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1.  Bacterial Lipopolysaccharide Augments Febrile-Range Hyperthermia-Induced Heat Shock Protein 70 Expression and Extracellular Release in Human THP1 Cells 
PLoS ONE  2015;10(2):e0118010.
Sepsis, a devastating and often lethal complication of severe infection, is characterized by fever and dysregulated inflammation. While infections activate the inflammatory response in part through Toll-like receptors (TLRs), fever can partially activate the heat shock response with generation of heat shock proteins (HSPs). Since extracellular HSPs, especially HSP70 (eHSP70), are proinflammatory TLR agonists, we investigated how exposure to the TLR4 agonist, bacterial lipopolysaccharide (LPS) and febrile range hyperthermia (FRH; 39.5°C) modify HSP70 expression and extracellular release. Using differentiated THP1 cells, we found that concurrent exposure to FRH and LPS as well as TLR2 and TLR3 agonists synergized to activate expression of inducible HSP72 (HSPA1A) mRNA and protein via a p38 MAP kinase-requiring mechanism. Treatment with LPS for 6 h stimulated eHSP70 release; levels of eHSP70 released at 39.5°C were higher than at 37°C roughly paralleling the increase in intracellular HSP72 in the 39.5°C cells. By contrast, 6 h exposure to FRH in the absence of LPS failed to promote eHSP70 release. Release of eHSP70 by LPS-treated THP1 cells was inhibited by glibenclamide, but not brefeldin, indicating that eHSP70 secretion occurred via a non-classical protein secretory mechanism. Analysis of eHSP70 levels in exosomes and exosome-depleted culture supernatants from LPS-treated THP1 cells using ELISA demonstrated similar eHSP70 levels in unfractionated and exosome-depleted culture supernatants, indicating that LPS-stimulated eHSP70 release did not occur via the exosome pathway. Immunoblot analysis of the exosome fraction of culture supernatants from these cells showed constitutive HSC70 (HSPA8) to be the predominant HSP70 family member present in exosomes. In summary, we have shown that LPS stimulates macrophages to secrete inducible HSP72 via a non-classical non-exosomal pathway while synergizing with FRH exposure to increase both intracellular and secreted levels of inducible HSP72. The impact of increased macrophage intracellular HSP70 levels and augmented secretion of proinflammatory eHSP70 in the febrile, infected patient remains to be elucidated.
PMCID: PMC4320107  PMID: 25659128
2.  Tracheal intubation in patients with cervical spine immobilization: A comparison of McGrath® video laryngoscope and Truview EVO2® laryngoscope 
Indian Journal of Anaesthesia  2014;58(3):269-274.
Background and Aims:
Literature suggests that glottic view is better when using McGrath® Video laryngoscope and Truview® in comparison with McIntosh blade. The purpose of this study was to evaluate the effectiveness of McGrath Video laryngoscope in comparison with Truview laryngoscope for tracheal intubation in patients with simulated cervical spine injury using manual in-line stabilisation.
This prospective randomised study was undertaken in operation theatre of a tertiary referral centre after approval from the Institutional Review Board. A total of 100 consenting patients presenting for elective surgery requiring tracheal intubation were randomly assigned to undergo intubation using McGrath® Video laryngoscope (n = 50) or Truview® (n = 50) laryngoscope. In all patients, we applied manual-in-line stabilisation of the cervical spine throughout the airway management. Statistical testing was conducted with the statistical package for the social science system version SPSS 17.0. Demographic data, airway assessment and haemodynamics were compared using the Chi-square test. A P < 0.05 was considered significant.
The time to successful intubation was less with McGrath video laryngoscope when compared to Truview (30.02 s vs. 38.72 s). However, there was no significant difference between laryngoscopic views obtained in both groups. The number of second intubation attempts required and incidence of complications were negligible with both devices. Success rate of intubation with both devices was 100%. Intubation with McGrath Video laryngoscope caused lesser alterations in haemodynamics.
Both laryngoscopes are reliable in case of simulated cervical spine injury using manual-in-line stabilisation with 100% success rate and good glottic view.
PMCID: PMC4090991  PMID: 25024468
McGrath video laryngoscope; simulated difficult airway; Truview laryngoscope
3.  A Central Role for Transcription Factor C/EBP-β in Regulating CD1d Gene Expression in Human Keratinocytes1 
CD1d is a nonclassical Ag-presenting molecule that presents glycolipid Ags to NKT cells that are involved in immune defense and tumor rejection. It also plays a role in immunoregulatory functions in the epidermis. The mechanisms controlling the expression of CD1d are not well understood. Therefore, we cloned the CD1d gene promoter and characterized its activities in primary human keratinocytes and other cell lines of epithelial origin. We found that a CCAAT box in the CD1d promoter is required for its expression in keratinocytes. We show here that transcription factor C/EBP-β binds to the CCAAT box in the CD1d promoter in vitro and in vivo. Consistent with these observations, deletion of the gene encoding for C/EBP-β caused a loss of CD1d expression. The in vivo regulation of CD1d has significant implications for the pathologic mechanisms of certain immunologic skin diseases in which NKT cells play a role, such as allergic contact dermatitis and psoriasis. Together, these data show a central role for C/EBP-β in regulating CD1d transcription.
