Role of active nitrogen molecules in progression of septic shock.

INTRODUCTION: Active nitrogen molecules are formed as a result of cell metabolism. They are essential for cell metabolism, but when produced in excess, they contribute to the pathogenesis of several disease processes. These nitrogen molecules play an important role in vascular instability of septic shock. This study was planned to detect the role of active nitrogen molecules in the progression of septic shock. MATERIALS AND METHODS: Blood samples were collected from 118 critically ill patients admitted in ICU and from 95 healthy relatives accompanying the patients. Patients were categorized into three groups: systemic inflammatory response syndrome (n = 54), sepsis (n = 35) and septic shock (n = 29). Plasma total nitrite (nitrites and nitrates), cytokines like tumour necrosis factor-α (TNF-α) and plasma lactate were measured to assess inflammatory activity and severity of septic shock. RESULTS: High plasma levels of nitrite and nitrate (No2-/No3-) were observed in critically ill patients (mean level 78.92 µmol/l in sepsis and 97.20 µmol/l in septic shock). Mean plasma TNF-α level in sepsis was 213.50 pg/ml and septic shock was 227.38 pg/ml. CONCLUSION: Plasma No2-/No3- and TNF-α levels were high in patients with sepsis and septic shock, which increased with severity of sepsis.

Optimization of ferric chloride induced thrombosis model in rats: effect of anti-platelet and anti-coagulant drugs.

Numerous studies on various animal species, with variability in the site of application and the concentration of ferric chloride (FeCl3) to induce intravascular thrombosis has prompted us to undertake the present study to obtain a threshold concentration of FeCl3 and validate this model by clinically used anti-thrombotic drugs. A small piece of filter paper, soaked in FeCl3 solution (10, 20, 40, 60 or 80%, w/v), was topically applied on the carotid artery of SD rats to measure the time till occlusion (TTO), to ascertain 20% as an optimal FeCl3 concentration. Anti-platelet drugs, aspirin (30 mg/kg), ticlopidine (200 mg/kg) or clopidogrel (30 mg/kg), administered by oral route for 3 days (once daily) or 4h (single dose) prior to the induction of thrombosis, showed the rank-order: clopidogrel>ticlopidine>aspirin based on TTO by 20% FeCl3. Anti-coagulant drug, heparin (10 U/kg, 30 U/kg and 100 U/kg, i.v) increased TTO in a dose dependent manner (18+/-1.7, 22+/-0.9 and 27+/-3.9 min respectively), while warfarin treated rats at 0.1 mg/kg or 0.3 mg/kg, (po for 5 days), exhibited TTO augmentation to 85+/-11.8 and 120+/-0 min respectively. The results obtained indicate that among the drugs tested warfarin and clopidogrel were most efficacious in this model, while rank order of the other anti-thrombotic drugs were heparin>ticlopidine>aspirin. The present study thus provides a simple, reproducible and well controlled methodology to induce thrombosis in rats by the topical application of 20% FeCl3 to assess the efficacy of new anti-thrombotic agents.

Antioxidant Status before and after Dietary Intervention in Cardiovascular Disease (CVD) Patients.

There is some evidence showing an inverse correlation between dietary sources including natural antioxidant vitamins and the risk of cardiovascular disease (CVD). The aim of this study was to evaluate the effect of dietary antioxidants on oxidative stress in CVD patients. This study was carried out on 31 CVD patients and 63 healthy individuals. Nutritional status and dietary antioxidant vitamins were assessed by 48-hour recall. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities as well as the levels of vitamins A, E, C, total antioxidant capacity (TAC) and malondialdehyde (MDA) were determined before and after serving fresh fruits and vegetables for 3 months. Before intervention intake, levels of vitamins A, E and C were significantly lower in patients than in normal individuals (P<0.001). The serum levels of vitamins A, E and C were significantly lower in the cases than in the control subjects. After intervention, the serum levels of vitamins A, E and C were increased significantly (P<0.0001). Similarly, the levels of TAC as well as the activities of SOD and GPx were found to increase by end of 3 months. In addition, a significant increase of TAC and a decrease in MDA levels were observed. In conclusion, the findings show that dietary supplementation improves the antioxidant defense system in CVD patients.

Establishment of a rat model of myocardial infarction with a high survival rate: A suitable model for evaluation of efficacy of stem cell therapy.

