Muzammil Ali

Pomegranate (Punica granatum) purified polyphenol extract inhibits influenza virus and has a synergistic effect with oseltamivir

Influenza epidemics cause numerous deaths and millions of hospitalizations each year. Because of the alarming emergence of resistance to anti-influenza drugs, there is a need to identify new naturally occurring antiviral molecules. We tested the hypothesis that pomegranate polyphenol extract (PPE) has anti-influenza properties. Using real time PCR, plaque assay, and TCID 50% hemagglutination assay, we have shown that PPE suppresses replication of influenza A virus in MDCK cells. PPE inhibits agglutination of chicken red blood cells (cRBC) by influenza virus and is virucidal. The single-cycle growth conditions indicated that independent of the virucidal effect PPE also inhibits viral RNA replication. PPE did not alter virus ribonucleoprotein (RNP) entry into nucleus or translocation of virus RNP from nucleus to cytoplasm in MDCK cells. We evaluated four major Polyphenols in PPE (ellagic acid, caffeic acid, luteolin, and punicalagin) and demonstrated that punicalagin is the effective, anti-influenza component of PPE. Punicalagin blocked replication of the virus RNA, inhibited agglutination of chicken RBCs by the virus and had virucidal effects. Furthermore, the combination of PPE and oseltamivir synergistically increased the anti-influenza effect of oseltamivir. In conclusion, PPE inhibited the replication of human influenza A/Hong Kong (H3N2) in vitro. Pomegranate extracts should be further studied for therapeutic and prophylactic potential especially for influenza epidemics and pandemics.

Influenza virus directly infects, inflames, and resides in the arteries of atherosclerotic and normal mice

OBJECTIVE Influenza can trigger heart attacks, and vaccination against influenza reduces the risk of cardiovascular events. Currently, it is believed that influenza virus in general does not disseminate to extra-pulmonary tissues. We assessed the vascular effects of influenza infection and whether the virus can directly infect atherosclerotic arteries in mice. METHODS/RESULTS We intranasally infected 4 different types of mice--atherosclerotic apo E-deficient (our primary model), LDL receptor knockout, C57BL/6, and outbred Swiss--with influenza A/HK (H3/N2) virus. On day 7 after infection, we cultured viable virus from lung, aorta, and heart tissue, but not from the blood of apo E-deficient mice. Immunofluorescence studies showed influenza A virus NP1 protein and real time polymerase chain reaction (PCR) assay showed RNA in the aorta of infected apo E-deficient mice. Infected mice had significantly higher blood levels of chemokines and cytokines than control mice. At the local level, gene expression for several chemokines and cytokines was increased and eNOS expression was decreased. Infected mice had a higher density of macrophages in plaque than did control mice. CONCLUSIONS We have shown for the first time that influenza virus can directly infect and reside in atherosclerotic arteries and that infection was associated with systemic and arterial-level pro-inflammatory changes.

Influenza and atherosclerosis: vaccination for cardiovascular disease prevention.

In both animal and human studies, strong prothrombotic and pro-inflammatory effects have been observed after influenza infection. Influenza is an important trigger for acute coronary syndromes, and it has been shown that in the US it may cause up to 90,000 deaths per year simply by triggering fatal myocardial infarctions. Multiple case-control and cohort studies have shown that the influenza vaccine has a marked protective effect against cardiovascular events, decreasing the incidence of these events by 20 – 70% in the settings of primary and secondary prevention. Although influenza vaccination is an extremely cost-effective method of cardiovascular protection and is recommended for all patients with cardiac diseases, it is largely underused in these patients. Therefore, increased efforts should be directed towards educating physicians and patients about the benefits of influenza vaccination in patients with coronary heart disease.

Increased oxidative stress in atherosclerosis-predisposed regions of the mouse aorta.

AIMS The localization of atherosclerotic lesions to predictable regions in mammalian arteries has been recognized for over a century. We sought to investigate the association between oxidative stress and regional susceptibility of the mouse aorta to atherosclerosis. MAIN METHODS En face confocal microscopy was employed to assess oxidative stress in the aortic intima of atherosclerosis-susceptible and protected regions of wild-type C57BL/6 mouse. Expression of reactive oxygen species and antioxidant producing genes were compared in endothelial cells from the susceptible and protected regions. KEY FINDINGS In vivo administration of redox-sensitive fluorescent dyes revealed an increase in the production of reactive oxygen species (ROS) in the atherosclerosis-susceptible regions relative to the protected regions. In contrast, Hoechst a redox-insensitive dye distributed evenly in the susceptible and protected regions. Accumulation of superoxide in the susceptible regions of the aorta was significantly blocked by the administration of Diphenyleneiodonium, a flavoprotein inhibitor. mRNA levels of superoxide-producing and scavenging enzymes were significantly increased in the regions predisposed to atherosclerosis. The regional difference in oxidative stress was at a lesser magnitude in BALB/c than the atherosclerosis-susceptible mouse (C57BL/6). SIGNIFICANCE Our study for the first time demonstrated an augmented oxidative stress in atherosclerosis-susceptible regions of the normal mouse aorta.