PMCID: PMC3891659  PMID: 19592659
4.  Hyperthermia Promotes and Prevents Respiratory Epithelial Apoptosis through Distinct Mechanisms 
Hyperthermia has been shown to confer cytoprotection and to augment apoptosis in different experimental models. We analyzed the mechanisms of both effects in the same mouse lung epithelial (MLE) cell line (MLE15). Exposing MLE15 cells to heat shock (HS; 42°C, 2 h) or febrile-range hyperthermia (39.5°C) concurrent with activation of the death receptors, TNF receptor 1 or Fas, greatly accelerated apoptosis, which was detectable within 30 minutes and was associated with accelerated activation of caspase-2, -8, and -10, and the proapoptotic protein, Bcl2-interacting domain (Bid). Caspase-3 activation and cell death were partially blocked by inhibitors targeting all three initiator caspases. Cells expressing the IκB superrepessor were more susceptible than wild-type cells to TNF-α–induced apoptosis at 37°C, but HS and febrile-range hyperthermia still increased apoptosis in these cells. Delaying HS for 3 hours after TNF-α treatment abrogated its proapoptotic effect in wild-type cells, but not in IκB superrepressor-expression cells, suggesting that TNF-α stimulates delayed resistance to the proapoptotic effects of HS through an NF-κB–dependent mechanism. Pre-exposure to 2-hour HS beginning 6 to16 hours before TNF-α treatment or Fas activation reduced apoptosis in MLE15 cells. The antiapoptotic effects of HS pretreatment were reduced in TNF-α–treated embryonic fibroblasts from heat shock factor-1 (HSF1)-deficient mice, but the proapoptotic effects of concurrent HS were preserved. Thus, depending on the temperature and timing relative to death receptor activation, hyperthermia can exert pro- and antiapoptotic effects through distinct mechanisms.
PMCID: PMC3547099  PMID: 22962066
heat shock; febrile-range hyperthermia; Fas; TNF; apoptosis
5.  Comparison of total versus partial revision of primary ventriculoperitoneal shunt failures 
Optimal surgical management of patients presenting with primary shunt failure in the era of neuroendoscopy remains complex. The value of replacing the entire shunt system as opposed to a single shunt component has not been assessed extensively.
In a retrospective study, the records of all patients who underwent their first shunt revision due to mechanical obstruction between September 2007 and December 2011 were reviewed. Shunt revisions were classified as total (entire shunt replaced) or partial (only malfunctioning component replaced). Patients having a minimum follow-up of 1 year after primary shunt revision were included in the study. Kaplan-Meier (shunt survival curves) and log-rank analysis were used to compare failure rates between partially and totally revised shunts.
Records of 62 patients in whom cause of primary shunt failure was obstruction (P roximal or distal) were analyzed retrospectively. At the end of follow-up period, 26 out of 28 partial revision group and 22 out of 34 total revision group had shunt failure. The median survival of the shunt in the partial revision and total revision groups was 60 and 270 days, respectively. The method (partial/total revision) related difference in shunt survival duration was statistically significant as shown by log-rank analysis (log-rank test value = 5.94 and P < 0.05).
Partial revision of shunt predisposes to accelerated shunt failure as compared with total revision in cases of obstructed ventriculoperitoneal shunt.
PMCID: PMC3740619  PMID: 23956943
Hydrocephalus; revision; ventriculoperitoneal shunt failure
6.  Physiological and biochemical effect of 24-epibrassinoslide on cold tolerance in maize seedlings 
Germination and early seedling growth are important for establishment of maize because maize is chilling sensitive crop and low temperature during early period of growth can be detrimental to subsequent crop growth and productivity. Therefore, it is important to protect maize seedling from cold stress. A study was conducted on induced cold tolerance by 24-epibrassinoslide (EBR) at the Indian Agricultural Research Institute, New Delhi, India. Maize seedlings were raised in green house condition (25/18 °C day-night temperatures). Ten days old seedlings were treated with EBR (0.0, 0.01, 0.1, 1.0 and 10 μM) and then divided into two sets, one set was kept in greenhouse (25/18 °C day-night temperatures) and another was transferred to net house (cold stress). Data on various morpho-physiological traits was recorded after 7, 14 and 21 days of treatment. Exogenous application of 1.0 μM EBR had significant effect on growth and morpho-physiological traits under both conditions. The maize seedlings treated with EBR were more tolerant to cold stress than the untreated one. Significant increase in plant height, dry matter accumulation, chlorophyll content, total soluble proteins and starch contents was observed under both conditions, however, the results were more pronounced under cold stress. 1.0 μ M concentration being the most effective under both conditions. Maintenance of high tissue water content, reduced membrane injury index, increased total chlorophyll, soluble sugar and protein content were taken as the possible indicators of EBR induced chilling tolerance.
