Devendra K. Agrawal

Morphological and biochemical characterization and analysis of apoptosis

Apoptosis is a form of programmed cell death serving physiologic and homeostatic functions. However, recent evidence implicating apoptosis in the etiology and pathophysiology of known human diseases, such as heart diseases, cancer, AIDS, and neurodegenerative and autoimmune diseases are continually surfacing. This has spawned the need for identifying which methods are the most effective and well accepted to decipher its presence in a variety of research settings. We have therefore detailed the morphology and biochemical features of apoptotic cell death, with an emphasis on discriminating it from necrosis. In addition, we describe specific and selective techniques which are optimal to target hallmark apoptotic features, such as microscopy, Annexin V labeling, in situ nick-end labeling (TUNEL), and DNA fragmentation analysis by gel electrophoresis and ELISA for oligonucleosome-sized DNA. The advantages and disadvantages of each technique are discussed, as well as their experimental importance relative to one another. The methods have been described in a stepwise fashion, and can readily be applied in the majority of cell systems. Whether working on the tissue or single cell level, these methods are highly effective in qualifying and quantifying apoptosis. The application of these methods in conjunction with molecular techniques can further delineate the underlying mechanisms of apoptosis.

Insulin-like growth factor-1 and TNF-α regulate autophagy through c- jun N-terminal kinase and Akt pathways in human atherosclerotic vascular smooth cells

A balance between programmed cell death and survival of vascular smooth muscle cells (VSMC) in the fibrous cap, which is primarily composed of VSMC and extracellular matrix, appears to best correlate with plaque instability or stability and is controlled by growth factors and cytokines. Autophagy is also involved in programmed cell death. We assessed the effect of TNF‐α and insulin‐like growth factor‐1 (IGF‐1) on the expression of autophagic genes, microtubule‐associated protein 1 light chain 3 (MAPLC‐3) and Beclin‐1 in VSMC isolated from atherosclerotic plaques. Transmission electron microscopy showed a significantly higher number of vacuolated cells in the TNF‐α‐treated VSMC and a markedly lower number in the IGF‐1‐treated VSMC when compared with the untreated control group. TNF‐α‐induced MAPLC‐3 mRNA expression through c‐jun N‐terminal kinase and protein kinase B pathways and induced Beclin‐1 protein expression through the c‐jun N‐terminal kinase pathway. Expression of MAPLC‐3 and Beclin‐1 correlated with autophagic cell death of plaque VSMC. IGF‐1 inhibited MAPLC‐3 mRNA transcripts through the Akt pathway. These findings suggest that the expression of autophagy genes can be influenced by IGF‐1 and TNF‐α through c‐jun N‐terminal kinase or Akt pathways and autophagy might be involved in the regulation of plaque stability.

In stent restenosis: bane of the stent era

The long term outcome of stent implantation is affected by a process called in stent restenosis (ISR). Multiple contributory factors have been identified, but clear understanding of the overall underlying mechanism remains an enigma. ISR progresses through several different phases and involves numerous cellular and molecular constituents. Platelets and macrophages play a central role via vascular smooth muscle cell migration and proliferation in the intima to produce neointimal hyperplasia, which is pathognomic of ISR. Increased extracellular matrix formation appears to form the bulk of the neointimal hyperplasia tissue. Emerging evidence of the role of inflammatory cytokines and suppressors of cytokine signalling make this an exciting and novel field of antirestenosis research. Activation of Akt pathway triggered by mechanical stretch may also be a contributory factor to ISR formation. Prevention of ISR appears to be a multipronged attack as no therapeutic “magic bullet” exists to block all the processes in one go.

