Molecular Biology provides a new interface between Ayurved & Modern Medicine.

It is a great honour to be asked to deliver NAMS Golden Jubilee Lecture. I will introduce the subject with a quote from Pandit Jawaharlal Nehru, India's first Prime Minister (who was also the first Honorary Fellow of NAMS India). After visiting the Central Institute of Research in Indigenous System of Medicine at Jamnagar on 2nd November 1955, he observed: “A fascinating inquiry is going on in this research institute and it may well lead to very fruitful results. The only right approach has to be one of Science, that is of experiment, trial and error. In whatever type of medicine we may deal with, we cannot profit by its study unless we apply the method of Science. Nothing should be taken for granted. Every thing should be tested and proved and then it becomes a part of scientific medicine - old and new.” Charak states “The Science of life shall never attain finality. Therefore, humility and relentless industry should characterize your every endeavour and approach to knowledge. The entire world consists of teachers for the wise and enemies for the fools. Therefore, knowledge, conducive to health, longevity, fame and excellence coming even from an unfamiliar source should be received, assimilated and utilized with earnestness”. Charak Samhita Viman Sthan 8, 14. These words of wisdom should continue to guide the 600,000 practitioners of Ayurveda in India today, as well as 400,000 practitioners of modern medicine who may not have familiarity with our glorious ancient scientific heritage (1).

Beyond reverse pharmacology: Mechanism-based screening of Ayurvedic drugs.

This paper reviews the pharmacology of Indian medicinal plants, starting with the historical background of European work on the subject beginning as early as the 17th century, and tracing its history through the work of Sen and Bose in the 1930’s, and Vakhil’s historic 1949 paper on Sarpaghanda. The often crucial role of patient feedback in early discoveries is highlighted, as is the time lag between proof of pharmacological action and identification of the active principle, and subsequent elucidation of mechanism of action. In the case of Indian plants in the 20th century this process sometimes took almost 50 years. Reserpine and its mechanisms are given in detail, and its current relevance to public health discussed. The foundation of present day methods of pharmacology is briefly presented so the complexity of methods used to identify properties of Ayurveda derived drugs like forskolin and baicalein, and their bioavailability, may be better appreciated. Ayurveda derived anti-oxidants and their levels of action, immuno-modulators, particularly with respect to the NF-kB pathway and its implications for cancer control, are all considered. The example of curcumin derived from turmeric is explained in more detail, because of its role in cancer prevention. Finally, the paper emphasizes the importance of Ayurveda’s concepts of rasayana as a form of dietary chemo-prevention; the significance of ahar, diet, in Ayurveda’s aspiration to prevent disease and restore health thus becomes clear. Understood in this light, Ayurveda may transcend pharmacology as a treatment paradigm.

Epigenetics - Gene silencing.

The discovery of the mechanism of RNA interference by ds RNA by Prof. Andrew Fire and Prof. Craig Mello in 1998, gave them the Nobel Prize in 2006. This discovery revealed a new mechanism for gene regulation through “gene silencing” at the transcriptional level (TGS) or at the post-transcriptional level (PTGS), which play a key role in many essential cellular processes. Today dsRNA is used as a powerful tool to experimentally elucidate the function of essentially any gene in a cell. The immense impact of the discovery of RNA interference (RNAi) on biomedical research and its novel medical applications in the future are reviewed in this article, with particular stress on therapeutic applications of radio-labeled antisense oligonucleotides (RASONs) for diagnosis and treatment of various cancers and neurodegenerative diseases by “gene silencing”. Antisense oligonucleotides (ASONs) can also modulate alternative splicing which 74% of all human genes undergo. The most effective targeting strategy employs simultaneous blocking SnRNP binding sites and splice junctions. Correction of splicing by ASONs can be used to silence mutations causing aberrant splicing as in thalassemia, Duchenne muscular dystrophy and cystic fibrosis. ©

Assessment of diastolic heart function--experience with 16-gated myocardial perfusion SPECT.