The most common rat model of myocardial infarction (MI) is by ligation of left anterior descending (LAD) coronary artery but it is associated with high mortality and large variations in the infarct size. We evolved certain innovations/modifications in the existing technique including immobilization of the heart without exteriorization, identification of the LAD by pressing it proximal to the site of ligation by an ear-bud, and subsequently its ligation 8 mm from its origin, no touch technique of the lungs during surgery, removal of air from the chest cavity prior to its closure using an in-house tubing, and deflation of the lungs before extubation. We induced MI in 24 Sprague- Dawley (SD) rats using these modifications and carried out post-MI evaluation of hemodynamic parameters, serum cardiac enzymes and histological studies upto 90 days using 13 sham operated and 3 healthy SD rats as controls. Three of the 24 rats (13%) died <24 hours of MI, but thereafter no mortality was observed till the follow-up period of 90 days. The infarct size was consistent in all the rats (21±4% of left ventricular area). This model with low early and no long-term mortality may be suitable for studying efficacy of stem cell therapy in MI, where a follow-up of at least 13 weeks is required to assess myocardial regeneration.

Allium sativum constituents: effect on free radical generation from rat neutrophils.

Reactive oxygen species formation or respiratory burst by the neutrophils helps to remove the invaded pathogens and thus constitute a major defense against pathogenic microorganisms. Production of these radicals by activated neutrophils at the site of inflammation however inflicts damage to the host tissue. Modulation of the neutrophil respiratory burst is therefore important in determining the balance between immune defense and host tissue injury during inflammatory conditions. Garlic extracts and various compounds isolated from garlic have been found to possess various activities, however no report is available on their effect on neutrophil free radical generation. The present study was therefore undertaken to evaluate the effect of garlic aqueous extract, alcoholic extract and various fractions on the free radical generation from neutrophils. Among the tested fractions, chloroform fraction of garlic seems to be very potent in attenuating the free radical generation from rat neutrophils, which could be beneficial in the inflammation associated pathological conditions.

Biochemical and molecular evaluation of neutrophil NOS in spontaneously hypertensive rats.

Resting neutrophils generate NO, while activation leads to the production of reactive oxygen and nitrogen species. Nowadays cardiovascular pathological conditions such as hypertension, cardiac ischemia, reperfusion and heart failure are associated with inflammation. This project explores the respiratory burst potential and NO generation status in the neutrophils, plasma, aorta, and kidneys from normotensive Wistar and spontaneously hypertensive rats (SHR). Total and protein associated nitrite content was quantitated using Griess reagent following cadmium reduction and mercuric chloride treatment respectively. NO and superoxide generation evaluated by Flowcytometry and peroxynitrite by spectrofluorimetric method. Expression of NOS isoforms was analyzed by RT-PCR. NO generation from SHR neutrophils was significantly augmented in comparison to normotensive counterparts. Neutrophils activated in response to arachidonic acid, PMA, fMLP or E. coli generated more superoxide radicals among SHR, and consequentially peroxynitrite. Expression of iNOS was significantly more in the SHR neutrophils, while that of nNOS remained unaffected. Results suggest that NO generated in SHR is utilized in scavenging superoxide radicals thereby limiting its bioavailability. Thus induction of NOS in neutrophils combined with augmented oxidative stress might influence its association with endothelium and contribute to inflammatory responses under hypertensive condition.

Nitrite and malondialdehyde content in cerebrospinal fluid of patients with Parkinson's disease.

Evidence from clinical and experimental studies supports the hypothesis of free radical-mediated damage of dopaminergic neurons in the pathology of Parkin's disease (PD). The present study was undertaken to evaluate the role of nitric oxide and oxidative stress in PD. Estimation of the stable metabolites of nitric oxide (NO, nitrite, nitrate) and malondialdehyde (MDA), an acceptable marker of lipid peroxidation, can provide indirect evidence of involvement of free radicals. Nitrite and malondialdehyde (MDA) levels were estimated in the lumbar cerebrospinal fluid (CSF) of 20 controls and 21 patients with PD. Nitrite and MDA content was not significantly altered in the CSF of PD patients as compared to the controls. Nitrite and MDA levels in CSF of PD patients exhibited no correlation with age, duration of disease, and severity of illness (measured by the Unified Parkinson's Disease Rating Score). There was no correlation between the CSF nitrite and MDA level. Findings of the present study do not provide evidence for the involvement of nitric oxide and oxidative stress in PD.

Contribution of genomics and proteomics in understanding the role of modifying factors in Parkinson's disease.