The regulatory role of TGF- β in airway remodeling in asthma

Both structural and inflammatory cells are capable of secreting transforming growth factor (TGF)‐β and expressing TGF‐β receptors. TGF‐β can induce multiple cellular responses including differentiation, apoptosis, survival and proliferation, and has been implicated in the development of several pathogenic conditions including cancer and asthma. Elevated levels of TGF‐β have been reported in the asthmatic airway. TGF‐β binds to its receptor complex and activates multiple pathways involving proteins such as Sma and Mad homologues, phosphatidylinositol‐3 kinase and the mitogen‐activated protein kinases, leading to the transcription of several genes. Cell type, cellular condition, and microenvironment, all play a role in determining which pathway is activated, which, in turn, is an indication of which gene is to be transcribed. TGF‐β has been shown to induce apoptosis in airway epithelial cells. A possible role for TGF‐β in the regulation of epithelial cell adhesion properties has also been reported. Enhancement of goblet cell proliferation by TGF‐β suggests a role in mucus hyper‐secretion. Elevated levels of TGF‐β correlate with subepithelial fibrosis. TGF‐β induces proliferation of fibroblast cells and their differentiation into myofibroblasts and extracellular matrix (ECM) protein synthesis during the development of subepithelial fibrosis. TGF‐β also induces proliferation and survival of and ECM secretion in airway smooth muscle cells (ASMCs), suggesting a possible cause of increased thickness of airway tissues. TGF‐β also induces the production and release of vascular endothelial cell growth factor and plasminogen activator inhibitor, contributing to the vascular remodeling in the asthmatic airway. Blocking TGF‐β activity inhibits epithelial shedding, mucus hyper‐secretion, angiogenesis, ASMC hypertrophy and hyperplasia in an asthmatic mouse model. Reduction of TGF‐β production and control of TGF‐β effects would be beneficial in the development of therapeutic intervention for airway remodeling in chronic asthma.

Immunobiology of Toll-like receptors: Emerging trends

Toll‐like receptors (TLR), a family of evolutionarily conserved pathogen recognition receptors, play pivotal role as primary sensors of invading pathogens. TLR identify molecular motifs of infectious agents (pathogen associated molecular patterns) and elicit an effective defensive response against them. Mammalian TLR derive their name from the Drosophila Toll protein, with which they share sequence similarity. So far, expression of 10 TLR is known in humans. The adaptor proteins, myeloid differentiation factor 88 and Toll IL‐1 receptor (TIR) domain containing adaptor inducing IFN‐β (TRIF) are the key players in the TLR signalling cascade leading to the activation of nuclear factor (NF)‐κB and interferon regulatory factor‐3, respectively. Targeted manipulation of the TLR signalling pathway has immense therapeutic potential and may eventually prove to be a boon in the development of innovative treatments for diverse disease conditions. There is accumulating evidence that TLR agonists have tremendous potential as novel therapeutic targets. In this review, we have discussed the immunobiology of TLR and emphasize significant advances made within the ever‐expanding field of TLR that provide intriguing insights efficacious in unravelling the complexities associated with TLR.

Airway Hyperresponsiveness, Late Allergic Response, and Eosinophilia Are Reversed with Mycobacterial Antigens in Ovalbumin-Presensitized Mice

Pretreatment with mycobacterial Ags has been shown to be effective in preventing allergic airway inflammation from occurring in a mouse model. Because most asthmatics are treated after the development of asthma, it is crucial to determine whether mycobacterial Ags can reverse established allergic airway inflammation in the presensitized state. Our hypothesis, based upon our previous findings, is that mycobacteria treatment in presensitized mice will suppress the allergic airway inflammation with associated clinical correlates of established asthma, with the noted exception of factors associated with the early allergic response (EAR). BALB/c mice sensitized and challenged with OVA were evaluated for pulmonary functions during both the EAR and late allergic response, and airway hyperresponsiveness to methacholine. Following this, sensitized mice were randomized and treated with placebo or a single dose (1 × 105 CFUs) of bacillus Calmette-Guérin (BCG) or Mycobacterium vaccae via nasal or peritoneal injection. One week later, the mice were rechallenged with OVA and methacholine, followed by bronchoalveolar lavage (BAL) and tissue collection. Mice treated with intranasal BCG were most significantly protected from the late allergic response (p < 0.02), airway hypersensitivity (p < 0.001) and hyperreactivity (p < 0.05) to methacholine, BAL (p < 0.05) and peribronchial (p < 0.01) eosinophilia, and BAL fluid IL-5 levels (p < 0.01) as compared with vehicle-treated, sensitized controls. Intranasal M. vaccae treatment was less effective, suppressing airway hypersensitivity (p < 0.01) and BAL eosinophilia (p < 0.05). No changes were observed in the EAR, BAL fluid IL-4 levels, or serum total and Ag-specific IgE. These data suggest that mycobacterial Ags (BCG≫M. vaccae) are effective in attenuating allergic airway inflammation and associated changes in pulmonary functions in an allergen-presensitized state.