BACKGROUND: Ischaemic heart disease (IHD) is a major health problem today. However the focus has shifted primarily to angiographically detecting epicardial vessel stenoses, and ways and means of surgically correcting the blocks. Patients are often not fully evaluated for cardiac function, and diastolic dysfunction of heart, which is often an earlier manifestation than systolic dysfunction, goes undetected. METHODS: Recent gamma cameras have better imaging quality due to attenuation correction with SPECT-CT. 121 patients underwent gated myocardial perfusion imaging (MPI) for suspected coronary artery disease (CAD). We studied the diastolic function of these patients by 16-gated SPECT MPI. RESULTS: 60% patients showed absence of inducible ischaemia on MPI, and hence further invasive procedures like angiography were prevented, 40% showed inducible ischaemia and had to be further evaluated and required intervention. Of all 121 patients, 10% had LV systolic and diastolic dysfunction whereas 66% had isolated diastolic dysfunction. 40% patients had no ischaemia, normal systolic function and only diastolic dysfunction. 40% of these cases had symptoms of chest heaviness/angina equivalent. CONCLUSION: Myocardial perfusion imaging is a useful modality for evaluating patients of suspected CAD and in addition to perfusion data, also provides functional assessment of systolic and diastolic function, which provides comprehensive information regarding patients' symptomatology and can guide further management.

Causation, prevention and reversal of vascular endothelial dysfunction.

Insulin Resistance along with endothelial dysfunction give rise to a constellation of syndromes designated as IRS/MBS metabolic syndrome. Endothelial dysfunction starts early in life much before the development of structural atherosclerosis. Recent insights into vascular biology enable us to understand the molecular mechanisms underlying endothelial dysfunction, and the scope and need for prevention of "pre-clinical" coronary atherosclerosis through lifestyle modification; diet, exercise and stress management. Diminished production of nitric oxide (NO) and/or increased inactivation of NO through oxidative stress (reactive oxygen species ROS and reactive nitrogen species (RNS) are the basis of endothelial dysfunction hence increasing the bioavailability of NO and decreasing its inactivation is the aim of prevention and reversal of endothelial dysfunction. Insulin regulates constitutive NOS gene expression in endothelial cells in vivo; vasodilation is an important component of Insulin-stimulated whole body glucose uptake. Successful strategies are: PPAR alpha and gamma agonists which increase NO production in endothelium; anti-oxidants such as vit. E and C; supplementation with L-arginine, tetrahydrobiopterin-BH4 or sepiapterin (precursor of BH4), SOD mimetic tempol, statins which apart from lowering cholesterol improve NO production, selective beta1 adrenoreceptor antagonists such as nebivolol; suppression of angiotensin-mediated endothelin production by ACE inhibitors and ATR blockers; CB1 receptor blockers, PKCb inhibitors, nitric oxide donors (glyceryl trinitrate and isosorbide dinitrate), dietary supplements of EPA/DHA and regular physical exercise and control of mental stress.

Fat and muscle component of body mass index (BMI): relation with hyperinsulinemia.

Currently there is tremendous interest in obesity and its harmful donsequences. Height, weight and body mass index (BMI) along with waist girth are routinely used parameters. One snag in the interpretation of BMI >25 as a measure of obesity is the assumption that the increase is mainly due to fat. This review emphasizes the importance of assessing the muscle component of BMI (by simple somatoscopy or somatotyping). 75 percent of Indian T2DM patients have a normal or low BMI, only 25 percent have BMI >25, wherein muscle mass also contributes as well as fat. Hyperinsulinemia is anabolic to both fat and muscle. Since skeletal muscle is a primary site of insulin resistance, greater the muscle mass, greater the importance of physical exercise to overcome the insulin resistance and greater the importance of dietary supplement of n3-PUFA to optimize the phospholipid composition of the muscle membrane (increasing membrane fluidity and thereby permitting longer residence of GLUT-4 in the plasma membrane). I propose three testable hypotheses: (1) Brown fat (FDG-PET imaging) and UCP2 and UCP3 expression in muscle are positively correlated with ectomorphy and mesomorphy, and negatively correlated with endomorphy and obesity. BAT is absent in obese people. (2) Indian T2DM patients with normal or low BMI have increased UCP2 and UCP3 expession in their muscle, as well as increased high molecular weight adiponectin which promote fatty acid oxidation and prevent obesity. (3) Indian T2DM with BMI >25 and obesity have dysfunction of UCP2 and UCP3. They have high leptin with leptin resistance (induced by hyperinsulinemia) and low adiponectin. There is inverse relationship between adipose mass and adiponectin production.