Parkinson's disease (PD) is a complex neurological disorder, characterized by selective degeneration of nigrostriatal dopaminergic neurons. It is a multi-factorial disease, contributed by a combination of age, genetic and environmental factors. Etiology of sporadic PD and mechanism underlying selective loss of dopaminergic neurons has not yet been clearly understood. Recent developments in genomics and proteomics have revolutionized the research on PD at genetic level. Differential gene expression patterns (DNA biochip technology), age-dependent complex genetic patterns (SNP genotyping), and protein expression profiles (proteomics) of PD patients have started providing the specific and rigorous molecular explanation and role of modifying factors in PD. Genomics and proteomics are further expected to help in developing biomarkers for diagnosis of early onset PD and also to develop valuable and potential therapeutic strategies for its treatment. In this review, we have discussed the progress made by genomics and proteomics, in understanding the role of modifying factors in PD.

A preliminary investigation into maximal expiratory pressures in some village children.

A clinical mercury sphygmomanometer was used to measure Maximal Expiratory Pressure (MEP) in 29 boys (mean age 8 +/- 1.4 yr) and 21 girls (mean age 7.6 +/- 1.5 yr) of a village in interior Maharashtra. The values of 70.6 +/- 13.4 mmHg SD for the boys and 61.9 +/- 18.9 mmHg for the girls were quite comparable to the respiratory pressures reported elsewhere in literature, even though the subjects were apparently poorly nourished. There was no statistical difference between the MEPs of boys and girls. The MEP was positively and significantly (P<0.01) correlated to height (r=0.51) and weight (r=0.05) in the boys. The MEP denoting respiratory muscle strength also correlated positively with handgrip power used to represent non-respiratory muscle strength (r=0.34) (P>0.05). The simple, reproducible method of measuring MEP as described may be useful for measuring this important physiological parameter at the bedside in children whose respiratory muscle function needs to be evaluated.

Nitric oxide synthase localization in the rat neutrophils: immunocytochemical, molecular, and biochemical studies.

Nitric oxide (NO) modulates diverse functions of polymorphonuclear neutrophils (PMNs), but localization of NO synthase (NOS) and identification of its interacting proteins remain the least defined. The present study discerns subcellular distribution of NOS and caveolin-1, a prominent NOS-interacting protein in rat PMNs. Localization of NOS was explored by confocal and immunogold electron microscopy, and its activity was assessed by L-[3H] arginine and 4,5-diaminofluorescein diacetate (DAF-2DA). Reverse transcriptase-polymerase chain reaction using NOS primers and Western blotting demonstrated the presence of neuronal NOS (nNOS) and inducible NOS (iNOS) in PMNs. Immunocytochemical studies exhibited distribution of nNOS and iNOS in cytoplasm and nucleus, and L-[3H] citrulline formation and DAF fluorescence confirmed NOS activity in both fractions. NOS activity correlated positively with calmodulin concentration in both of the fractions. nNOS and iNOS colocalized with caveolin-1, as evidenced by immunocytochemical and immunoprecipitation studies. The results thus provide first evidence of nNOS and iNOS in the nuclear compartment and suggest NOS interaction with caveolin-1 in rat PMNs.

Involvement of nitric oxide in neurodegeneration: a study on the experimental models of Parkinson's disease.

The present study was undertaken to explore involvement of nitric oxide (NO) in the experimental models of Parkinson's disease. Neurodegeneration was induced by unilateral injections of 6-hydroxydopamine (6-OHDA) or lipopolysaccharide (LPS) in the right striatum. Lesions were functionally evaluated by amphetamine-induced asymmetrical behaviour and by decrease in the tyrosine hydroxylase (TH) immunostaining. An induction in the expression of iNOS and augmentation in nitrite content was observed in both the models. The extent of increase in iNOS expression was, however, different but the elevation in the nitrite content was comparable in both the models. The increase in iNOS expression inversely correlated with the tyrosine hydroxylase (TH) immunolabeling. Animals pretreated with a NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), exhibited complete protection against amphetamine induced rotations in both the models. Thus, augmented NO availability subsequent to iNOS induction seems to play an important role in the initial phase of neurodegeneration.

Lung functions with spirometry: an Indian perspective--I. Peak expiratory flow rates.