PMCID: PMC3550514  PMID: 23814437
24-epibrassinoslide; Cold tolerance; Maize; Glycine betaine; Total chlorophyll; Membrane injury index
7.  Febrile-Range Hyperthermia Modifies Endothelial and Neutrophilic Functions to Promote Extravasation 
Acute respiratory distress syndrome (ARDS) is a neutrophil (polymorphonuclear leukocyte; PMN)–driven lung injury that is associated with fever and heat-stroke, and involves approximately 40% mortality. In murine models of acute lung injury (ALI), febrile-range hyperthermia (FRH) enhanced PMN accumulation, vascular permeability, and epithelial injury, in part by augmenting pulmonary cysteine-x-cysteine (CXC) chemokine expression. To determine whether FRH increases chemokine responsiveness within the lung, we used in vivo and in vitro models that bypass the endogenous generation of chemokines. We measured PMN transalveolar migration (TAM) in mice after intratracheal instillations of the human CXC chemokine IL-8 in vivo, and of IL-8–directed PMN transendothelial migration (TEM) through human lung microvascular endothelial cell (HMVEC-L) monolayers in vitro. Pre-exposure to FRH increased in vivo IL-8–directed PMN TAM by 23.5-fold and in vitro TEM by 7-fold. Adoptive PMN transfer demonstrated that enhanced PMN TAM required both PMN donors and recipients to be exposed to FRH, suggesting interdependent effects on PMNs and endothelium. FRH exposure caused the activation of extracellular signal–regulated kinase (ERK) and p38 mitogen–activated protein kinase in lung homogenates and circulating PMNs, with an associated increase in HSP27 phosphorylation and stress-fiber formation. The inhibition of these signaling pathways with U0126 and SB203580 blocked the effects of FRH on PMN extravasation in vivo and in vitro. Collectively, these results (1) demonstrate that FRH augments chemokine-directed PMN extravasation through direct effects on endothelium and PMNs, (2) identify ERK and p38 signaling pathways in the effect, and (3) underscore the complex effects of physiologic temperature change on innate immune function and its potential consequences for lung injury.
PMCID: PMC3380289  PMID: 22281986
ARDS; neutrophil; endothelium; febrile-range hyperthermia; p38 MAP kinase
8.  Antioxidant Enzymes and Lipid Peroxidation in Type 2 Diabetes Mellitus Patients with and without Nephropathy 
Oxidative stress has been considered to be a pathogenic factor of diabetic complications including nephropathy. There are many controversies and limited studies regarding the antioxidant enzymes in diabetic nephropathy.
This study was to evaluate the levels of antioxidant enzymes and lipid peroxidation in Type-2 Diabetes Mellitus (DM) patients with and without nephropathy.
Materials and Methods:
The study included 90 age and sex matched subjects. Blood samples of all subjects were analyzed for all biochemical and oxidative stress parameters.
The malondialdehyde (MDA) levels and catalase (CAT) activity were significantly increased and reduced glutathione (GSH) levels and activities of glutathione peroxidase (GPx) and glutathione reductase (GR) were significantly decreased in Type-2 DM with and without nephropathy as compared to controls and also in Type-2 DM with nephropathy as compared to Type-2 DM without nephropathy. There were an excellent positive correlation of glycohemoglobin (HbA1c) with MDA and a good negative correlation of GPx with GSH in controls. There were positive correlations of GR, CAT, and superoxide dismutase (SOD) with MDA in Type-2 diabetes patients with nephropathy.
Intensity of oxidative stress in Type-2 diabetic patients with nephropathy is greater when compared with Type-2 diabetic patients without nephropathy as compared to the controls.
PMCID: PMC3632026  PMID: 23626958
Antioxidant enzymes; Glycosylated hemoglobin and diabetic nephropathy; Lipid profile; Malondialdehyde; Non-insulin dependent diabetes mellitus; Reduced glutathione
9.  Removal of self expandable metallic airway stent: A rare case report 
Covered self expandable metallic airway stents (SEMS) have been used for benign tracheal stenosis, post intubation tracheal stenosis, tracheal burn or trauma, tracheo-broncho-malacia, and extrinsic compression of trachea. Their placement is considered to be permanent, with open surgery the only way to remove the stent, though there are few cases reports of their removal with the bronchoscope, but the complications after their removal are very high. In our patient, one and a half years after placement of SEMS, she developed cough with dyspnoea, video bronchoscopy showed stenosis above the level of stent with granulation tissue inside the stent, stent fracture in lower part and stent migration to right main bronchus, thus she had all conceivable complications of stent placement. The stent was removed with the help of rigid bronchoscope under general anaesthesia. She was discharged the following day. The case is being reported because it was unique in having all the possible complications of stent placement, and rare as we could take out the stent in Toto. Thirdly, the stent could be removed without any complication.