Association of adipocytokines (leptin, adiponectin TNF-alpha), insulin and proinsulin with diabetes--the Mumbai Obesity Project [MOP].

Asian Indians have a unique phenotype characterized by increased abdominal obesity and visceral fat despite low body mass index [BMI]. Though studies have indicated some adipocytokines to be associated with diabetes and obesity in Indians, there are virtually no studies relating adipocytokines and proinsulin with diabetes and obesity in Asian Indians. In this study we looked at adipocytokines--leptin, adiponectin and tumour necrosis factor-a [TNF-alpha] and insulin and proinsulin in subjects with diabetes and obesity. Thirty five diabetic subjects and 50 healthy controls were recruited for the study. Leptin [p=0.002J and adiponectin levels [p=0.011] were lower and proinsulin values higher [p<0.001] in diabetic subjects compared to non-diabetic subjects. In addition, leptin [p<0.001] and proinsulin [p<0.001] were higher and adiponectin [p<0.001] lower, in obese subjects compared to non-obese subjects. TNF-alpha failed to show any significant difference between the study groups. Leptin and proinsulin showed a significant and positive correlation with BMI [p<0.001] and waist circumference [p<0.001]. Adiponectin showed an inverse correlation with BMI [p=0.050] and waist circumference [p=0.002]. Proinsulin showed a significant negative association with adiponectin [p=0.002]. Logistic regression analysis revealed leptin to be negatively associated [Odds ratio [OR]: 0.864, 95% confidence interval [95% CI]: 0.775 -0.963, p=0.008] and proinsulin [OR: 1.567, 95% CI: 1.246-1.971, p<0.001] to be positively associated with diabetes even after adjusting for age, gender and BMI. Leptin [OR: 1.365, 95% CI: 1.170-1.592, p<0.001] and proinsulin [OR: 1.617, 95% CI: 1.218 -2.147, p=0.001] showed a significant positive association with obesity, while adiponectin [OR: 0.927, 95% CI: 0.865 - 0.995, p=0.035] had a significant inverse association. Linear regression analysis revealed that adiponectin is inversely associated with proinsulin even after the addition of age, gender and diabetes status [beta= -0.61, p=0.033] into the model. In conclusion, in urban Asian Indians in western India, proinsulin levels showed a positive association, while leptin and adiponectin showed a negative association with diabetes. With regard to obesity, leptin and proinsulin had a positive association, while adiponectin had a negative association. Proinsulin levels showed an inverse association with adiponectin indicating a possible link between insulin secretion and insulin resistance.

Prognostic value of 99mTc-Sestamibi stress Myocardial Perfusion Single Photon Emission Computed Tomography (SPECT) in ischemic heart disease.