Cellular, molecular and immunological mechanisms in the pathophysiology of vein graft intimal hyperplasia

Coronary artery disease, leading to myocardial infarction and ischaemia, affects millions of persons and is one of the leading causes of morbidity and mortality worldwide. Invasive techniques such as coronary artery bypass grafting are used to alleviate the sequelae of arterial occlusion. Unfortunately, restenosis or occlusion of the grafted conduit occurs over a time frame of months to years with a gradual reduction in patency, especially in vein grafts. The events leading to intimal hyperplasia (IH) formation involve numerous cellular and molecular components. Various cellular elements of the vessel wall are involved as are leucocyte–endothelial interactions that trigger the coagulation cascade leading to localized thrombus formation. Subsequent phenotypic modification of the medial smooth muscle cells and their intimal migration is the basis of the lesion formation that is thought to be propagated by an immune‐mediated reaction. Despite intense scrutiny, the pathophysiology of IH remains an enigma. Although several growth factors, cytokines and numerous other biomolecules have been implicated and their relationship to prohyperplasia pathways such as the phosphatidyl‐inositol 3‐kinase (PI3K)‐Akt pathway has been established, many pieces of the puzzle are still missing. An in‐depth understanding of early vein graft adaptation and progression is necessary to improve the long‐term prognosis and develop more effective therapeutic measures. In this review, we have critically evaluated and summarized the literature to elucidate and interlink the numerous established and emerging factors that play a key role in the development of IH leading to vein graft restenosis.

DNA methylation in breast and colorectal cancers

DNA methylation is one of several epigenetic changes observed in cells. Aberrant methylation of tumor suppressor genes, proto-oncogenes, and vital cell cycle genes has led many scientists to investigate the underlying cellular mechanisms of DNA methylation under normal and pathological conditions. Although DNA methylation is necessary for normal mammalian embryogenesis, both hypo- and hypermethylation of DNA are frequently observed in carcinogenesis and other pathological disorders. DNA hypermethylation silences the transcription of many tumor suppressor genes, resulting in immortalization of tumor cells. The reverse process, demethylation and restoration of normal functional expression of genes, is augmented by DNA methylation inhibitors. Recent studies suggest that DNA hypomethylation may also control gene expression and chromosomal stability. However, the roles of and relationship between hypomethylation and hypermethylation are not well understood. This review provides a brief overview of the mechanism of DNA methylation, its relationship to extrinsic stimulation including dietary intake and aging, and of abnormally methylated DNA in breast and colorectal cancers, which could be used as prognostic and diagnostic markers.

Vitamin D and inflammatory diseases

Beyond its critical function in calcium homeostasis, vitamin D has recently been found to play an important role in the modulation of the immune/inflammation system via regulating the production of inflammatory cytokines and inhibiting the proliferation of proinflammatory cells, both of which are crucial for the pathogenesis of inflammatory diseases. Several studies have associated lower vitamin D status with increased risk and unfavorable outcome of acute infections. Vitamin D supplementation bolsters clinical responses to acute infection. Moreover, chronic inflammatory diseases, such as atherosclerosis-related cardiovascular disease, asthma, inflammatory bowel disease, chronic kidney disease, nonalcoholic fatty liver disease, and others, tend to have lower vitamin D status, which may play a pleiotropic role in the pathogenesis of the diseases. In this article, we review recent epidemiological and interventional studies of vitamin D in various inflammatory diseases. The potential mechanisms of vitamin D in regulating immune/inflammatory responses in inflammatory diseases are also discussed.

Pathogenesis of allergic airway inflammation

Advances have been made in defining the mechanisms for the control of allergic airway inflammation in response to inhaled antigens. Several genes, including ADAM33, DPP10, PHF11, GPRA, TIM-1, PDE4D, OPN3, and ORMDL3, have been implicated in the pathogenesis and susceptibility to atopy and asthma. Growing evidence associates asthma with a systemic propensity for allergic T-helper type 2 cytokines. Disordered coagulation and fibrinolysis also exacerbate asthma symptoms. Balance among functionally distinct dendritic cell subsets contributes to the outcome of T-cell-mediated immunity. Allergen-specific T-regulatory cells play a pivotal role in the development of tolerance to allergens and immune suppression. The major emphasis on immunotherapy for asthma during the past decade has been to direct the immune response to a type 1 response, or immune tolerance. In this review, we discuss the current information on the pathogenesis of allergic airway inflammation and potential immunotherapy, which could be beneficial in the treatment of airway inflammation, allergy, and asthma.