PMCID: PMC3644838  PMID: 23661920
Granulation tissue inside the stent; stenosis above the stent; stent fracture; stent migration
10.  Decrease in Antioxidant Status of Plasma and Erythrocytes from Geriatric Population 
Disease markers  2012;33(6):303-308.
Background: Ageing is associated with an accumulation of free radical damage, which leads to physiological and clinical modifications. The study aims to find out the status of lipid profile, antioxidant enzymes, malondialdehyde in geriatric population.
Patients/methods: The study was conducted on 150 subjects (75 healthy control between the ages of 20–30 years and 75 elderly subjects between ages of 50–70 years as cases). The following parameters were analyzed using the standard reference methods: lipid profile, reduced glutathione, glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase and malondialdehyde.
Results: The present study was conducted to estimate the oxidative stress parameters in geriatric population. Highly significant increase in total cholesterol (TC), triglyceride (TG), LDL-cholesterol (LDL-C), VLDL-cholesterol (VLDL-C), malondialdehyde, catalase and decrease in high density lipoprotein cholesterol (HDL-C), reduced glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase was observed in geriatrics when compared with their younger counterparts.
Conclusion: This study concluded that there is enhanced oxidative stress and decreased antioxidant defence in geriatrics as compared to younger subjects which could play an important role in ageing. Dyslipidemia has become one of the important risk factors for the increasing prevalence of cardiovascular diseases. There is lack of awareness on the relationship between blood lipids and the risk of cardiovascular diseases in geriatric population. The strategy of early prevention should be adopted against dyslipidemia.
PMCID: PMC3810699  PMID: 23089922
Oxidative stress; malondialdehyde; antioxidants; geriatric population
11.  Proboscis Lateralis: Case Report and Overview 
We present a rare case of the congenital malformation of the nose in a 10-month-old baby.
PMCID: PMC3146664  PMID: 22754832
Proboscis lateralis; Malformation of the nose
12.  Role of magnesium sulfate in neuroprotection in acute ischemic stroke 
To study the effect of intravenous magnesium sulfate infusion on clinical outcome of patients of acute stroke.
Materials and Methods:
Sixty consecutive cases of acute ischemic stroke hospitalised within 24 h of an episode of stroke were taken as subjects. All subjects underwent a computed tomography head, and those found to have evidence of bleed/space-occupying lesions were excluded from the study. The subjects taken up for the study were divided into two groups of 30 subjects each. Both the groups received the standard protocol management for acute ischemic stroke. Subjects of Group 1 additionally received intravenous magnesium sulfate as initial 4 g bolus dose over 15 min followed by 16 g as slow infusion over the next 24 h. In all the subjects of the two study groups, serum magnesium levels were estimated at the time of admission (Day 0), Day 1 and Day 2 of hospitalization using an atomic absorption spectrometer.
Statistical Analysis Used:
Scandinavian stroke scores were calculated on Day 3, day of discharge and Day 28. Paired t-test was employed for comparison of stroke scores on Day 3, day of discharge and Day 28 within the same group and the unpaired t-test was used for the intergroup comparison, i.e. comparison of stroke scores of control group with corresponding stroke scores of magnesium group.
Comparison of stroke scores on Day 3 and day of discharge, on the day of discharge and Day 28 and on Day 3 and Day 28 in the magnesium group produced a t-value of 5.000 and P <0.001, which was highly significant. However, the comparison of the mean stroke scores between the magnesium and the control groups on Day 3, day of discharge and Day 28 yielded a P-value of >0.05, which was not significant.
The study failed to document a statistical significant stroke recovery in spite of achieving a significant rise in serum magnesium level, more than that necessary for neuroprotection, with an intravenous magnesium sulfate regime.
PMCID: PMC3424793  PMID: 22919188
Ischemic stroke; magnesium sulfate; neuroprotection
13.  Distinct, Gene Specific Effect of Heat Shock on Heat Shock Factor-1 Recruitment and Gene Expression of CXC Chemokine Genes 
Cytokine  2011;54(1):61-67.