AIM: To study prognostic value of 99mTc-Sestamibi stress Myocardial Perfusion Single Photon Emission Computed Tomography (SPECT) in suspected or diagnosed ischemic heart disease, in an urban Indian population. METHODS: Eight hundred and eighty one patients with clinically suspected or diagnosed ischemic heart disease who underwent 99mTc-MIBI stress-rest Myocardial Perfusion SPECT (MPS) between 1st February 2001 to October 2002 were followed up for 14 +/- 2 months after the scan by questionnaire and telephonic interview with queries about cardiac death, myocardial infarct (hard events) and admission for unstable angina, CABG and PTCA (soft events). Patients were classified into pre-test: low, intermediate or high risk subsets based on clinical risk factors and ECG criteria. They were then reclassified based on MPS scan into post-test: high, intermediate and low risk subsets. Subsequent cardiac event rate was compared in the three subsets. A 12-lead ECG was an integral component of the stress MPS evaluation. RESULTS: MPS changed (1) the pre-test low risk category in 114 out of 613 patients to intermediate and 102 to high risk; (2) pre-test intermediate risk in 110 patients out of 163 to low risk and 19 patients to high risk (3) pre-test high risk category in 56 patients out of 105 to low risk and 28 to intermediate risk (total change 429 out of 881 patients). The hard cardiac event rate at one year was less than 0.5% in low risk, 2.3% in intermediate risk and 4.2% in high risk group. CONCLUSIONS: 99mTc-Sestamibi stress SPECT MPS thus provided incremental information for prognostic evaluation of patients with suspected or diagnosed coronary artery disease by assessing the effect of ischemic burden on LV function. This incremental information is crucial since coronary arteriography alone is not enough for prognosis and management decisions. Patients with a normal or low risk MPS have generally a benign prognosis with a low annual hard cardiac event rate of 0.5%. Future challenge is to identify high risk subsets within this group, with CT coronary calcium score > or = 100 and inflammation markers such as high hsCRP so that more aggressive secondary preventive measures can be instituted to prevent future hard cardiac events.

Free radicals and antioxidants in human health: current status and future prospects.

Free radicals and related species have attracted a great deal of attention in recent years. They are mainly derived from oxygen (reactive oxygen species/ROS) and nitrogen (reactive nitrogen species/RNS), and are generated in our body by various endogenous systems, exposure to different physicochemical conditions or pathophysiological states. Free radicals can adversely alter lipids, proteins and DNA and have been implicated in aging and a number of human diseases. Lipids are highly prone to free radical damage resulting in lipid peroxidation that can lead to adverse alterations. Free radical damage to protein can result in loss of enzyme activity. Damage caused to DNA, can result in mutagenesis and carcinogenesis. Redox signaling is a major area of free radical research that is attracting attention. Nature has endowed us with protective antioxidant mechanisms- superoxide dismutase (SOD), catalase, glutathione, glutathione peroxidases and reductase, vitamin E (tocopherols and tocotrienols), vitamin C etc., apart from many dietary components. There are epidemiological evidences correlating higher intake of components/ foods with antioxidant abilities to lower incidence of various human morbidities or mortalities. Current research reveals the different potential applications of antioxidant/free radical manipulations in prevention or control of disease. Natural products from dietary components such as Indian spices and medicinal plants are known to possess antioxidant activity. Newer and future approaches include gene therapy to produce more antioxidants in the body, genetically engineered plant products with higher level of antioxidants, synthetic antioxidant enzymes (SOD mimics), novel biomolecules and the use of functional foods enriched with antioxidants.

Beneficial effects of yoga lifestyle on reversibility of ischaemic heart disease: caring heart project of International Board of Yoga.

OBJECTIVES: Yoga based lifestyle modifications have been earlier shown to be beneficial in coronary artery disease in a small number of patients. We evaluated the role of lifestyle modification based on Yoga techniques, stress management and dietary modifications in retardation of coronary artery disease. METHODS: This prospective, controlled, open trial included angiographically proven coronary artery disease patients (71 patients in study group and 42 patients in control group). They were assessed clinically, by biochemical parameters, stress myocardial perfusion and function studies and coronary angiography and on psychological parameters. The study group patients were given a family based Yoga Programme which included, control of risk factors, dietary modifications and stress management for a period of one year. The patients were assessed at baseline, at frequent intervals and at the end of one year. RESULTS: At the end of one year of yoga training, statistical significant changes (P<0.05) were found in serum total cholesterol (reduction by 23.3% in study group patients as compared to 4.4% in controls); serum LDL cholesterol (reduction of 26% in study group patients as compared to 2.6% in the control group), regression of disease (43.7% of study group patients v/s 31% control group on MPI and 70.4% of study group v/s 28% of control group on angiography) arrest of progression (46.5% study group v/s 33.3% control group on MPI) and progression (9.9% of study group vs 35.7% of controls on MPI, 29.6% of study group v/s 60.0% of controls on angiography). At the end of the study improvement in anxiety scores was concordant with the improvement seen in the MPI. No untoward effects of the therapy were observed. CONCLUSION: Yoga based lifestyle modifications help in regression of coronary lesions and in improving myocardial perfusion. This is translated into clinical benefits and symptomatic improvement.