Oligonol a low molecular weight polyphenol of lychee fruit extract inhibits proliferation of influenza virus by blocking reactive oxygen species-dependent ERK phosphorylation

The emergence of resistance to anti-influenza drugs calls for the search for new antiviral molecules with different resistance profiles. Polyphenolic compounds are found in various plants and have antiviral and antioxidative properties. We tested the hypothesis that oligonol, a lychee fruit-derived low molecular weight polyphenol, possesses anti-influenza effects by inhibiting phosphorylation of extracellular-signal-regulated kinases (ERK). Real time PCR, plaque assay, and immunofluorescence techniques were used to study the effects of oligonol on proliferation of influenza virus. Oligonol inhibits influenza virus proliferation by blocking attachment of the virus to MDCK cells and by suppression of nuclear export of influenza virus ribonucleoprotein (RNP). Infection of MDCK cells with influenza virus leads to an increase in production of reactive oxygen species (ROS) and induction of a ROS-dependent ERK phosphorylation. Inhibition of ERK activation by a dominant negative mutant of ERK or N-acetyl-cysteine (NAC) leads to inhibition of influenza RNP nuclear export. Phorbol 12-myristate 13-acetate (PMA) induces ROS production, ERK phosphorylation and enhances influenza proliferation in MDCK cells. Oligonol and NAC inhibit PMA-induced ERK phosphorylation and ROS production. Our studies suggest that the underlying mechanism for the inhibitory effect of oligonol on influenza virus RNP nuclear export is blocking of ROS-dependent induction of ERK phosphorylation.

Inhibition of MLC phosphorylation restricts replication of influenza virus--a mechanism of action for anti-influenza agents.

Influenza A viruses are a severe threat worldwide, causing large epidemics that kill thousands every year. Prevention of influenza infection is complicated by continuous viral antigenic changes. Newer anti-influenza agents include MEK/ERK and protein kinase C inhibitors; however, the downstream effectors of these pathways have not been determined. In this study, we identified a common mechanism for the inhibitory effects of a significant group of anti-influenza agents. Our studies showed that influenza infection activates a series of signaling pathways that converge to induce myosin light chain (MLC) phosphorylation and remodeling of the actin cytoskeleton. Inhibiting MLC phosphorylation by blocking RhoA/Rho kinase, phospholipase C/protein kinase C, and HRas/Raf/MEK/ERK pathways with the use of genetic or chemical manipulation leads to the inhibition of influenza proliferation. In contrast, the induction of MLC phosphorylation enhances influenza proliferation, as does activation of the HRas/Raf/MEK/ERK signaling pathway. This effect is attenuated by inhibiting MLC phosphorylation. Additionally, in intracellular trafficking studies, we found that the nuclear export of influenza ribonucleoprotein depends on MLC phosphorylation. Our studies provide evidence that modulation of MLC phosphorylation is an underlying mechanism for the inhibitory effects of many anti-influenza compounds.

Lipoprotein-associated phospholipase A2: a cardiovascular risk predictor and a potential therapeutic target

Lipoprotein-associated phospholipase A2 (Lp-PLA2), present in the circulation and in atherosclerotic plaque, is an inflammatory marker with potential use as a predictor of cardiovascular risk and as a therapeutic target. Although Lp-PLA2 is associated with both LDL and HDL, it is important to determine whether Lp-PLA2 has a predominantly pro- or anti-atherogenic effect. Increasing evidence suggests a proatherogenic role for Lp-PLA2. Epidemiologic and clinical evidence suggests Lp-PLA2 is an independent predictor of risk and may be superior to other inflammatory markers owing to its specificity and minimal biovariation. Lp-PLA2 inhibitors currently being investigated in clinical trials are promising novel anti-inflammatory agents with a specificity for the vascular bed and a potential for decreasing plaque vulnerability.