The heat shock (HS) response, a phylogenetically conserved ubiquitous response to stress, is generally characterized by the induced expression of heat shock protein (HSP) genes. Our earlier studies showed that the stress-activated transcription factor, heat shock factor-1 (HSF1), activated at febrile range or HS temperatures also modified expression of non-HSP genes including cytokine and chemokine genes. We also showed by in silico analysis that 28 among 29 human and mouse CXC chemokine genes had multiple putative heat shock response elements (HSEs) present in their gene promoters. To further determine whether these potential HSEs were functional and bound HSF1, we analyzed the recruitment of HSF1 to promoters of 5 human CXC chemokine genes (CXCL-1, 2, 3, 5 and 8) by chromatin immunoprecipitation (ChIP) assay and analyzed the effect of HS exposure on tumor necrosis factor-α (TNFα)-induced expression of these genes in human lung epithelial-like A549 cells. HSF1 ChIP analysis showed that HSF1 was recruited to all but one of these CXC chemokine genes (CXCL-3) and HS caused a significant increase in recruitment of HSF1 to one or multiple HSEs present in the promoters of CXCL-1, 2, 5 and 8 genes. However, the effect of HS exposure on expression of these genes showed a variable gene-specific effect. For example, CXCL8 expression was markedly enhanced (p<0.05) whereas CXCL5 expression was significantly repressed (p<0.05) in cells exposed to HS coincident with TNFα stimulation. In contrast, expression of CXCL1 and CXCL2, despite HSF1 recruitment to their promoters, was not affected by HS exposure. Our results indicate that some, if not all, putative HSEs present in the CXC chemokine gene promoters are functional and recruit HSF1 in vivo but the effects on gene expression are variable and gene specific. We speculate, the physical proximity and interactions of other transcription factors and co-regulators with HSF1 could be critical to determining the effects of HS on the expression of these genes.
PMCID: PMC3048923  PMID: 21266308
Hyperthermia; stress; TNF; A549; IL-8; transcription
14.  Response of mice to continuous 5-day passive hyperthermia resembles human heat acclimation 
Cell Stress & Chaperones  2010;16(3):297-307.
Chronic repeated exposure to hyperthermia in humans results in heat acclimation (HA), an adaptive process that is attained in humans by repeated exposure to hyperthermia and is characterized by improved heat elimination and increased exercise capacity, and acquired thermal tolerance (ATT), a cellular response characterized by increased baseline heat shock protein (HSP) expression and blunting of the acute increase in HSP expression stimulated by re-exposure to thermal stress. Epidemiologic studies in military personnel operating in hot environments and elite athletes suggest that repeated exposure to hyperthermia may also exert long-term health effects. Animal models demonstrate that coincident exposure to mild hyperthermia or prior exposure to severe hyperthermia can profoundly affect the course of experimental infection and injury, but these models do not represent HA. In this study, we demonstrate that CD-1 mice continuously exposed to mild hyperthermia (ambient temperature ~37°C causing ~2°C increase in core temperature) for 5 days and then exposed to a thermal stress (42°C ambient temperature for 40 min) exhibited some of the salient features of human HA, including (1) slower warming during thermal stress and more rapid cooling during recovery and (2) increased activity during thermal stress, as well as some of the features of ATT, including (1) increased baseline expression of HSP72 and HSP90 in lung, heart, spleen, liver, and brain; and (2) blunted incremental increase in HSP72 expression following acute thermal stress. This study suggests that continuous 5-day exposure of CD-1 mice to mild hyperthermia induces a state that resembles the physiologic and cellular responses of human HA. This model may be useful for analyzing the molecular mechanisms of HA and its consequences on host responsiveness to subsequent stresses.
PMCID: PMC3077225  PMID: 21080137
Heat acclimation; Mouse model; Heat shock protein; Acquired thermal tolerance
15.  Prostaglandin E2 Potentiates Heat Shock-Induced Heat Shock Protein 72 Expression in A549 Cells 
The heat shock (HS) response is an important cytoprotective response comprising expression of heat shock proteins (HSP) and orchestrated by the heat/stress-induced transcription factor, heat shock factor-1 (HSF-1). Previous studies suggest that the activation threshold and magnitude of the HS response may be modified by treatment with arachidonic acid (AA). We analyzed the effect of exogenous AA and its metabolites, PGE2, LTD4, and 15-HETE on HSF-1-dependent gene expression in A549 human respiratory epithelial-like cells. When added at 1 μM, PGE2 much more than LTD4, but not 15-HETE increased activity of a synthetic HSF-1-dependent reporter after HS exposure (42°C for 2h), but had no effect in the absence of HS. Exposing A549 cells to HS stimulated release of PGE2 and treatment with the cyclooxygenase inhibitor, ibuprofen, reduced HS-induced HSF-1-dependent transcription. PGE2 increased HS-induced HSP72 mRNA and protein expression but EMSA and Western blot analysis failed to show an effect on HSF-1 DNA binding activity or post-translational modification. In summary, we showed that HS stimulates generation of PGE2, which augments generation of HSPs. The clinical consequences of this pathway have yet to be determined.
PMCID: PMC2919605  PMID: 20382255
Heat shock; HSP-72; Prostaglandin E2; Arachidonic Acid
16.  Hypercapnia resulting from a faulty co-axial (Bain) circuit 
Indian Journal of Anaesthesia  2011;55(4):402-404.
The Bain co-axial circuit is fully established in general anaesthesia practice. A major concern is the potential malfunctioning of the circuit due to avulsion of the inner fresh gas delivery tube at the machine end of the circuit. The following case report presents a case in which a patient connected to the Bain circuit developed severe hypercapnia in the early intraoperative period due to the above mentioned defect.