Hypertension: molecular approach.

The so-called essential hypertension is not a single entity but a mixed bag with several polygenic quantitative traits acting in concert in different combinations in different individuals. This review collates all published information from different centres using different approaches to identify candidate genes in human hypertension. 1) gene targeting approach in animal models of HT (Smithies and Maeda, 1995); 2) identification of 874 candidate SNPs in 75 candidate genes for human HT (Halushka et al, 1999); 3) comparative genomic approach translating QTLs between rat and human HT, to identify 26 chromosome regions on 16 autosomes (Stoll M et al, 2000); 4) Ten centimorgan genome-wide scan done on 2010 affected sibling pairs drawn from 1599 severely hypertensive families (Caulfield et al, 2003). The molecular mechanisms of various molecules involved in the homeostasis of blood pressure are discussed. NO, O2, PG12, EDHF, endothelin, IL-6, selectin, phospholipase A2G1B, BH4, SOD, IRS-1, adrenomedullin, PAMP, CGRP, ANP, bradykinin and bombesin; adducin alpha, beta, gamma, SAH, renin, angiotensinogen. angiotensin II, aldosterone CYP11B1, mineralocorticoid receptors, 11betaHSD, DBH, PNMT, beta2adrenoreceptors, and genes related to ion transport-sodium-lithium cotransporters, ENaC, NaCl cotransporters NKCC2, KCNJ and NaKATPase. Altered gene expression in fetus due to maternal malnutrition also "programmes" for adult hypertension.

Treadmill ECG test combined with myocardial perfusion imaging for evaluation of coronary artery disease: analysis of 340 cases.

AIM: Using Coronary arteriogram as the gold standard, stress ECG (Treadmill Test - TMT) has a sensitivity of 68% and specificity (77%) for the detection of coronary artery disease (CAD). Stress myocardial perfusion imaging (MPI) with Tc-99m Sestamibi gated SPECT has a sensitivity of 85% and specificity of 90%. The aim of the study was to ascertain if the combined use of the two physiologic tests will raise the predictive value for the presence or absence of physiologically significant CAD to 100%. METHODS AND MATERIAL: Three hundred and fourty patients (200 with suspected and 140 proved CAD) were studied with the same day rest and stress protocol. A rest MPI image was acquired with 8 mCi Tc-99m Sestamibi, followed by TMT; at the peak exercise 20 mCi tracer was injected and post-stress MPI image was acquired after 1 hour. 12-lead ECG at rest and during stress and recovery period was analyzed. RESULTS: Out of 200 patients with a prior probability of CAD 40-50% (151 with pain in chest, 81 with shortness of breath on exertion and 68 asymptomatic high risk for CAD--more than 5/9 risk factors), a normal stress MPI result in 150 patients excluded the probability of physiologically significant CAD. Fifty patients with abnormal stress MPI were refered for coronary arteriography. Stress ECG had 17% "false negative" and 23% "false positive" compared to stress MPI. In this group out of 140 known CAD, (56 post-infarct, 52 post-CABG and 32 post-PTCA), all sent for evaluation of ischemic symptoms, MPI documented 101 infarcts (fixed defects with no wall motion and thickening), 20 of them were "silent" (with no history of previous infarct) ECG did not help in picking them up. 58/101 infarcts had only fixed defects while 43/101 were accompanied by reversible ischaemia in same or other vascular territories. Thirty nine patients showed only reversible ischaemia without any infarct. Risk stratification was possible based on the extent and severity of the perfusion defects and number of territories in which defects were seen, rest LVEF, size of LV and transient dilation CONCLUSIONS: Combined stress ECG and stress MPI perform "gate keeper" function for referral for angiography, as well as for risk stratification of those who already have coronary angiograms. Decisions for revascularization should be based on combined evaluation--a shift from stenosis- based to ischaemic--based evaluation. Success or failure of revascularization was also documented by this evaluation.