Peak expiratory flow rate is an effective measure of effort dependent airflow. It is relatively a simple procedure, and may be carried out in the field using portable instruments. The average PEFR of healthy young Indian males and females is around 500 and 350 lpm respectively. The PEFR reaches a peak at about 18-20 years, maintains this level up to about 30 years in males, and about 40 years in females, and then declines with age. Common regression equations for Indians enveloping major studies from various parts of the country have been formulated. Indian PEFR values compare favourably with other ethnic groups such as Americans and Europeans.

Lung functions with spirometry: an Indian perspective--II: on the vital capacity of Indians.

Spirometry has been used in India since 1929 to evaluate vital capacity. The mean value for this parameter has changed slightly for the better over about eight decades. It is currently recorded at about 21.8 ml/cm height for males and about 18 ml/cm height for females, the difference between the two sexes being statistically significant throughout the period studied. The vital capacity reaches its peak at about 30 years of age in both Indian men and women and declines there after. There is no significant statistical difference in the vital capacities of subjects from different regions of India. Composite regressions have been generated for use as reference equations for estimating. Vital capacity of Indians is lower than that of Caucasians, but the age related decline is much greater for Caucasians.

Heart rate and blood pressure responses of left-handers and right-handers to autonomic stressors.

We hypothesized that cerebral dominance may contribute to differences in cardio-vascular responses of right-handers (RH) and left-handers (LH) to autonomic stressors. We tested this hypothesis by exposing 14 RH, and 14 LH males to category I tests in which the hand and cerebral cortex were involved in performing the test viz.--i) Cold pressor test (CPT), ii) Handgrip dynamometry (HGD) and; category II (no use of hand)--i) Orthostatic Tolerance Test (OTT), ii) Valsalva Manuever (VM), iii) Controlled Breathing Test for sinus arrhythmia (SA) in a random sequence, and measured their heart rate (HR/min) and blood pressure (MAP mmHg). All subjects had similar resting HR and MAP values, and responded to the category I interventions with increased HR and BP. The absolute HR values of LH and RH did not differ significantly during the interventions. However, the increase in HR from control induced by the CPT, and the HGD was greater for LH (P<0.05). Also, LH showed a greater decrease in HR and MAP in the recovery phase (P<0.05). The VAS scores for degree of discomfort during the CPT were similar for both the groups. During the OTT, the increase in HR was more in RH (P<0.05). The Valsalva ratios for LH and RH were similar. Our findings suggest that the autonomic control over the cardio-vascular system may be different in LH and RH, and that this imbalance could be attributable to a variation in cerebral dominance.

The antifilarial activity of a marine red alga, Botryocladia leptopoda, against experimental infections with animal and human filariae.

The antifilarial activity of the marine red alga Botryocladia leptopoda against rodent and human lymphatic filarial parasites is described. The animal filarial species included Litomosoides sigmodontis and Acanthocheilonema viteae maintained in cotton rats and Mastomys coucha, respectively, while a subperiodic strain of the human lymphatic filarial parasite Brugia malayi was maintained in M. coucha. The crude extract and its hexane fraction brought about a marked reduction in the peripheral microfilarial level in both of the rodent filarial parasites L. sigmodontis and A. viteae. The microfilaricidal effect began slowly from day 8 or 15 after initiation of treatment and increased with time with a very high efficacy at the end of the observation period against both rodent filariids. The microfilaricidal efficacy was, however, not as prominent in the case of B. malayi. The antifilarial activity, which occurred in the hexane fraction, exerted action at a much lower dose. The product killed a significant proportion of A. viteae and L. sigmodontis adult parasites. In the case of B. malayi, although the macrofilaricidal efficacy was much less than that of the rodent parasites, it (hexane fraction) caused sterilization of a significant proportion of the surviving female parasites. The present findings indicate the possibility of developing an adulticidal and female sterilizing agent against filarial parasites from a marine red alga.

Baylis-Hillman reaction assisted parallel synthesis of 3,5-disubstituted isoxazoles and their in vivo bioevaluation as antithrombotic agents.

The solution-phase parallel synthesis involving reactions of Baylis-Hillman products of 3-substituted-5-isoxazolecarbaldehydes with nucleophiles and their in vivo antithrombotic evaluations are described along with the results of in vitro platelet aggregation inhibition assay of a few compounds. Results of the detailed evaluation of one of the compounds as an inhibitor of platelet aggregation are also presented.

Neutrophil-free radical generation and enzymatic antioxidants in migraine patients.