PMCID: PMC3190520  PMID: 22013262
Bain circuit; complication; general anaesthesia; hypercapnia
17.  Febrile range temperature represses TNF-α gene expression in LPS-stimulated macrophages by selectively blocking recruitment of Sp1 to the TNF-α promoter 
Cell Stress & Chaperones  2010;15(5):665-673.
We have previously shown that exposure to febrile-range temperature (FRT, 39.5°C) reduces LPS-induced TNF-α transcription in mouse macrophages through at least two mechanisms: (1) by directly recruiting heat shock factor-1 (HSF-1) to a heat shock response element present in the TNF-α 5′-UTR and (2) by markedly reducing LPS-induced recruitment of NFκB-p65 to the κB enhancer (at −510) in the TNF-α gene. In the present study, we used EMSA and chromatin immunoprecipitation assays to further analyze the complex effects of FRT on the recruitment of transcription factors and co-activators on the TNF-α gene in LPS-stimulated RAW 264.7 mouse macrophages. Our results showed that in FRT-exposed RAW cells, HSF-1 was recruited only to the 5′-UTR site, and no additional interaction was evident in the TNF-α gene up to 1,300 nt upstream of the transcription start site. Similarly, FRT exposure selectively reduced LPS-induced NFκB-p65 recruitment to the κB enhancer site at −510 without affecting the other three κB enhancer sites present in the TNF-α 5′-flanking sequence. Finally, we found that FRT exposure abrogated LPS-stimulated recruitment of Sp1 to the proximal TNF-α promoter without any change in associated histone H3 acetylation around the TNF-α promoter and despite a marked increase in the total intra-nuclear Sp1 DNA binding activity. In conclusion, our studies further emphasize the complex and redundant control of TNF-α transcription and identify additional potential mechanisms through which FRT exposure may reduce TNF-α expression by selectively modifying gene-specific recruitment of transcription factors to the proximal TNF-α promoter.
PMCID: PMC3006616  PMID: 20221720
Fever; Hyperthermia; TNF-α; Repression; Sp1
18.  A comparative study of efficacy of propofol auto-co-induction versus midazolam propofol co-induction using the priming principle 
Indian Journal of Anaesthesia  2010;54(6):558-561.
Application of priming principle is well documented in relation to the use of muscle relaxants. The aim of the present study was to evaluate the efficacy of priming technique in relation to induction agents. Clinical efficacy in terms of dose reduction and alteration in peri-intubation haemodynamics was compared in propofol auto-co-induction and midazolam propofol co-induction groups along with a control group. The study was carried out in 90 patients scheduled for upper abdominal surgery, who were randomly divided into three equal groups. Group I received 0.5 mg/kg propofol IV (20% of the pre-calculated induction dose), group II received 0.05 mg/kg IV midazolam and group III received 3 ml of normal saline. This was followed by IV induction with propofol 2 minutes later in all the three groups at a predetermined rate till the bispectral index value of 45 was attained. The results showed a significant decrease in induction dose requirement in both the groups but haemodynamic stability during induction and intubation was more in propofol auto-co-induction group.
PMCID: PMC3016578  PMID: 21224975
Priming principle; propofol; auto-co-induction; bispectral index
19.  Core temperature correlates with expression of selected stress and immunomodulatory genes in febrile patients with sepsis and noninfectious SIRS 
Cell Stress & Chaperones  2009;15(1):55-66.
Environmental hyperthermia and exercise produce extensive changes in gene expression in human blood cells, but it is unknown whether this also happens during febrile-range hyperthermia. We tested the hypothesis that heat shock protein (HSP) and immunomodulatory stress gene expression correlate with fever in intensive care unit patients. Whole blood messenger RNA was obtained over consecutive days from 100 hospitalized patients suffering from sepsis or noninfectious systemic inflammatory response syndrome (SIRS) as defined by conventional criteria. The most abnormal body temperature in the preceding 24 h was recorded for each sample. Expression analysis was performed using the Affymetrix U133 chip. ANCOVA followed by correlation analysis was performed on a subset of 278 prospectively identified sequences of interest. Temperature affected expression of 60 sequences, either independently or as a function of clinical diagnosis. Forty-eight of these (representing 38 genes) were affected by temperature only, including several HSPs, transcription factors heat shock factor (HSF)-1 and HSF-4, cellular adhesion molecules such as ICAM1/CD54 and JAM3, toll receptors TLR-6 and TLR-7, ribosomal proteins, and a number of molecules involved in inflammatory pathways. Twelve sequences demonstrated temperature-dependent responses that differed significantly between patients with sepsis and noninfectious SIRS: CXCL-13; heat shock proteins DNAJB12 and DNAJC4; the F11 receptor; folate hydrolase 1; HSF-2; HSP 70 proteins HSPA1A, HSPA1B, and HSPA1L; interleukin 8; lipopolysaccharide binding protein; and prostaglandin E synthase. Febrile-range temperatures achieved during sepsis and noninfectious SIRS correlate with detectable changes in stress gene expression in vivo, suggesting that fever can activate HSP gene expression and modify innate immune responses. For some genes, it appears that clinical condition can alter temperature-sensitive gene expression. Collectively, these data underscore the potential importance of body temperature in shaping the immune response to infection and injury.
