.05), protein kinase C (p < 0.001) and intracellular calcium (p < 0.001). A fall in CD4/CD8 ratio was observed on reperfusion. These changes during CABG show that ischemia/reperfusion leads to a release of an array of pro-inflammatory mediators of tissue injury, which could lead to pathophysiological changes. Hence the study suggests the need of some protective therapies against these inflammatory markers.