The present study was undertaken to elucidate the role of circulating neutrophils if any in oxidative stress in migraine by evaluating free radical generation and activities of enzymatic antioxidants in the blood in 55 patients with migraine and 60 healthy controls. Free radical generation was assessed by flow cytometry, while activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) was estimated in blood polymorphonuclear neutrophils (PMNs) by standard procedures. Platelet SOD was also measured. No significant change was found in free radical generation and in the activity of catalase, SOD and GPx in migraine patients. Univariate analysis of PMN catalase level revealed that migraineurs with a positive family history had significantly lower catalase activity compared with those with a negative family history. No correlation was found in the activity of antioxidant enzymes with age, duration of disease, time since last attack and headache index. The platelet SOD also did not show any significant change in patients of migraine without aura. Platelet aggregation in the presence or absence of PMNs was also not altered significantly. Thus the findings of the present study suggest that neutrophils are not the cause of oxidative stress observed in migraine patients.

Role of ascorbic acid in the modulation of inhibition of platelet aggregation by polymorphonuclear leukocytes.

OBJECTIVES: We investigated the modulatory effect of ascorbate on the inhibition of platelet aggregation response by polymorphonuclear leukocytes (PMNs) and characterized the mechanism of the inhibitory response. BACKGROUND: PMNs have been reported to play a significant role in vascular homeostasis by releasing various factors including short-lived reactive oxygen species (ROS) and nitric oxide (NO). NO prevents the activation of circulating platelets and plays a significant role in hemostasis. In addition, PMNs also have the capacity to store very high concentrations of ascorbate. The physiological implications of storing such high concentrations of an antioxidant by a cell-releasing free radicals is unknown, viz. a viz. hemostatic regulation. METHODS: ADP-induced aggregation in human, monkey and rat platelet-rich plasma (PRP) was monitored in the presence of PMNs treated with varying concentrations of ascorbate/dehydroascorbate. NO generation from rat and human PMNs treated with ascorbate was monitored on a FACS Calibur flow cytometer and intraplatelet cyclic guanosine 3',5'-monophosphate (cGMP) levels was also measured. RESULTS: PMNs induced a cell number and time-dependent inhibition of ADP-induced aggregation. The PMNs dependent inhibition was enhanced significantly at 30 min by ascorbate (300 microM). Ascorbate seemed to exert its effects through its oxidized product, dehydroascorbate, as the effects was prevented in the presence of D-glucose (10 mM). Dehydroascorbate elicited significant potentiation of the PMNs induced inhibitory responses and these effects were mediated by the release of NO and subsequent activation of platelet guanylyl cyclase. Flow cytometry experiments with human and rat PMNs confirmed the release of NO and the elevated platelet cGMP levels confirmed NO-mediated activation of guanylyl cyclase. CONCLUSIONS: Ascorbate in circulation seems to prevent the activation of platelets by enhancing the release of antiaggregatory NO, from neighbouring or cohabitant PMNs. The ascorbate effect is mediated through its conversion to dehydroascorbate, subsequently, gets taken up by the cell and converted back to ascorbate. Intracellular ascorbate potentiates the release of NO from the PMNs and subsequently activates guanylyl cyclase in the platelets.

Cerebrospinal fluid nitrite and malondialdehyde levels in patients with motor neuron disease.

Nitric oxide (NO) mediated oxidative damage may be involved in the pathogenesis of neuronal degeneration in motor neuron disease (MND). The present study was undertaken to evaluate the role of NO and oxidative stress in MND by estimating nitrite and malondialdehyde (MDA) levels in the cerebrospinal fluid (CSF) in 22 patients of MND and 20 control subjects suffering from neurological disorders not known to affect NO metabolism. There was no significant change in the CSF nitrite and MDA levels in MND. The nitrite and MDA levels did not have any significant correlation with age, duration of illness, or severity of disease. Univariate analysis of the clinical features in patients with MND and the nitrite levels revealed that two patients with a positive family history had significantly higher CSF nitrite levels as compared to those with a negative family history. There was no correlation between the CSF nitrite and MDA levels. Results of the present study did not indicate significant alterations in the MDA and NO levels in the CSF of MND patients. However, involvement of NO in MND with positive family history is suggested by the results obtained.

Isoxazole-based derivatives from Baylis-Hillman chemistry: assessment of preliminary hypolipidemic activity.

The synthesis of isoxazole-based derivatives utilizing Baylis-Hillman chemistry and results of their preliminary bioevaluation as hypolipidemic agents in triton model are described.