PMCID: PMC2866972  PMID: 19496026
Heat shock; Fever; Hyperthermia; Sepsis; SIRS; Microarray; mRNA; Whole blood; Gene expression; Gene chip
20.  Spindle cell carcinoma of head and neck: an immunohistochemical and molecular approach to its pathogenesis 
Journal of Clinical Pathology  2006;60(5):472-475.
Spindle cell carcinoma (SpCC) is a rare microscopic type of cancer of the mouth and oropharynx. Although SpCC is thought to arise from squamous cell carcinoma (SCC), it carries a worse prognosis.
To find out the difference in immunohistochemical expression of cytokeratin, vimentin and smooth‐muscle actin, and mutational alterations in the K‐ras oncogene between the two tumours, in an attempt to characterise SpCC.
Immunohistochemical analysis was performed by standard avidin–biotin complex method in 35 cases each of SpCCs and SCCs. DNA extracted from paraffin wax‐embedded tumours was used for PCR followed by single‐strand conformation polymorphism for mutational analysis of K‐ras exon 1 and exon 2.
In the SpCC group, cytokeratin positivity was significantly higher in epithelial areas (52.2%) than in spindle cell areas (16.1%), whereas vimentin was more positive in spindle cell areas (18.7%) than epithelial areas (2.7%). Cells intermediate between epithelial and spindle cell areas were consistently positive for both cytokeratin and vimentin. Cytokeratin was found to be significantly more positive in SCC (72.6%) than the squamous component and spindle cell component of SpCC. In this study, no mutation was detected in the K‐ras gene of either the SpCC or SCC group.
The spindle cell component of SpCC is intermixed with cells that are morphologically mesenchymal but express dual antigen‐positivity characteristic of epithelial (cytokeratin) and mesenchymal (vimentin) cells. These, possibly, are cells in transition suggesting that SpCC may be a sarcomatous metaplasia of SCC.
PMCID: PMC1994537  PMID: 16731596
21.  Indoxacarb poisoning: A rare presentation as methemoglobinaemia 
Indian Journal of Anaesthesia  2010;54(3):239-241.
Indoxacarb is a broad-spectrum non-organophosphorus oxidiazine insecticide widely used in farming. Once absorbed it acts on sodium channels and blocks the flow of sodium ions. We report a case of indoxacarb poisoning in a farmer following suicidal consumption, manifested as unconsciousness, cyanosis and stationary SpO2 values. Methemoglobinaemia was suspected on clinical presentation which was successfully managed with inj. methylene blue and other symptomatic and supportive treatment.
PMCID: PMC2933484  PMID: 20885872
Indoxacarb; methemoglobinaemia; methylene blue
22.  Hyperthermia in the febrile range induces HSP72 expression proportional to exposure temperature but not to HSF-1 DNA-binding activity in human lung epithelial A549 cells 
Cell Stress & Chaperones  2009;14(5):499-508.
Expression of heat shock proteins (HSPs) is classically activated at temperatures above the physiologic range (≥42°C) via activation of the stress-activated transcription factor, heat shock factor-1 (HSF-1). Several studies suggest that less extreme hyperthermia, especially within the febrile range, as occurs during fever and exertional/environmental hyperthemia, can also activate HSF-1 and enhance HSP expression. We compared HSP72 protein and mRNA expression in human A549 lung epithelial cells continuously exposed to 38.5°C, 39.5°C, or 41°C or exposed to a classic heat shock (42°C for 2 h). We found that expression of HSP72 protein and mRNA increased linearly as incubation temperature was increased from 37°C to 41°C, but increased abruptly when the incubation temperature was raised to 42°C. A similar response in luciferase activity was observed using A549 cells stably transfected with an HSF-1-responsive luciferase reporter plasmid. However, activation of intranuclear HSF-1 DNA-binding activity was comparable at 38.5°C, 39.5°C, and 41°C and only modestly greater at 42°C but the mobility of HSF1 protein on a denaturing gel was altered with increasing exposure temperature and was distinctly different at 42°C. These findings indicate that the proportional changes in HSF-1-dependent HSP72 expression at febrile-range temperatures are dependent upon exposure time and temperature but not on the degree of HSF-1 DNA-binding activity. Instead, HSF-1-mediated HSP expression following hyperthermia and heat shock appears to be mediated, in addition to HSF-1 activation, by posttranslational modifications of HSF-1 protein.
PMCID: PMC2728283  PMID: 19221897
Hyperthermia; Fever; Heat shock; HSF-1; HRE promoter; HSP72
23.  Febrile-range hyperthermia accelerates caspase-dependent apoptosis in human neutrophils1 
Human neutrophilic polymorphonuclear leukocytes (PMNs) are central to innate immunity and are responsible for clearance of pathogens. PMNs undergo a tightly regulated apoptosis program that allows for timely clearance of PMNs without extravasation of toxic intracellular contents. We investigated the rate of spontaneous apoptosis of human peripheral blood PMNs cultured at basal (37°C) and febrile-range (39.5°C) temperatures (FRT). We found that PMN apoptosis is accelerated at FRT, reaching ~90% completion by 8 h at 39.5°C vs. 18 h at 37°C based on morphologic criteria. Caspase-8 activation peaked within 15 minutes of PMN exposure to FRT and subsequent activation of caspase-3 and -9, cleavage of the BH3 only protein Bid, and mitochondrial release of cytochrome c were also greater in FRT-exposed PMNs. Inhibition of caspase-3, -8, and -9 conferred comparable protection from apoptosis in FRT-exposed PMNs. These results demonstrate that exposure to FRT enhances caspase-8 activation and subsequent mitochondrial-dependent and mitochondrial–independent apoptosis pathways. The PMN survival factors G-CSF, GM-CSF, and IL-8, each prolonged PMN survival at 37°C and 39.5°C, but did reduce the difference in survival at the two temperatures. In a mouse model of intratracheal endotoxin-induced alveolitis, co-exposure to FRT (core temperature ~39.5°C) doubled the proportion of bronchoalveolar PMNs undergoing apoptosis compared with euthermic mice. This process may play an important role in limiting inflammation and tissue injury during febrile illnesses.
PMCID: PMC2829976  PMID: 18684954
Neutrophils; fever; apoptosis; caspase; hyperthermia
24.  Heat Shock Co-Activates Interleukin-8 Transcription 
The heat shock (HS) response is a phylogenetically ancient cellular response to stress, including heat, that shifts gene expression to a set of conserved HS protein (HSP) genes. In our earlier studies, febrile-range hyperthermia (FRH) not only activated HSP gene expression, but also increased expression of CXC chemokines in mice, leading us to hypothesize that the CXC chemokine family of genes might be HS-responsive. To address this hypothesis we analyzed the effect of HS on the expression of IL-8/CXCL-8, a member of the human CXC family of ELR+ chemokines. HS markedly enhanced TNF-α–induced IL-8 secretion in human A549 respiratory epithelial-like cells and in primary human small airway epithelial cells. IL-8 mRNA was also up-regulated by HS, but the stability of IL-8 mRNA was not affected. TNF-α–induced reporter activity of an IL-8 promoter construct IL8−1471/+44-luc stably transfected in A549 cells was also enhanced by HS. Electrophoretic mobility and chromatin immunoprecipitation assays showed that the stress-activated transcription factor heat shock factor-1 (HSF-1) binds to at least two putative heat shock response elements (HSE) present in the IL-8 promoter. Deletional reporter constructs lacking either one or both of these sites showed reduced HS responsiveness. Furthermore, depletion of HSF-1 using siRNA also reduced the effects HS on TNF-α–induced IL-8 expression, demonstrating that HSF-1 could also act to regulate IL-8 gene transcription. We speculate that during evolution the CXC chemokine genes may have co-opted elements of the HS response to amplify their expression and enhance neutrophil delivery during febrile illnesses.
PMCID: PMC2542457  PMID: 18367728
neutrophil; hyperthermia; IL-8; chemokine; heat shock factor-1
25.  Comparison of Clinical Performance of I-Gel™ with LMA—Proseal™ in Elective Surgeries 
Indian Journal of Anaesthesia  2009;53(3):302-305.
Sixty ASA grade I & II adult patients of either sex were randomly assigned into two groups. Group I (n=30) for I-gel and Group P (n=30) for LMA – ProSeal. We assessed the airway sealing pressure, ease of insertion, success rate of insertion, ease of gastric tube placement, airway trauma by post operative blood staining of the device, tongue, lip and dental trauma, hoarseness, regurgitation / aspiration and cost effectiveness. Although the airway sealing pressure was higher with Group P (29.6 cm H2O) than with Group I (25.27 cm H20) (p < 0.05), but the airway sealing pressure of Group I was very well within the normal limit to prevent aspiration. The ease of insertion was more with Group I (29/30) than with Group P (25/30) (p < 0.05). The success rate of first attempt of insertion and ease of gastric tube placement was more with Group I (p > 0.05). Blood staining of the device & tongue, lip and dental trauma was more with Group P (p >0.05). There was no evidence of bronchospasm, laryngospasm, regurgitation, aspiration or hoarseness in either group.
To conclude I-gel is a novel supraglottic device with an acceptable airway sealing pressure (25.27 cm H2O). It is easier to insert, requires less attempts of insertion, has easier gastric tube placement and is less traumatic as compared to LMA-ProSeal.
PMCID: PMC2900120  PMID: 20640137
I-gel; LMA – ProSeal; Airway sealing pressure

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